The thylakoid lumen protease Deg1 is involved in the repair of photosystem II from photoinhibition in Arabidopsis

Deg1 is a Ser protease peripherally attached to the lumenal side of the thylakoid membrane. Its physiological function is unknown, but its localization makes it a suitable candidate for participation in photoinhibition repair by degradation of the photosystem II reaction center protein D1. We transformed Arabidopsis thaliana with an RNA interference construct and obtained plants with reduced levels of Deg1. These plants were smaller than wild-type plants, flowered earlier, were more sensitive to photoinhibition, and accumulated more of the D1 protein, probably in an inactive form. Two C-terminal degradation products of the D1 protein, of 16 and 5.2 kD, accumulated at lower levels compared with the wild type. Moreover, addition of recombinant Deg1 to inside-out thylakoid membranes isolated from the mutant could induce the formation of the 5.2-kD D1 C-terminal fragment, whereas the unrelated proteases trypsin and thermolysin could not. Immunoblot analysis revealed that mutants containing less Deg1 also contain less FtsH protease, and FtsH mutants contain less Deg1. These results suggest that Deg1 cooperates with the stroma-exposed proteases FtsH and Deg2 in degrading D1 protein during repair from photoinhibition by cleaving lumen-exposed regions of the protein. In addition, they suggest that accumulation of Deg1 and FtsH proteases may be coordinated.     

Cooperative D1 Degradation in the Photosystem II Repair Mediated by Chloroplastic Proteases in Arabidopsis

Light energy constantly damages photosynthetic apparatuses, ultimately causing impaired growth. Particularly, the sessile nature of higher plants has allowed chloroplasts to develop unique mechanisms to alleviate the irreversible inactivation of photosynthesis. Photosystem II (PSII) is known as a primary target of photodamage. Photosynthetic organisms have evolved the so-called PSII repair cycle, in which a reaction center protein, D1, is degraded rapidly in a specific manner. Two proteases that perform processive or endopeptidic degradation, FtsH and Deg, respectively, participate in this cycle. To examine the cooperative D1 degradation by these proteases, we engaged Arabidopsis (Arabidopsis thaliana) mutants lacking FtsH2 (yellow variegated2 [var2]) and Deg5/Deg8 (deg5 deg8) in detecting D1 cleaved fragments. We detected several D1 fragments only under the var2 background, using amino-terminal or carboxyl-terminal specific antibodies of D1. The appearance of these D1 fragments was inhibited by a serine protease inhibitor and by deg5 deg8 mutations. Given the localization of Deg5/Deg8 on the luminal side of thylakoid membranes, we inferred that Deg5/Deg8 cleaves D1 at its luminal loop connecting the transmembrane helices C and D and that the cleaved products of D1 are the substrate for FtsH. These D1 fragments detected in var2 were associated with the PSII monomer, dimer, and partial disassembly complex but not with PSII supercomplexes. It is particularly interesting that another processive protease, Clp, was up-regulated and appeared to be recruited from stroma to the thylakoid membrane in var2, suggesting compensation for FtsH deficiency. Together, our data demonstrate in vivo cooperative degradation of D1, in which Deg cleavage assists FtsH processive degradation under photoinhibitory conditions.     

Phosphorylation of photosystem II core proteins prevents undesirable cleavage of D1 and contributes to the fine‐tuned repair of photosystem II

Photosystem II (PSII) is a primary target for light‐induced damage in photosynthetic protein complexes. To avoid photoinhibition, chloroplasts have evolved a repair cycle with efficient degradation of the PSII reaction center protein, D1, by the proteases FtsH and Deg. Earlier reports have described that phosphorylated D1 is a poor substrate for proteolysis, suggesting a mechanistic role for protein phosphorylation in PSII quality control, but its precise role remains elusive. STN8, a protein kinase, plays a central role in this phosphorylation process. To elucidate the relationship between phosphorylation of D1 and the protease function we assessed in this study the involvement of STN8, using Arabidopsis thaliana mutants lacking FtsH2 [yellow variegated2 (var2)] and Deg5/Deg8 (deg5 deg8). In support of our presumption we found that phosphorylation of D1 increased more in var2. Furthermore, the coexistence of var2 and stn8 was shown to recover the delay in degradation of D1, resulting in mitigation of the high vulnerability to photoinhibition of var2. Partial D1 cleavage fragments that depended on Deg proteases tended to increase, with concomitant accumulation of reactive oxygen species in the mutants lacking STN8. We inferred that the accelerated degradation of D1 in var2 stn8 presents a tradeoff in that it improved the repair of PSII but simultaneously enhanced oxidative stress. Together, these results suggest that PSII core phosphorylation prevents undesirable cleavage of D1 by Deg proteases, which causes cytotoxicity, thereby balancing efficient linear electron flow and photo‐oxidative damage. We propose that PSII core phosphorylation contributes to fine‐tuned degradation of D1.     

A chloroplast DegP2 protease performs the primary cleavage of the photodamaged D1 protein in plant photosystem II

Although light is the ultimate substrate in photosynthesis, it can also be harmful and lead to oxidative damage of the photosynthetic apparatus. The main target for light stress is the central oxygen-evolving photosystem II (PSII) and its D1 reaction centre protein. Degradation of the damaged D1 protein and its rapid replacement by a de novo synthesized copy represent the important repair mechanism of PSII crucial for plant survival under light stress conditions. Here we report the isolation of a single-copy nuclear gene from Arabidopsis thaliana, encoding a protease that performs GTP-dependent primary cleavage of the photodamaged D1 protein and hence catalysing the key step in the repair cycle in plants. This protease, designated DegP2, is a homologue of the prokaryotic Deg/Htr family of serine endopeptidases and is associated with the stromal side of the non-appressed region of the thylakoid membranes. Increased expression of DegP2 under high salt, desiccation and light stress conditions was measured at the protein level.     

The thylakoid protease Deg1 is involved in photosystem‐II assembly in Arabidopsis thaliana

DegP proteases have been shown to possess both chaperone and protease activities. The proteolytic activities of chloroplast DegP‐like proteases have been well documented. However, whether chloroplast Deg proteases also have chaperone activities has remained unknown. Here we show that chloroplast Deg1 also has chaperone activities, like its Escherichia coli ortholog DegP. Transgenic plants with reduced levels of Deg1 accumulated normal levels of different subunits of the major photosynthetic protein complexes, but their levels of photosystem‐II (PSII) dimers and supercomplexes were reduced. In vivo pulse‐chase protein labeling experiments showed that the assembly of newly synthesized proteins into PSII dimers and supercomplexes was impaired, although the synthesis rate of chloroplast proteins was unaffected in the transgenic lines. Protein overlay assays provided direct evidence that Deg1 interacts with the PSII reaction center protein D2. These results suggest that Deg1 assists the assembly of the PSII complex, probably through interaction with the PSII reaction center D2 protein.     

Deg proteases and their role in protein quality control and processing in different subcellular compartments of the plant cell

Degradation of periplasmic proteins (Deg)/high temperature requirement A (HtrA) proteases are ATP-independent serine endopeptidases found in almost every organism. Database searches revealed that 16 Deg paralogues are encoded by the genome of Arabidopsis thaliana, six of which were experimentally shown to be located in chloroplasts, one in peroxisomes, one in mitochondria and one in the nucleus. Two more Deg proteases are predicted to reside in chloroplasts, five in mitochondria (one of them with a dual chloroplastidial/mitochondrial localization) and the subcellular location of one protein is uncertain. This review summarizes the current knowledge on the role of Deg proteases in maintaining protein homeostasis and protein processing in various subcompartments of the plant cell. The chloroplast Deg proteases are the best examined so far, especially with respect to their role in the degradation of photodamaged photosynthetic proteins and in biogenesis of photosystem II (PSII). A combined action of thylakoid lumen and stroma Deg proteases in the primary cleavage of photodamaged D1 protein from PSII reaction centre is discussed on the basis of a recently resolved crystal structure of plant Deg1. The peroxisomal Deg protease is a processing enzyme responsible for the cleavage of N-terminal peroxisomal targeting signals (PTSs). A. thaliana mutants lacking this enzyme show reduced peroxisomal β-oxidation, indicating for the first time the impact of protein processing on peroxisomal functions in plants. Much less data is available for mitochondrial and nuclear Deg proteases. Based on the available expression data we hypothesize a role in general protein quality control and during acquired heat resistance.     

High light stimulates Deg1-dependent cleavage of the minor LHCII antenna proteins CP26 and CP29 and the PsbS protein in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The chloroplast Deg1 protein performs proteolytic cleavage of the photodamaged D1 protein of the photosystem II (PSII) reaction center, PSII extrinsic subunit PsbO and the soluble electron carrier plastocyanin. Using biochemical, immunological and mass spectrometry approaches we showed that the heterogeneously expressed Deg1 protease from Arabidopsis thaliana can be responsible for the degradation of the monomeric light-harvesting complex antenna subunits of PSII (LHCII), CP26 and CP29, as well as PSII-associated PsbS (CP22/NPQ4) protein. The results may indicate that cytochrome b ₆ protein and two previously unknown thylakoid proteins, Ptac16 and an 18.3-kDa protein, may be the substrates for Deg1. The interaction of Deg1 with the PsbS protein and the minor LHCII subunits implies its involvement in the regulation of both excess energy dissipation and state transition adaptation processes.     

Analysis of the changes of electron transfer and heterogeneity of photosystem II in Deg1-reduced Arabidopsis plants

Deg1 protease functions in protease and chaperone of PSII complex components, but few works were performed to study the effects of Deg1 on electron transport activities on the donor and acceptor side of PSII and its correlation with the photoprotection of PSII during photoinhibition. Therefore, we performed systematic and comprehensive investigations of electron transfers on the donor and acceptor sides of photosystem II (PSII) in the Deg1-reduced transgenic lines deg1-2 and deg1-4. Both the maximal quantum efficiency of PSII photochemistry (F_v/F_m) and the actual PSII efficiency (Φ_PSII) decreased significantly in the transgenic plants. Increases in nonphotochemical quenching (NPQ) and the dissipated energy flux per reaction center (DI₀/RC) were also shown in the transgenic plants. Along with the decreased D1, CP47, and CP43 content, these results suggested photoinhibition under growth light conditions in transgenic plants. Decreased Deg1 caused inhibition of electron transfer on the PSII reducing side, leading to a decline in the number of Q_B-reducing centers and accumulation of Q_B-nonreducing centers. The Tm of the Q band shifted from 5.7 °C in the wild-type plant to 10.4 °C and 14.2 °C in the deg1-2 and deg1-4 plants, respectively, indicating an increase in the stability of S₂Q_A^¯ in transgenic plants. PSIIα in the transgenic plants largely reduced, while PSIIβ and PSIIγ increased with the decline in the Deg1 levels in transgenic plants suggesting PSIIα centers gradually converted into PSIIβ and PSIIγ centers in the transgenic plants. Besides, the connectivity of PSIIα and PSIIβ was downregulated in transgenic plants. Our results reveal that downregulation of Deg1 protein levels induced photoinhibition in transgenic plants, leading to loss of PSII activities on both the donor and acceptor sides in transgenic plants. These results give a new insight into the regulation role of Deg1 in PSII electron transport.

     

The thylakoid protease Deg2 is involved in stress-related degradation of the photosystem II light-harvesting protein Lhcb6 in Arabidopsis thaliana

? The thylakoid protease Deg2 is a serine-type protease peripherally attached to the stromal side of the thylakoid membrane. Given the lack of knowledge concerning its function, two T-DNA insertion lines devoid of Deg2 were prepared to study the functional importance of this protease in Arabidopsis thaliana. ? The phenotypic appearance of deg2 mutants was studied using a combination of stereo and transmission electron microscopy, and short-stress-mediated degradation of apoproteins of minor light-harvesting antennae of photosystem II (PSII) was analysed by immunoblotting in the mutants in comparison with wild-type plants. ? Deg2 repression produced a phenotype in which reduced leaf area and modified chloroplast ultrastructure of older leaves were the most prominent features. In contrast to the wild type, the chloroplasts of second-whorl leaves of 4-wk-old deg2 mutants did not display features typical of the early senescence phase, such as undulation of the chloroplast envelope and thylakoids. The ability to degrade the photosystem II light-harvesting protein Lhcb6 apoprotein in response to brief high-salt, wounding, high-temperature and high-irradiance stress was demonstrated to be impaired in deg2 mutants. ? Our results suggest that Deg2 is required for normal plant development, including the chloroplast life cycle, and has an important function in the degradation of Lhcb6 in response to short-duration stresses.     

The family of Deg/HtrA proteases: from Escherichia coli to Arabidopsis

In the genomic era, an increasing number of protease genes have been identified in various organisms. During the last few years, many of these proteases have been characterized using biochemical as well as molecular biological techniques. However, neither the precise location nor the physiological substrates of these enzymes has been identified in many cases, including the Deg/HtrA proteases, a family of serine-type ATP-independent proteases. This family has become especially interesting for many researchers following the determination of the crystal structures of an Escherichia coli and a human Deg/HtrA protease. A breakthrough in photosynthesis research has revealed that a Deg/HtrA protease of Arabidopsis thaliana is involved in the degradation of the D1 protein of photosystem II following photoinhibition. In this review, the available data on Deg/HtrAs of different organisms are compared with those from the photoautotroph cyanobacterium Synechocystis sp. PCC 6803 and the plant Arabidopsis thaliana .     

Crystal Structure of Arabidopsis Deg2 Protein Reveals an Internal PDZ Ligand Locking the Hexameric Resting State

Eukaryotic organelles have developed elaborate protein quality control systems to ensure their normal activity, among which Deg/HtrA proteases play an essential role. Plant Deg2 protease is a homologue of prokaryotic DegQ/DegP proteases and is located in the chloroplast stroma, where its proteolytic activity is required to maintain the efficiency of photosynthetic machinery during stress. Here, we demonstrate that Deg2 exhibits dual protease-chaperone activities, and we present the hexameric structure of Deg2 complexed with co-purified peptides. The structure shows that Deg2 contains a unique second PDZ domain (PDZ2) following a conventional PDZ domain (PDZ1), with PDZ2 orchestrating the cage assembly of Deg2. We discovered a conserved internal ligand for PDZ2 that mediates hexamer formation and thus locks the protease in the resting state. These findings provide insight into the diverse modes of PDZ domain-mediated regulation of Deg proteases.     

The E3 ligase AtCHIP ubiquitylates FtsH1, a component of the chloroplast FtsH protease, and affects protein degradation in chloroplasts

The Arabidopsis E3 ligase AtCHIP was found to interact with FtsH1, a subunit of the chloroplast FtsH protease complex. FtsH1 can be ubiquitylated by AtCHIP in vitro, and the steady-state level of FtsH1 is reduced in AtCHIP-over-expressing plants under high-intensity light conditions, suggesting that the ubiquitylation of FtsH1 by AtCHIP might lead to the degradation of FtsH1 in vivo. Furthermore, the steady-state level of another subunit of the chloroplast FtsH protease complex, FtsH2, is also reduced in AtCHIP-over-expressing plants under high-intensity light conditions, and FtsH2 interacts physically with AtCHIP in vivo, suggesting the possibility that FtsH2 is also a substrate protein for AtCHIP in plant cells. A substrate of FtsH protease in vivo, the photosystem II reaction center protein D1, is not efficiently removed by FtsH in AtCHIP-over-expressing plants under high-intensity light conditions, supporting the assumption that FtsH subunits are substrates of AtCHIP in vivo, and that AtCHIP over-expression may lead to a reduced level of FtsH in chloroplasts. AtCHIP interacts with cytosolic Hsp70 and the precursors of FtsH1 and FtsH2 in the cytoplasm, and Hsp70 also interacts with FtsH1, and these protein-protein interactions appear to be increased under high-intensity light conditions, suggesting that Hsp70 might be partly responsible for the increased degradation of the substrates of Hsp70, such as FtsH1 and FtsH2, in AtCHIP-over-expressing plants under high-intensity light conditions. Therefore, AtCHIP, together with Hsp70, may play an important role in protein quality control in chloroplasts.     

The role of the FtsH and Deg proteases in the repair of UV-B radiation-damaged Photosystem II in the cyanobacterium Synechocystis PCC 6803

The photosystem two (PSII) complex found in oxygenic photosynthetic organisms is susceptible to damage by UV-B irradiation and undergoes repair in vivo to maintain activity. Until now there has been little information on the identity of the enzymes involved in repair. In the present study we have investigated the involvement of the FtsH and Deg protease families in the degradation of UV-B-damaged PSII reaction center subunits, D1 and D2, in the cyanobacterium Synechocystis 6803. PSII activity in a DeltaFtsH (slr0228) strain, with an inactivated slr0228 gene, showed increased sensitivity to UV-B radiation and impaired recovery of activity in visible light after UV-B exposure. In contrast, in DeltaDeg-G cells, in which all the three deg genes were inactivated, the damage and recovery kinetics were the same as in the WT. Immunoblotting showed that the loss of both the D1 and D2 proteins was retarded in DeltaFtsH (slr0228) during UV-B exposure, and the extent of their restoration during the recovery period was decreased relative to the WT. However, in the DeltaDeg-G cells the damage and recovery kinetics of D1 and D2 were the same as in the WT. These data demonstrate a key role of FtsH (slr0228), but not the Deg proteases, for the repair of PS II during and following UV-B radiation at the step of degrading both of the UV-B damaged D1 and D2 reaction center subunits.     

Differential turnover of the photosystem II reaction centre D1 protein in mesophyll and bundle sheath chloroplasts of maize

Photoinhibition is caused by an imbalance between the rates of the damage and repair cycle of photosystem II D1 protein in thylakoid membranes. The PSII repair processes include (i) disassembly of damaged PSII-LHCII supercomplexes and PSII core dimers into monomers, (ii) migration of the PSII monomers to the stroma regions of thylakoid membranes, (iii) dephosphorylation of the CP43, D1 and D2 subunits, (iv) degradation of damaged D1 protein, and (v) co-translational insertion of the newly synthesized D1 polypeptide and reassembly of functional PSII complex. Here, we studied the D1 turnover cycle in maize mesophyll and bundle sheath chloroplasts using a protein synthesis inhibitor, lincomycin. In both types of maize chloroplasts, PSII was found as the PSII-LHCII supercomplex, dimer and monomer. The PSII core and the LHCII proteins were phosphorylated in both types of chloroplasts in a light-dependent manner. The rate constants for photoinhibition measured for lincomycin-treated leaves were comparable to those reported for C3 plants, suggesting that the kinetics of the PSII photodamage is similar in C3 and C4 species. During the photoinhibitory treatment the D1 protein was dephosphorylated in both types of chloroplasts but it was rapidly degraded only in the bundle sheath chloroplasts. In mesophyll chloroplasts, PSII monomers accumulated and little degradation of D1 protein was observed. We postulate that the low content of the Deg1 enzyme observed in mesophyll chloroplasts isolated from moderate light grown maize may retard the D1 repair processes in this type of plastids.     

The role of the FtsH and Deg proteases in the repair of UV-B radiation-damaged Photosystem II in the cyanobacterium Synechocystis PCC 6803

The photosystem two (PSII) complex found in oxygenic photosynthetic organisms is susceptible to damage by UV-B irradiation and undergoes repair in vivo to maintain activity. Until now there has been little information on the identity of the enzymes involved in repair. In the present study we have investigated the involvement of the FtsH and Deg protease families in the degradation of UV-B-damaged PSII reaction center subunits, D1 and D2, in the cyanobacterium Synechocystis 6803. PSII activity in a ΔFtsH (slr0228) strain, with an inactivated slr0228 gene, showed increased sensitivity to UV-B radiation and impaired recovery of activity in visible light after UV-B exposure. In contrast, in ΔDeg-G cells, in which all the three deg genes were inactivated, the damage and recovery kinetics were the same as in the WT. Immunoblotting showed that the loss of both the D1 and D2 proteins was retarded in ΔFtsH (slr0228) during UV-B exposure, and the extent of their restoration during the recovery period was decreased relative to the WT. However, in the ΔDeg-G cells the damage and recovery kinetics of D1 and D2 were the same as in the WT. These data demonstrate a key role of FtsH (slr0228), but not the Deg proteases, for the repair of PS II during and following UV-B radiation at the step of degrading both of the UV-B damaged D1 and D2 reaction center subunits.     

Possible compensatory role among chloroplast proteases under excess-light stress condition

The reaction center protein D1 of photosystem II (PSII), known as a primary target of photodamage, is repaired efficiently by the PSII repair cycle, to cope with constant photooxidative damage. Recent studies of Arabidopsis show that the endo-type Deg protease and the exo-type FtsH proteases cooperatively degrade D1 in the PSII repair in vivo. It is particularly interesting that we observed upregulation of Clp and SppA proteases when FtsH was limited in the mutant lacking FtsH2. To examine how the complementary functions of chloroplastic proteases are commonly regulated, we undertook a high-light stress on wild-type Arabidopsis leaves. The result that wild type leaves also showed increased levels of these proteases upon exposure to excessively strong illumination not only revealed the importance of FtsH and Deg in the PSII repair, but also implied cooperation among chloroplastic proteases under chronic stress conditions.     

The serine protease HhoA from Synechocystis sp. strain PCC 6803: substrate specificity and formation of a hexameric complex are regulated by the PDZ domain

Enzymes of the ATP-independent Deg serine endopeptidase family are very flexible with regard to their substrate specificity. Some family members cleave only one substrate, while others act as general proteases on unfolded substrates. The proteolytic activity of Deg proteases is regulated by PDZ protein interaction domains. Here we characterized the HhoA protease from Synechocystis sp. strain PCC 6803 in vitro using several recombinant protein constructs. The proteolytic activity of HhoA was found to increase with temperature and basic pH and was stimulated by the addition of Mg(2+) or Ca(2+). We found that the single PDZ domain of HhoA played a critical role in regulating protease activity and in the assembly of a hexameric complex. Deletion of the PDZ domain strongly reduced proteolysis of a sterically challenging resorufin-labeled casein substrate, but unlabeled beta-casein was still degraded. Reconstitution of the purified HhoA with total membrane proteins isolated from Synechocystis sp. wild-type strain PCC 6803 and a DeltahhoA mutant resulted in specific degradation of selected proteins at elevated temperatures. We concluded that a single PDZ domain of HhoA plays a critical role in defining the protease activity and oligomerization state, combining the functions that are attributed to two PDZ domains in the homologous DegP protease from Escherichia coli. Based on this first enzymatic study of a Deg protease from cyanobacteria, we propose a general role for HhoA in the quality control of extracytoplasmic proteins, including membrane proteins, in Synechocystis sp. strain PCC 6803.     

The Stromal Chloroplast Deg7 Protease Participates in the Repair of Photosystem II after Photoinhibition in Arabidopsis

Light is the ultimate source of energy for photosynthesis; however, excessive light leads to photooxidative damage and hence reduced 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 and degraded. However, it is still largely unknown whether degradation of D1 and other PSII proteins involves previously uncharacterized proteases. Here, we show that Deg7 is peripherally associated with the stromal side of the thylakoid membranes and that Deg7 interacts directly with PSII. Our results show that Deg7 is involved in the primary cleavage of photodamaged D1, D2, CP47, and CP43 and that this activity is essential for its function in PSII repair. The double mutants deg5 deg7 and deg8 deg7 showed no obvious phenotypic differences under normal growth conditions, but additive effects were observed under high light. These results suggest that Deg proteases on both the stromal and luminal sides of the thylakoid membranes are important for the efficient PSII repair in Arabidopsis (Arabidopsis thaliana).Chloroplasts of higher plants carry out one of the most important biochemical reactions: the capture of light energy and its conversion into chemical energy. Although light is the ultimate source of energy for photosynthesis, it can also be harmful to plants. Light-induced loss of photosynthetic efficiency, which is generally termed as photoinhibition, limits plant growth and lowers productivity, especially when combined with other abiotic stresses.The main target of photoinhibition is PSII, which catalyzes the light-dependent water oxidation concomitantly with oxygen production (for review, see Prasil et al., 1992; Aro et al., 1993; Adir et al., 2003). In higher plants, PSII consists of more than 20 subunits, including the reaction center D1 and D2 proteins, cytochrome (Cyt) b₅₅₉, the light-harvesting chlorophyll a-binding proteins CP47 and CP43, the oxygen-evolving 33-kD protein (PsbO), and several low molecular mass proteins (Nelson and Yocum, 2006). The PSII reaction center D1 protein has been identified among PSII proteins as the primary target of light-induced damage (Kyle et al., 1984; Mattoo et al., 1984; Ohad et al., 1984; Adir et al., 1990), but several studies have shown that the D2, CP47, and CP43 proteins are degraded under photoinhibitory conditions (Schuster et al., 1988; Yamamoto and Akasaka, 1995; Jansen et al., 1999; Adir et al., 2003). Moreover, several small PSII subunits, such as PsbH, PsbW, and Cyt b₅₅₉, were also found to be frequently replaced within PSII (Hagman et al., 1997; Ortega et al., 1999; Bergantino et al., 2003). Evidence for the involvement of two families of proteases, FtsH and Deg, in the degradation of the D1 protein in thylakoids of higher plants has been recently described (Lindahl et al., 1996, 2000; Bailey et al., 2002; Sakamoto et al., 2003; Silva et al., 2003; Kapri-Pardes et al., 2007; Sun et al., 2007a, 2007b). However, it is still largely unknown whether degradation of D1 and other PSII proteins involves previously uncharacterized proteases.DegP (or HtrA) proteases were initially identified based on the fact that they are required for the survival of Escherichia coli at high temperatures and for the degradation of abnormal periplasmic proteins (Lipinska et al., 1988; Strauch and Beckwith, 1988). DegP is an ATP-independent Ser endopeptidase, and it contains a trypsin-like protease domain at the N terminus, followed by two PDZ domains (Gottesman, 1996; Pallen and Wren, 1997; Clausen et al., 2002). PDZ domains 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., 1996; Harris and Lim, 2001). DegP switches between chaperone and protease functions in a temperature-dependent manner. The chaperone function dominates at low temperatures, and DegP becomes proteolytically active at elevated temperatures (Spiess et al., 1999). Crystal structures of different members of the DegP protein family (Krojer et al., 2002; Li et al., 2002; Kim et al., 2003; Wilken et al., 2004) have revealed the structure-function relationship of these PDZ-containing proteases. Trimeric DegP is the functional unit, and the hexameric DegP is formed via the staggered association of trimers (Clausen et al., 2002; Kim and Kim, 2005). At normal growth temperatures, the active site of the protease is located within the chamber of hexameric DegP, which is not accessible to the substrates. However, at high temperatures, conformational changes induce the activation of the protease function (Krojer et al., 2002). Recent studies have shed light on the substrate binding-induced formation of larger oligomeric complexes of DegP (Jiang et al., 2008; Krojer et al., 2008).In Arabidopsis (Arabidopsis thaliana), 16 genes coding for DegP-like proteases have been identified, and at least seven gene products are predicted to be located in chloroplasts (Kieselbach and Funk, 2003; Huesgen et al., 2005; Adam et al., 2006; Sakamoto, 2006; Kato and Sakamoto, 2009). Based on proteomic data, four Deg proteases have been shown to be localized to the chloroplast (Peltier et al., 2002; Schubert et al., 2002) and functionally characterized. Deg1, Deg5, and Deg8 are located in thylakoid lumen, and Deg2 is peripherally associated with the stromal side of thylakoid membranes (Itzhaki et al., 1998; Haußühl et al., 2001; Sun et al., 2007a). Recombinant DegP1, now renamed Deg1, has been shown to be proteolytically active toward thylakoid lumen proteins such as plastocyanin and PsbO of PSII in vitro (Chassin et al., 2002). A 5.2-kD C-terminal fragment of the D1 protein was detected in vitro after incubation of recombinant Deg1 with inside-out thylakoid membranes. In transgenic plants with reduced levels of Deg1, fewer of its 16- and 5.2-kD degradation products were observed (Kapri-Pardes et al., 2007). Deg5 and Deg8 form a dodecameric complex in the thylakoid lumen, and recombinant Deg8 is able to degrade the photodamaged D1 protein of PSII in an in vitro assay (Sun et al., 2007a). The 16-kD N-terminal degradation fragment of the D1 protein was detected in wild-type plants but not in a deg5 deg8 double mutant after high-light treatment. The deg5 deg8 double mutant showed increased sensitivity to high light and high temperature in terms of growth and PSII activity compared with the single mutants deg5 and deg8, suggesting that Deg5 and Deg8 have overlapping functions in the primary cleavage of the CD loop of the D1 protein (Sun et al., 2007a, 2007b). In vitro analysis has demonstrated that recombinant stroma-localized Deg2 was also shown to be involved in the primary cleavage of the DE loop of the D1 protein (Haußühl et al., 2001). However, analysis of a mutant lacking Deg2 suggested that Deg2 may not be involved in D1 degradation in vivo (Huesgen et al., 2006).Here, we have expressed and purified a recombinant DegP protease, His-Deg7. In vitro experiments showed that His-Deg7 is proteolytically active toward the PSII proteins D1, D2, CP43, and CP47. In vivo analyses of a deg7 mutant revealed that the mutant is more sensitive to high light stress than the wild-type plants. We demonstrated that Deg7 is a chloroplast stroma protein associated with the thylakoid membranes and that it interacts with PSII, which suggests that it can cleave the stroma-exposed region of substrate proteins. Our results also provide evidence that Deg7 is important for maintaining PSII function.