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Pp150722 1263.1273The Stromal Chloroplast Deg7 Protease Participates inthe Repair of Photosystem II after Photoinhibitionin Arabidopsis1[W][OA] Xuwu Sun, Tingjiao Fu, Ning Chen, Jinkui Guo, Jinfang Ma, Meijuan Zou, Congming Lu, and Lixin Zhang* Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy ofSciences, Beijing 100093, China Light is the ultimate source of energy for photosynthesis; however, excessive light leads to photooxidative damage and hencereduced photosynthetic efficiency, especially when combined with other abiotic stresses. Although the photosystem II (PSII)reaction center D1 protein is the primary target of photooxidative damage, other PSII core proteins are also damaged anddegraded. However, it is still largely unknown whether degradation of D1 and other PSII proteins involves previouslyuncharacterized proteases. Here, we show that Deg7 is peripherally associated with the stromal side of the thylakoidmembranes and that Deg7 interacts directly with PSII. Our results show that Deg7 is involved in the primary cleavage ofphotodamaged D1, D2, CP47, and CP43 and that this activity is essential for its function in PSII repair. The double mutants deg5deg7 and deg8 deg7 showed no obvious phenotypic differences under normal growth conditions, but additive effects wereobserved under high light. These results suggest that Deg proteases on both the stromal and luminal sides of the thylakoidmembranes are important for the efficient PSII repair in Arabidopsis (Arabidopsis thaliana).
Chloroplasts of higher plants carry out one of the mass proteins (Nelson and Yocum, 2006). The PSII most important biochemical reactions: the capture of reaction center D1 protein has been identified among light energy and its conversion into chemical energy.
PSII proteins as the primary target of light-induced Although light is the ultimate source of energy for damage (Kyle et al., 1984; Mattoo et al., 1984; Ohad photosynthesis, it can also be harmful to plants.
et al., 1984; Adir et al., 1990), but several studies have Light-induced loss of photosynthetic efficiency, which shown that the D2, CP47, and CP43 proteins are is generally termed as photoinhibition, limits plant degraded under photoinhibitory conditions (Schuster growth and lowers productivity, especially when com- et al., 1988; Yamamoto and Akasaka, 1995; Jansen et al., bined with other abiotic stresses.
1999; Adir et al., 2003). Moreover, several small PSII The main target of photoinhibition is PSII, which subunits, such as PsbH, PsbW, and Cyt b catalyzes the light-dependent water oxidation con- found to be frequently replaced within PSII (Hagman comitantly with oxygen production (for review, see et al., 1997; Ortega et al., 1999; Bergantino et al., 2003).
Prasil et al., 1992; Aro et al., 1993; Adir et al., 2003). In Evidence for the involvement of two families of pro- higher plants, PSII consists of more than 20 subunits, teases, FtsH and Deg, in the degradation of the D1 including the reaction center D1 and D2 proteins, protein in thylakoids of higher plants has been re- cytochrome (Cyt) b , the light-harvesting chlorophyll cently described (Lindahl et al., 1996, 2000; Bailey a-binding proteins CP47 and CP43, the oxygen-evolv- et al., 2002; Sakamoto et al., 2003; Silva et al., 2003; ing 33-kD protein (PsbO), and several low molecular Kapri-Pardes et al., 2007; Sun et al., 2007a, 2007b).
However, it is still largely unknown whether degra- dation of D1 and other PSII proteins involves previ- This work was supported by the Major State Basic Research Development Program (grant no. 2009CB118500), the National Nat- ously uncharacterized proteases.
ural Science Foundation of China (grant nos. 30725003 and DegP (or HtrA) proteases were initially identified 30870182), and the Frontier Project of Knowledge Innovation Engi- based on the fact that they are required for the survival neering of the Chinese Academy of Sciences (grant no. KJCX2–SW– of Escherichia coli at high temperatures and for the deg- radation of abnormal periplasmic proteins (Lipinska * Corresponding author; e-mail email@example.com.
et al., 1988; Strauch and Beckwith, 1988). DegP is an The author responsible for distribution of materials integral to the ATP-independent Ser endopeptidase, and it contains a findings presented in this article in accordance with the policy trypsin-like protease domain at the N terminus, fol- described in the Instructions for Authors (www.plantphysiol.org) is: lowed by two PDZ domains (Gottesman, 1996; Pallen Lixin Zhang (firstname.lastname@example.org).
and Wren, 1997; Clausen et al., 2002). PDZ domains The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a sub- appear to be important for complex assembly and substrate binding through three or four residues in the C terminus of their target proteins (Doyle et al., Plant PhysiologyÒ, March 2010, Vol. 152, pp. 1263–1273, www.plantphysiol.org Ó 2010 American Society of Plant Biologists 1996; Harris and Lim, 2001). DegP switches between may not be involved in D1 degradation in vivo chaperone and protease functions in a temperature- (Huesgen et al., 2006).
dependent manner. The chaperone function dominates Here, we have expressed and purified a recombi- at low temperatures, and DegP becomes proteolytically nant DegP protease, His-Deg7. In vitro experiments active at elevated temperatures (Spiess et al., 1999).
showed that His-Deg7 is proteolytically active toward Crystal structures of different members of the DegP the PSII proteins D1, D2, CP43, and CP47. In vivo protein family (Krojer et al., 2002; Li et al., 2002; Kim analyses of a deg7 mutant revealed that the mutant is et al., 2003; Wilken et al., 2004) have revealed the more sensitive to high light stress than the wild-type structure-function relationship of these PDZ-containing plants. We demonstrated that Deg7 is a chloroplast proteases. Trimeric DegP is the functional unit, and the stroma protein associated with the thylakoid mem- hexameric DegP is formed via the staggered association branes and that it interacts with PSII, which suggests of trimers (Clausen et al., 2002; Kim and Kim, 2005). At that it can cleave the stroma-exposed region of sub- normal growth temperatures, the active site of the strate proteins. Our results also provide evidence that protease is located within the chamber of hexameric Deg7 is important for maintaining PSII function.
DegP, which is not accessible to the substrates. How-ever, at high temperatures, conformational changesinduce the activation of the protease function (Krojeret al., 2002). Recent studies have shed light on the substrate binding-induced formation of larger oligo- Deg7 Is a Chloroplast Stromal Protein meric complexes of DegP (Jiang et al., 2008; Krojer et al.,2008).
Deg7 was predicted to be located in chloroplasts by In Arabidopsis (Arabidopsis thaliana), 16 genes cod- LOCtree. To determine the subcellular localization, the ing for DegP-like proteases have been identified, and N-terminal 243 amino acids of Deg7 were fused to the at least seven gene products are predicted to be located N terminus of the synthetic GFP under the control of in chloroplasts (Kieselbach and Funk, 2003; Huesgen the 35S promoter with a S65T mutation. The Deg7-GFP et al., 2005; Adam et al., 2006; Sakamoto, 2006; Kato fusion proteins were transiently expressed in proto- and Sakamoto, 2009). Based on proteomic data, four plasts, and GFP fluorescence of Deg7-GFP fusion Deg proteases have been shown to be localized to the proteins was colocalized with the chloroplastic chlo- chloroplast (Peltier et al., 2002; Schubert et al., 2002) rophyll, which is consistent with the signals observed and functionally characterized. Deg1, Deg5, and Deg8 when the GFP was fused to the transit peptide of are located in thylakoid lumen, and Deg2 is peripher- FtsH11 (Sakamoto et al., 2003). When the targeting ally associated with the stromal side of thylakoid signals of the fibrillarin and FRO1 proteins from membranes (Itzhaki et al., 1998; Haußu¨hl et al., 2001; Arabidopsis were fused to GFP (Cai et al., 2009), Sun et al., 2007a). Recombinant DegP1, now renamed GFP signals were located in the nucleus and mito- Deg1, has been shown to be proteolytically active chondria, respectively (Supplemental Fig. S1). Thus, toward thylakoid lumen proteins such as plastocyanin these results indicate that Deg7 is targeted to the and PsbO of PSII in vitro (Chassin et al., 2002). A 5.2- kD C-terminal fragment of the D1 protein was de- Next, intact chloroplasts were isolated, separated tected in vitro after incubation of recombinant Deg1 into chloroplast stroma and thylakoid membrane frac- with inside-out thylakoid membranes. In transgenic tions, and then subjected to immunoblot analysis with plants with reduced levels of Deg1, fewer of its 16- a specific antibody against Deg7. The purity of chlo- and 5.2-kD degradation products were observed roplasts was confirmed by immunoblot analysis using (Kapri-Pardes et al., 2007). Deg5 and Deg8 form a the antibodies against the nuclear histone H1 protein, dodecameric complex in the thylakoid lumen, and the mitochondrial NDUFS1 protein, the cytosolic recombinant Deg8 is able to degrade the photodam- DHAR2 protein, and the thylakoid membrane protein aged D1 protein of PSII in an in vitro assay (Sun et al., LHCII (Supplemental Fig. S2). The identities of the 2007a). The 16-kD N-terminal degradation fragment of different fractions were further verified by immuno- the D1 protein was detected in wild-type plants but blot analyses with the antibodies against the thylakoid not in a deg5 deg8 double mutant after high-light membrane protein LHCII, the peripheral protein treatment. The deg5 deg8 double mutant showed in- Deg2, and the stromal protein RbcL (Fig. 1A). To creased sensitivity to high light and high temperature determine the localization of Deg7, polyclonal antise- in terms of growth and PSII activity compared with the rum was raised and immunoblot analysis of chloro- single mutants deg5 and deg8, suggesting that Deg5 plast protein showed the specificity of the Deg7 and Deg8 have overlapping functions in the primary antibody (Supplemental Fig. S3). We found that Deg7 cleavage of the CD loop of the D1 protein (Sun et al., is located in chloroplasts and that the majority of 2007a, 2007b). In vitro analysis has demonstrated that Deg7 is present in chloroplast stromal fractions (Fig.
recombinant stroma-localized Deg2 was also shown to 1A). To further investigate whether Deg7 is a periph- be involved in the primary cleavage of the DE loop of eral or an intrinsic membrane protein, the thylakoid the D1 protein (Haußu¨hl et al., 2001). However, anal- membranes were incubated with trypsin. Protease ysis of a mutant lacking Deg2 suggested that Deg2 digestion assays showed that Deg7 could be degraded Plant Physiol. Vol. 152, 2010
PSII Protein Degradation by DEG7 protein and is associated with the membranes. Next, toexamine the strength of Deg7's membrane association,we treated the thylakoid membranes with salts andchaotropic agents. Washing the membranes with 0.25M NaCl did not release the Deg7 from the membranes,but Deg7 was barely detectable after washing themembranes with 0.2 M Na CO , 1 As a control, we found that the integral membraneprotein CP47 was not released from the membranes bysuch treatments. High-light treatment increased theamount of Deg7 associated with the thylakoid mem-branes, and the levels of Deg7 in the stromal fractionsremained almost unchanged after exposure to high-light treatment (Fig. 1C). This may reflect an increasedexpression of Deg7 after high-light treatment.
Proteolytic Activity and Physiological Targets of Deg7 The increased association of Deg7 with the mem- branes suggested that it might function in the degra-dation of photosynthetic proteins. To examine theproteolytic activity of the recombinant His-taggedDeg7, we incubated it with a mixture of a-, b-, and k-forms of casein. b-Casein is the preferred substratefor assaying bacterial DegP activity under in vitroconditions (Lipinska et al., 1990). b-Casein was effi-ciently degraded by the fractions containing overex-pressed Deg7, and the a- and k-forms remainedunchanged under our experimental conditions (Sup-plemental Fig. S4). More than 50% of the b-casein wasdegraded within the first 30 min. These results indi-cated that the recombinant Deg7 was proteolyticallyactive.
Next, we examined whether Deg7 is proteolytically active toward photosynthetic proteins. Since the DegPprotease has been shown to degrade damaged ormisfolded proteins (Gottesman, 1996; Pallen andWren, 1997; Clausen et al., 2002), the thylakoid mem- Figure 1. Immunolocalization and expression of Deg7. A, Intact branes used in this experiment were first subjected to chloroplasts were separated into stroma and thylakoid membranes.
high-light treatment (1,800 mmol m22 s21) for 90 min at Proteins in each fraction were resolved by SDS-PAGE, and the location 0°C. Treatment with high light causes irreversible of Deg7 was determined by immunoblot analysis. B, The thylakoid oxidative protein damage, which subsequently in- membranes from mature Arabidopsis leaves were treated with trypsin duces conformational changes in proteins, making for 30 min at 25°C or incubated with 250 mM NaCl, 200 mM Na2CO3, them vulnerable to proteolytic degradation (Prasil M urea, and 0.05% Triton X-100 for 30 min at 4°C. After these treatments, the thylakoid proteins were separated by SDS-PAGE et al., 1992; Aro et al., 1993; Andersson and Aro, and immunodetected with anti-Deg7, anti-CF1b, anti-PsbO, and anti- 2001). The thylakoid membranes isolated from leaves CP47 antibodies. CF1b, PsbO, and CP47 were used as markers.
exposed to high light were treated with 50 mmol Membrane preparations that had not been subjected to any of these EDTA to inhibit the endogenous FtsH proteases. They treatments were used as controls. The experiments were repeated five were then incubated with or without recombinant times independently, and similar results were obtained. Results of a Deg7 in the dark at 37°C. In the absence of recombi- representative experiment are shown. C, Expression of Deg7 associated nant Deg7, the D1, D2, CP43, and CP47 protein levels with the thylakoid membranes. The detached leaves, floating adaxial remained unchanged and no degradation products side up on water, were illuminated with a photon flux density of 1,800 were detected (Fig. 2A). However, when recombinant mmol m22 s21 for 1, 2, and 4 h. Then, the thylakoid and stroma proteins Deg7 was added to isolated thylakoid membranes, the were prepared, and the proteins were subjected to SDS-PAGE andimmunoblot analyses.
amounts of full-length D1, D2, CP43, and CP47 pro-teins decreased with time, and polypeptides of 20 kDfor D1, 29 kD for D2, 19 kD for CP43, and 19 kD for by trypsin, while the lumen protein PsbO was pro- CP47 were immunodetected using antibodies raised tected from trypsin treatment (Fig. 1B). These results against the DE loop of the D1 protein, D2 protein, indicate that the Deg7 protein is a chloroplast stroma CP43 protein, and CP47 protein, respectively (Fig. 2A).
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using isolated PSII and anti-CP47 for calibration (datanot shown), which showed a large excess of PSII overprotease.
Functions of PDZ Domains with Respect to the Activitiesof Deg7 To determine the function of the PDZ domains in regulating the proteolytic activities of Deg7, we ex-pressed a series of Deg7 proteases that contain zero totwo PDZ domains in vitro (Supplemental Figs. S6–S8).
Regardless of the presence of either one or two PDZdomains in the truncated Deg7 protein, the levels ofPSII core proteins D1, D2, CP43, and CP47 decreasedand the specific degradation fragments of their corre-sponding proteins were not detected. When the trun-cated Deg7 without any PDZ domains was incubatedwith the thylakoid membranes, the amounts of notonly PSII core proteins D1, D2, CP43, and CP47 butalso LHCII, PsaA/B, Cyt f, and CF1b were reduced.
Association of Deg7 with PSII The proteolytic activity of Deg7 toward PSII core proteins suggests that Deg7 is associated with PSII. Totest this possibility, the thylakoid protein complexesseparated on a blue native gel were further subjectedto denaturing SDS-PAGE and immunoblot analysisusing specific antibodies. Our results showed that theDeg7 protein and PSII complex comigrated on the gel(Supplemental Fig. S9). Next, pull-down experimentswere performed with recombinant Deg7 fused to anN-terminal His tag. The purified His-Deg7 fusionprotein was incubated with n-dodecyl b-D-maltoside(DM)-solubilized thylakoid membranes. After wash- Figure 2. Degradation of photodamaged PSII proteins by recombinant ing nickel-nitrilotriacetic acid agarose (Ni-NTA) resin Deg7. A, High-light-treated thylakoid membranes (10 mg of chloro- with buffer, the bound proteins were separated by phyll) were incubated with (+Deg7) or without (2Deg7) purified SDS-PAGE and examined by immunoblot analysis recombinant Deg7 (0.5 mg), and the degradation of thylakoid proteins (Fig. 3). The PSII proteins D1, D2, and CP47 were was detected by SDS-PAGE and immunoblot analyses with anti-D1, detected when the His-Deg7 fusion protein was used anti-D2, anti-CP43, anti-CP47, anti-Cyt f, anti-CF1b, anti-PsaA/B, andanti-LHCII antibodies. Molecular mass markers (kD) are to the left. B,Thylakoid membranes isolated from wild-type (WT) and deg7 leaveswere exposed to high-light illumination for 2 h. The thylakoid proteinswere separated by SDS-PAGE and immunodetected with antibodiesraised against the D1, D2, CP43, and CP47 proteins. The degradationfragments are indicated with arrows. Similar results were obtained intwo additional independent experiments. Results from a representativeexperiment are shown.
Immunoblot analysis with anti-Cyt f, anti-LHCII, anti-CF1b, and anti-PsaA/B antibodies showed that thelevels of these proteins remained almost constant in the presence of recombinant Deg7 (Fig. 2A). When the Interaction of Deg7 with PSII. Ten micrograms of the Deg7- His tag fusion protein coupled to Ni-NTA resin was incubated with 100 thylakoid membranes were isolated from the plants mg of DM-solubilized thylakoid membranes. Bound proteins were that were not exposed to high-light treatment, the eluted, separated by SDS-PAGE, and subjected to immunoblot analysis photosynthetic proteins were not degraded by recom- with anti-PsaA/B, anti-D1, anti-D2, anti-CP47, anti-Cyt f, and anti- binant Deg7 (Supplemental Fig. S5). The relative mole CF1b antibodies. Similar results were obtained in two additional ratio of Deg7 and PSII was estimated to be about 1:10 Plant Physiol. Vol. 152, 2010
PSII Protein Degradation by DEG7 in the assay. In contrast, the PSI protein PsaA/B, the Increased Sensitivity of the deg7 Mutants to High Light protein Cyt f, and the ATP synthase protein CF1b were not detected when the solubilized thyla- To determine whether Deg7 is involved in photo- koid membrane and the resin were incubated in the inhibition and repair processes, the F /F presence or absence of His-Deg7 (Fig. 3).
sured in the wild-type and deg7 plants under high-lightillumination (1,800 mmol m22 s21). In the absence oflincomycin, within 2 h of illumination at a lightintensity of 1,800 mmol m22 s21, F /F declined in the T-DNA Insertion Mutants of deg7 wild-type and mutant leaves to about 53% and 39%, To study the in vivo function of Deg7, we obtained respectively, of the dark-adapted values (Fig. 5A).
Arabidopsis lines from the SALK collection. The deg7 These results clearly demonstrate the increased pho- mutant line (SALK_075584) contains a T-DNA inser- tosensitivity of the mutants. In the presence of linco- tion within the 1,417-bp sequence downstream of the mycin, the decline of F /F in the wild-type leaves was ATG codon, and this was confirmed by PCR and more rapid and continued until F /F subsequent sequencing of the amplified products proached about 10% of the dark-adapted values (Fig.
(Supplemental Fig. S10, A and B). Reverse transcrip- 5A). Interestingly, in the presence of lincomycin, the tion (RT)-PCR analysis showed that expression of the decline in F /F in the deg7 mutants was similar to that Deg7 gene was not detectable (Supplemental Fig.
observed in the wild-type leaves during the same S10C). Further immunoblot analyses revealed that photoinhibitory light treatment (Fig. 5A). Since linco- Deg7 protein was not detectable in the deg7 mutant mycin blocks the repair of PSII by inhibiting de novo and that the levels of Deg7 in the complemented plants protein synthesis in the chloroplast, these results sug- were comparable to those in the wild-type plants gest that the wild-type and mutant leaves display (Supplemental Fig. S10D). When grown under 120 similar rates of PSII photoinhibition.
mmol m22 s21 light, the growth rates of the deg7 To establish whether the high susceptibility of the mutants were comparable to those of the wild type deg7 mutants to photoinhibition is related to PSII (Fig. 4A). Chlorophyll fluorescence analysis showed protein turnover, we analyzed the PSII protein con- that the maximal photochemical efficiency of PSII tents of the thylakoid membranes in the wild-type and (F /F ) was similar between the deg7 plants (0.82 6 mutant plants under high-light illumination in the 0.02) and wild-type plants (0.83 6 0.01).
presence of lincomycin. Under high-light illumination To examine the steady-state levels of thylakoid and in the presence of this inhibitor, the levels of the proteins, immunoblot analyses were performed with PSII proteins D1, D2, CP43, and CP47 gradually de- antibodies raised against specific subunits of the pho- clined in the deg7 and wild-type plants, but the rate of tosynthetic protein complexes (Supplemental Fig.
reduction was slower in the mutant (Fig. 5B). The S11). Our results showed that levels of the thylakoid levels of PsbO, another PSII protein, and the PSI proteins, including D1, D2, LHCII, PsbO, and CP43 of protein PsaA/B were found to be relatively stable in PSII, PsaA/B of PSI, Cyt f of the Cyt b f complex, and both the wild-type and mutant plants.
CF1b of ATP synthase, were not altered in the deg7 Next, we performed an in vivo labeling experiment mutant. The levels of Deg1, Deg5, Deg8, and FtsH to follow the turnover of newly synthesized chloro- were also not changed in the mutant.
plast proteins (Fig. 5C). Our results showed that the Figure 4. Phenotypes of deg5, deg7, deg8, deg2 deg7, deg5 deg7, and deg8 deg7 mutants and wild-type (WT) plants. A, Four-week-old deg5, deg7, deg8, deg2 deg7, deg5 deg7, deg8 deg7, and deg7 complemented mutants and wild-type plants grownunder a photon flux density of 120 mmol m22 s21. B, Two-week-old deg5, deg7, deg8, deg2 deg7, deg5 deg7, deg8 deg7, anddeg7 complemented mutants and wild-type plants grown in a growth chamber under a photon flux density of 120 mmol m22 s21and then transferred to a greenhouse for another 2 weeks with a maximum photon flux density at noon of about 1,000 mmolm22 s21.
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synthesis rates of the PSII core proteins D1, D2, CP47,and CP43, the PSI reaction center proteins PsaA/B,and the ATP synthase CF1a/b were similar betweenthe wild-type and mutant plants. The PSII core pro-teins D1, D2, CP47, and CP43 showed increased sta-bility in the mutant compared with the wild-typeplants. The protein synthesis rates in pulse labeling for20 min reflect the differences between the synthesisand degradation rates of photosynthetic proteins.
Thus, the net synthesis rate of PSII reaction centerprotein D1 in the mutant was lower than that of thewild type, since the rate of D1 degradation was slowedin deg7.
To further analyze the physiological function of Deg7, photoinhibition was carried out with isolatedthylakoid membranes to which 50 mmol EDTA hadbeen added in order to inactivate the endogenous FtsHproteases. We found that the 20-kD polypeptide of D1,the 29-kD polypeptide of D2, the 19-kD polypeptide ofCP43, and the 19-kD polypeptide of CP47 were de-tected in the samples from wild-type plants follow-ing photoinhibition but not in samples from the deg7mutant. An 18-kD polypeptide from D1 was detectedboth in the wild-type and mutant plants (Fig. 2B),which may be due to the function of lumen-localizedDeg5 and Deg8 (Sun et al., 2007a).
Since Deg7 is involved in protection against photo- inhibition, we analyzed the phenotypes of the mutantsduring growth under high irradiance. For this pur-pose, we transferred the wild-type and deg7 mutantplants that were grown initially at 120 mmol m22 s21 toa greenhouse with a maximum intensity of 1,000 mmolm22 s21 at noon. Our results showed that when ex-posed to high light, the growth of the deg7 mutantwas inhibited compared with the wild-type plants(Fig. 4B).
Generation of deg2 deg7, deg5 deg7, and deg8 deg7 DoubleMutants and Their Growth under High Light Three double mutants were constructed, deg2 deg7, deg5 deg7, and deg8 deg7, to test the physiologicalimportance of stromal and luminal Deg proteases inphotosynthesis. The growth rates and PSII activities ofdeg2 deg7, deg5 deg7, and deg8 deg7 were comparableto those of wild-type plants when grown at 120 mmol tected with specific antibodies directed against D1, D2, CP43, CP47,PsbO, and PsaA/B proteins. X-ray films were scanned and analyzedusing an AlphaImager 2200 documentation and analysis system. The Figure 5. Changes in PSII activity and protein levels. A, The Fv/Fm was percentage protein levels shown below the lanes were estimated by measured for detached leaves from wild-type (WT) plants (squares) and comparison with levels found in corresponding samples taken at time deg7 mutant plants (circles) following high-light illumination (1,800 0. Similar results were obtained in two additional independent exper- mmol m22 s21) in the absence (white symbols) or presence (black iments. C, Pulse-chase analysis of chloroplast proteins. Two-week-old symbols) of lincomycin (Lin). B, Immunoblot analysis of thylakoid leaves of the wild-type and deg7 mutant plants were labeled for 20 min proteins following high-light illumination. Thylakoid membranes were followed by a chase after 1 or 4 h at 120 mmol m22 s21. Thylakoid isolated from the wild-type and deg7 mutant leaves during exposure to membranes with equal amounts of chlorophyll were isolated and high-light illumination in the presence of lincomycin for 1, 2, or 4 h.
separated by SDS-PAGE. The resolved proteins were visualized by The thylakoid proteins were separated by SDS-PAGE and immunode- Plant Physiol. Vol. 152, 2010 PSII Protein Degradation by DEG7 m22 s21 (Fig. 4A). However, when exposed to high 5B). Pulse-chase labeling experiments also revealed light, the growth of the mutants was slower than that that the turnover rates of newly synthesized PSII core of the wild-type plants, and the extent of the pheno- proteins were slowed in the deg7 mutant (Fig. 5C).
type in the deg5 deg7 and deg8 deg7 double mutants was These results suggest that Deg7 is involved in the more pronounced than in the single mutants, while degradation of PSII core proteins.
that of deg2 deg7 was similar to that of the deg7 mutant Deciphering the physiological roles of specific pro- (Fig. 4B). Moreover, some of the mutant leaves ex- teases is intimately related to the identification of their hibited symptoms of yellowing in the deg5 deg7 and substrates. The D1 protein of the PSII reaction center deg8 deg7 double mutants (Fig. 4B).
has been identified as the substrate of characterizedchloroplast Deg proteases in vivo. This protein issusceptible to proteolytic cleavage at its lumen-exposed domains (the AB and CD loops and the C terminus) by Deg1 (Kapri-Pardes et al., 2007) and theDeg5-Deg8 complex (Sun et al., 2007a). The detection The adverse effect of high light on plant growth in of a C-terminal D1 fragment of about 20 kD in in vitro the deg7 mutant indicates the physiological impor- experiments suggests that Deg7 catalyzes the cleavage tance of Deg7 for photoprotection under high-light of the photodamaged D1 protein at the stromal loop illumination (Figs. 4 and 5). Such a role for Deg7 is also connecting the B and C transmembrane helices (Fig. 2).
in agreement with our observation of a dramatic Another stroma-localized Deg2 protein was shown to increase in the levels of proteins associated with the be involved in the initial cleavage of the DE loop of the thylakoid membranes under high light based on im- D1 protein in vitro (Haußu¨hl et al., 2001); however, this munoblot analysis (Fig. 1). There was no apparent possibility is not supported in vivo, since the deg2 difference in growth between wild-type and mutant mutant showed a similar extent of PSII inactivation plants under normal growth conditions at 120 mmol and a similar D1 protein turnover rate to that in the m22 s21, which also reflected the similar F /F ratios.
wild-type plants (Huesgen et al., 2006). Since Deg2 and When the wild-type plants were subjected to high- Deg7 are located in chloroplast stroma, we analyzed a light treatment, the PSII activity was reduced for the deg2 deg7 double mutant, and the deg2 deg7 and deg7 duration of treatment in the absence of lincomycin, mutants showed similar degrees of sensitivity to high and the reduction of PSII activity became more pro- light in terms of growth (Fig. 4) and PSII activity nounced in the presence of lincomycin (Fig. 5). The (Supplemental Fig. S12). Thus, Deg2, unlike Deg7, PSII activity in the deg7 mutant showed enhanced seems to have little effect on PSII repair in vivo.
sensitivity to high-light treatment in the absence of Although the PSII reaction center protein D1 is best lincomycin compared with the wild-type plants, while characterized as the main target of photodamage, the the rate of PSII photoinhibition was similar in the other PSII proteins D2, CP47, and CP43 are occasion- mutant and wild-type plants in the presence of the ally damaged and degraded, especially in response protein synthesis inhibitor lincomycin (Fig. 5). These to increasing light intensity (Schuster et al., 1988; results indicate that under high light, the repair of PSII Yamamoto and Akasaka, 1995; Jansen et al., 1999; was perturbed in the mutant. It is likely that the Adir et al., 2003). The detection of the 29-kD fragment increased sensitivity of PSII to high light in the mutant of D2 indicates that the cleavage site of photodamaged could be ascribed to the impairment of the PSII repair D2 is located at its N terminus. The observation of the 19-kD fragments of CP47 and CP43 after treatment of The repair of PSII involves the degradation and thylakoid membranes with recombinant Deg7 sug- removal of damaged PSII proteins and their subse- gests that cleavage may take place at the stromal loop quent replacement with newly synthesized copies connecting transmembrane domains D and E.
(Prasil et al., 1992; Aro et al., 1993; Andersson and The PDZ domains in E. coli DegP proteases have Aro, 2001). The degradation of photodamaged PSII been implicated in the formation of oligomers and also proteins is inherent to the repair cycle of PSII after in the regulation of proteolytic activity (Doyle et al., photoinhibition. Removal of photooxidatively dam- 1996; Harris and Lim, 2001). It is interesting that Deg7 aged PSII proteins and subsequent replacement with harbors three PDZ domains. The presence of three newly synthesized copies represents an efficient repair PDZ domains raises questions about their functions.
cycle. However, the detailed mechanisms of the repair Truncated Deg7 containing either one or two PDZ of PSII are far from being understood. Impairment of domains was also able to degrade PSII core proteins, the PSII repair cycle could result from the perturbation but it did not generate specific fragments. The trun- of either protein synthesis or degradation. Our pulse- cated Deg7 that lacked any PDZ domain lost its labeling experiments showed that the synthesis rates specificity for the substrate and was proteolytically of the PSII core proteins D1, D2, CP47, and CP43 were active not only toward PSII core proteins but also not affected in the mutant (Fig. 5C). Instead, exami- toward other photosynthetic proteins (Supplemental nation of the degradation of PSII core proteins showed Figs. S6–S8). These results highlight the importance of that the decrease in the levels of PSII core proteins was PDZ domains as regulators of proteolytic activity that slower in the mutant than in the wild-type plants (Fig.
recognize and discriminate between substrates.
Plant Physiol. Vol. 152, 2010 The function of Deg7 in the degradation of photo- the D1 protein (Komenda et al., 2006). These studies damaged PSII core proteins suggests its possible in- suggest a possible function of FtsH in the degradation teraction with PSII. The results presented in Figure 3 of other PSII core proteins. Considering the functional and Supplemental Figure S9 strongly suggest that mode of FtsH, it is likely that other PSII core proteins Deg7 associates with PSII complexes but not with may also be cleaved by peptidases in order to gener- other photosynthetic complexes such as PSI, Cyt b f, or ate limited numbers of distinct fragments, followed ATP synthase. This association is consistent with the by their complete digestion by the processive ATP- physiological role of Deg7 in the repair of damage to dependent FtsH protease.
PSII caused by photoinhibition. The finding of anassociation between a protease and a complex con-taining its substrates is intriguing. Our results suggestthat, at least in the case of PSII (which is known to MATERIALS AND METHODS undergo continuous cycles of assembly and repair), protein complexes might be equipped with compo-nents that are essential for their repair.
Wild-type and mutant Arabidopsis (Arabidopsis thaliana ecotype Columbia) The distribution of Deg proteases on both the stro- plants were grown in soil under short-day conditions (10-h-light/14-h-darkcycles) with a photon flux density of 120 mmol m22 s21 at a constant mal and luminal sides of the thylakoid membranes temperature of 22°C. The T-DNA insertion line SALK_075584 (Deg7; might have implications for the efficient degradation At3g03380) was obtained from the T-DNA-transformed Arabidopsis collection of damaged D1 protein. It is thus reasonable to spec- from the Arabidopsis Biological Resource Center (Ohio State University).
ulate that Deg7 functions cooperatively with lumen- Homozygous deg7 mutant plants were identified by PCR analyses using thefollowing gene-specific and T-DNA-specific primers: LP (5#-GGTACGTT- localized Deg1, Deg5, and Deg8, generating more CAGTGACACACCC-3#), RP (5#-CAAAGCTTTCTAGGGGTGCTC-3#), and cleavage sites on both sides of the membranes. This T-DNA LB (5#-GCGTGGACCGCTTGCTGCAACT-3#). The precise location of may increase the efficiency of D1 degradation. In mito- the T-DNA insertion was determined by sequencing the PCR products.
chondria, the degradation of membrane proteins was Confirmation of null mutants was carried out by RT-PCR using the same greatly assisted by the cooperation of proteases func- primer set described above for Deg7. Equal cDNA loading in each sample wasmonitored by RT-PCR analysis of the expression level of actin (5#-AACTGG- tioning on both sides of the membranes (Leonhard GATGATATGGAGAA-3# and 5#-CCTCCAATCCAGACACTGTA-3#). The et al., 2000). The single mutants deg5, deg7, and deg8 double mutants deg2 deg7, deg5 deg7, and deg7 deg8 were obtained by PCR all showed decreased growth compared with the wild- screening of an F2 population from crossed single mutant lines.
type plants under high light, although these mutantsdid not show any apparent differences in terms Complementation of the deg7 mutant of phenotype under normal growth conditions at The cDNA containing the coding region of Deg7 was amplified with the 120 mmol m22 s21 (Fig. 4; Sun et al., 2007a), which following primers, which include XhoI and KpnI restriction sites at their indicates that the Deg proteases are important for 5# ends to facilitate cloning: sense primer (5#-GACCCGGGGGAGAT- efficient PSII repair. The additive functions of Deg CAAATGGGAGATCC-3#) and antisense primer (5#-GGGGTACCTTACTG- proteases were also demonstrated by the phenotypes CAAGGCTTTCAATATA-3#). The resulting fragment was cloned into the SamI of the deg5 deg7 and deg8 deg7 double mutants grown and KpnI sites of pSN1301 under the control of the cauliflower mosaic virus35S promoter. The plasmid pSN1301-Deg7 was transformed into Agrobacte- under high light.
rium tumefaciens strain C58 via electroporation and introduced into the Since Deg proteases are endopeptidases that can homozygous deg7 plants (Clough and Bent, 1998). Transformant plants were cleave intermembrane peptide regions exposed to the selected on medium containing half-strength Murashige and Skoog salt mix, stromal or the luminal side, it is unlikely that PSII 50 mg mL21 hygromycin, and 0.8% agar. The resistant plants were transferredto soil to grow to maturity, and their transgenic status was further confirmed proteins are completely degraded by Deg proteases.
by PCR and immunoblot analyses.
The creation of D1 degradation intermediates mayprovide additional sites for the initiation of proteoly- sis. The function of FtsH in degrading E. coli integralmembrane proteins involves relocation of regions of Detached mature leaves were floated adaxial side up on water and substrates from one side to the other (Ito and Akiyama, exposed to a photon flux density of 1,800 mmol m22 s21, and chlorophyll 2005). It may be that Deg proteases from both sides of fluorescence was measured using a PAM-2000 fluorometer (Walz). Thetemperature of the water was kept at 22°C during photoinhibition treatments.
the membranes enhance the efficiency of D1 degrada- Synthesis of chloroplast-encoded proteins was blocked by incubating de- tion by increasing the number of D1 degradation tached leaves with their petioles submersed in 1 mM lincomycin solution at an intermediates that are accessible by FtsH. The Arabi- irradiance of 20 mmol m22 s21 for 3 h prior to photoinhibitory light treatment.
dopsis var2 mutant lacking FtsH2 exhibited more To investigate the effects of high irradiance on plant growth, we transferred 2-week-old Arabidopsis plants grown in a growth chamber under a photon increased sensitivity to high-light treatment than the flux density of 120 mmol m22 s21 to a greenhouse under sunlight illumination deg7 mutant (Supplemental Fig. S12), which suggests for another 2 weeks. In the greenhouse, the maximum photon flux density at that FtsH2 is predominantly involved in the PSII repair noon was about 1,000 mmol m22 s21 and average day/night temperatures cycle. The light-dependent degradation of D1 and D2 were 25°C/22°C.
was slowed in the Arabidopsis var2 mutant (Baileyet al., 2002). In Synechocystis species PCC6803, the FtsH Thylakoid Membrane Preparation protease slr0228, which shows the highest similarity to Thylakoid membranes were isolated essentially as described by Zhang Arabidopsis FtsH2, has been shown to play a more et al. (1999). Briefly, Arabidopsis leaves were homogenized by a mortar and general role in the removal of PSII proteins other than pestle in an ice-cold isolation buffer (400 mM Suc, 50 mM HEPES-KOH, pH 7.8, Plant Physiol. Vol. 152, 2010 PSII Protein Degradation by DEG7 10 mM NaCl, and 2 mM MgCl ), filtered through two layers of cheesecloth, and Proteolytic Degradation Assays centrifuged at 5,000g for 10 min. The thylakoid pellets were resuspended inthe isolation buffer and then centrifuged at 5,000g for 10 min. The thylakoids The proteolytic activity was assayed by incubating Deg7 with 15-mg were finally suspended in isolation buffer, and their chlorophyll levels were mixtures of casein (a-, b-, and k-casein; Sigma-Aldrich) in standard reaction mixtures including 0.5 mg of purified Deg7 in a 30-mL solution containing 50mM Tris-HCl, pH 7.6. The mixtures were incubated for 0, 15, 30, 45, and 60 minat 37°C, separated by SDS-PAGE, and stained with Coomassie Brilliant Blue.
SDS-PAGE and Immunoblot Analysis For the in vitro degradation assay, wild-type Arabidopsis thylakoid membranes were illuminated at 1,800 mmol m22 s21 for 90 min at 4°C and The protein sample was mixed with the same volume of 23 SDS sample centrifuged at 15,000g for 15 min. The collected thylakoids were washed with buffer (125 mM Tris-HCl, pH 6.8, 20% [w/v] glycerol, 4% SDS, 5% b-mercap- 1.0 M CaCl2 to reduce endogenous Deg7 activity before the addition of toethanol, and 0.1% bromphenol blue) for 60 min and layered onto 15% SDS recombinant Deg7 or its derivatives and resuspended in 300 mM sorbitol and polyacrylamide gels containing 6 M urea (Laemmli, 1970). After electropho- 10 mM HEPES-KOH, pH 8.0. After incubation of purified recombinant Deg7 resis, gels were stained with Coomassie Brilliant Blue. For immunoblot protein with the thylakoid membranes at 37°C in the dark for various amounts analysis, the proteins resolved by SDS-PAGE were blotted onto nitrocellulose of time, the samples were subjected to SDS-PAGE and immunoblot analyses.
membranes and reacted with specific antibodies, and the signals werevisualized with the enhanced chemiluminescence method.
Primary antibodies used in this study were as follows: (1) a D1-specific In Vivo Labeling of Chloroplast Proteins antibody against the oligopeptide NYGYKFGQE containing the amino acids234 to 242 in the DE loop of the D1 protein of Synechocystis species PCC 6803; In vivo chloroplast protein labeling was carried out essentially according to (2) a D2-specific antibody against the oligopeptide NTFRAFNPTQAEETYS Meurer et al. (1998). Leaves of 2-week-old Arabidopsis plants were preincu- containing the amino acids 231 to 246 in the DE loop of the D2 protein of bated for 30 min in the presence of 20 mg mL21 cycloheximide, which blocks Arabidopsis; (3) an anti-peptide antibody specific for E. coli FtsH, which is the synthesis of nucleus-encoded proteins. Then, the leaves were radiolabeled potentially cross-reactive with all Synechocystis species PCC 6803 FtsH homo- with 1 mCi mL21 [35S]Met (specific activity . 1,000 Ci mmol21; Amersham logs; (4) a Cyt f antibody raised against the amino acids 146 to 257 in the Cyt f Pharmacia Biotech) at 120 mmol m22 s21 in the presence of 20 mg mL21 protein of Arabidopsis; (5) a CF1b antibody raised against the amino acids 206 cycloheximide for 20 min at 22°C, followed by a chase of 1 or 4 h in buffer to 387 in the CF1b protein of Arabidopsis; (6) a PsaA/B antiserum raised containing 10 mM cold Met. Afterward, the leaves were collected, the thyla- against the amino acids 3 to 77 in the N terminus of the PsaA protein of koid membranes were isolated, and the proteins were separated by SDS- Arabidopsis; (7) a CP43 antibody raised against the amino acids 330 to 449 in PAGE. For autoradiography, gels were stained, dried, and exposed to x-ray the C terminus of the CP43 protein of Arabidopsis; (8) a CP47 antibody raised against the amino acids 330 to 475 in the C terminus of the CP47 protein ofArabidopsis; (9) a LHCII antibody raised against the amino acids 25 to 154 inthe LHCB3 protein of Arabidopsis; (10) a Deg1 antibody raised against the GFP Fusion Constructs for Transient Expression amino acids 315 to 436 in the C terminus of the Deg1 protein of Arabidopsis; (11) a Deg5 antibody raised against the amino acids 91 to 211 in the N terminusof the Deg5 protein of Arabidopsis; and (12) a Deg8 antibody raised against A fragment encoding the N-terminal amino acids 1 to 243 of Deg7 the amino acids 297 to 423 in the C terminus of the Deg8 protein of was amplified by PCR using the following primers: 5#-GCGTCGACAT- GGGAGATCCGTTGGAGAG-3# and 5#-GGCCATGGTTAACGCCCTAACA-ACTCG-3#. The PCR product was cloned into the SalI and NcoI sites ofexpression vector pUC18-35S-sGFP to generate a fusion protein with the GFP Antiserum Production and Immunolocalization Studies as a reporter in the C terminus. The transit peptide (amino acids 1–245) of theFtsH11, the N-terminal part (amino acids 1–282) of AtFbr1, and the entire The His-Deg7 fusion protein was purified on a Ni-NTA resin matrix, and coding region of FRO1 were used as chloroplast, nuclear, and mitochondrial polyclonal antibodies were raised in rabbit with the purified antigens. The controls, respectively (Sakamoto et al., 2003; Cai et al., 2009). Isolation of the intracellular localization of Deg7 was determined essentially according to Arabidopsis protoplast and polyethylene glycol-mediated transfection were Lennartz et al. (2001). The Arabidopsis membranes were suspended to a final performed as described by Sakamoto et al. (2003). The cells with GFP signals concentration of 50 mg chlorophyll mL21 in 10 mM HEPES-KOH, pH 8.0, 10 were examined using a confocal laser scanning microscope (LSM510; Carl M sorbitol, and 1 mM phenylmethylsulfonyl fluoride supplemented with 250 mM NaCl, 200 mM Na2CO3, 1 M CaCl2, 6 M urea, and0.05% Triton X-100 at 0°C for 30 min or 0.05 mg mL21 trypsin at 25°C for 30min. Membrane fractions without supplements were used as a control. After Sequence data from this article can be found in the GenBank/EMBL data treatment, the membranes were pelleted at 100,000g for 2 h at 4°C, quickly libraries under accession number At3g03380 (DEG7).
washed with isolation buffer, and used for SDS-PAGE and immunoblotanalysis.
Supplemental Data Recombinant Expression of Deg7 and Pull-Down Assays The following materials are available in the online version of this article.
Full-length Deg7 cDNA was cloned into the pET28a plasmid and trans- Supplemental Figure S1. Subcellular localization of the Deg7 protein.
formed into BL21 cells. The expression of the His-Deg7 fusion protein was Supplemental Figure S2. Immunoblot analysis of the purity of isolated induced by isopropylthio-b-D-galactoside (0.4 mM) for 2 h, and the overex- pressed protein was purified using a Ni-NTA resin matrix. The purifiedprotein was renatured through a Sephadex G-75 column by eluting with 20 Supplemental Figure S3. Immunoblot analysis of chloroplast proteins with the specific Deg7 antibody.
2PO4, pH 7.8, buffer, and the identity of the protein was confirmed by immunoblot analyses with specific antibodies.
Supplemental Figure S4. Engineering of the Deg7 deletion construct and For protein pull-down assays, thylakoid membranes (100 mg of chloro- proteolytic activity of Deg7 with b-casein.
phyll) were solubilized with 1% (w/v) DM in 20% (w/v) glycerol, 25 mMBisTris-HCl, pH 7.0, and 1 mM phenylmethylsulfonyl fluoride for 15 min at Supplemental Figure S5. Degradation of photosynthetic proteins by a 4°C, then centrifuged at 10,000g for 10 min. The supernatant obtained after recombinant Deg7 protein.
centrifugation was incubated with His-Deg7 coupled to Ni-NTA resin. After Supplemental Figure S6. Degradation of photodamaged PSII proteins by incubation overnight with constant rotation at 4°C, the beads were washed a recombinant Deg7 protein containing two PDZ domains.
five times with 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 1 mM EDTA buffer,and the bound proteins were eluted with SDS-PAGE sample buffer. The eluted Supplemental Figure S7. Degradation of photodamaged PSII proteins by a proteins were resolved by SDS-PAGE followed by immunoblot analyses.
recombinant Deg7 protein containing one PDZ domain.
Plant Physiol. Vol. 152, 2010 Supplemental Figure S8. Degradation of photodamaged PSII proteins by a in cyanobacteria and chloroplasts of higher plants. Physiol Plant 123: recombinant Deg7 protein lacking PDZ domains.
Huesgen PF, Schuhmann H, Adamska I (2006) Photodamaged D1 protein Supplemental Figure S9. Immunoblot analysis of the association of Deg7 is degraded in Arabidopsis mutants lacking the Deg2 protease. FEBS Lett with photosynthetic protein complexes after blue native/SDS-PAGE.
Supplemental Figure S10. Identification of the deg7 mutant.
Ito K, Akiyama Y (2005) Cellular functions, mechanism of action, and regulation of FtsH protease. Annu Rev Microbiol 59: 211–231 Supplemental Figure S11. Analysis of thylakoid proteins from deg7 Itzhaki H, Naveh L, Lindahl M, Cook M, Adam Z (1998) Identification and mutant and wild-type plants.
characterization of DegP, a serine protease associated with the luminal Supplemental Figure S12. Changes in F /F side of the thylakoid membrane. J Biol Chem 273: 7094–7098 m of wild-type, deg7, deg2 deg7, and ftsh2 plants following high-light illumination.
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