The deg proteases protect Synechocystis sp. PCC 6803 during heat and light stresses but are not essential for removal of damaged D1 protein during the photosystem two repair cycle

Members of the DegP/HtrA (or Deg) family of proteases are found widely in nature and play an important role in the proteolysis of misfolded and damaged proteins. As yet, their physiological role in oxygenic photosynthetic organisms is unclear, although it has been widely speculated that they participate in the degradation of the photodamaged D1 subunit in the photosystem two complex (PSII) repair cycle, which is needed to maintain PSII activity in both cyanobacteria and chloroplasts. We have examined the role of the three Deg proteases found in the cyanobacterium Synechocystis sp. PCC 6803 through analysis of double and triple insertion mutants. We have discovered that these proteases show overlap in function and are involved in a number of key physiological responses ranging from protection against light and heat stresses to phototaxis. In previous work, we concluded that the Deg proteases played either a direct or an indirect role in PSII repair in a glucose-tolerant version of Synechocystis 6803 (Silva, P., Choi, Y. J., Hassan, H. A., and Nixon, P. J. (2002) Philos. Trans. R. Soc. Lond. B Biol. Sci. 357, 1461-1467). In this work, we have now been able to demonstrate unambiguously, using a triple deg mutant created in the wild type strain of Synechocystis 6803, that the Deg proteases are not obligatory for PSII repair and D1 degradation. We therefore conclude that although the Deg proteases are needed for photoprotection of Synechocystis sp. PCC 6803, they do not play an essential role in D1 turnover and PSII repair in vivo.     

FtsH is involved in the early stages of repair of photosystem II in Synechocystis sp PCC 6803

When plants, algae, and cyanobacteria are exposed to excessive light, especially in combination with other environmental stress conditions such as extreme temperatures, their photosynthetic performance declines. A major cause of this photoinhibition is the light-induced irreversible photodamage to the photosystem II (PSII) complex responsible for photosynthetic oxygen evolution. A repair cycle operates to selectively replace a damaged D1 subunit within PSII with a newly synthesized copy followed by the light-driven reactivation of the complex. Net loss of PSII activity occurs (photoinhibition) when the rate of damage exceeds the rate of repair. The identities of the chaperones and proteases involved in the replacement of D1 in vivo remain uncertain. Here, we show that one of the four members of the FtsH family of proteases (cyanobase designation slr0228) found in the cyanobacterium Synechocystis sp PCC 6803 is important for the repair of PSII and is vital for preventing chronic photoinhibition. Therefore, the ftsH gene family is not functionally redundant with respect to the repair of PSII in this organism. Our data also indicate that FtsH binds directly to PSII, is involved in the early steps of D1 degradation, and is not restricted to the removal of D1 fragments. These results, together with the recent analysis of ftsH mutants of Arabidopsis, highlight the critical role played by FtsH proteases in the removal of damaged D1 from the membrane and the maintenance of PSII activity in vivo.     

FtsH-mediated repair of the photosystem II complex in response to light stress

A common feature of light stress in plants, algae, and cyanobacteria is the light-induced damage to the photosystem II complex (PSII), which catalyses the photosynthetic oxidation of water to molecular oxygen. A repair cycle operates to replace damaged subunits within PSII, in particular, the D1 reaction centre polypeptide, by newly synthesized copies. As yet the molecular details of this physiologically important process remain obscure. A key aspect of the process that has attracted much attention is the identity of the protease or proteases involved in D1 degradation. The results are summarized here of recent mutagenesis experiments that were designed to assess the functional importance of the DegP/HtrA and FtsH protease families in the cyanobacterium Synechocystis sp. PCC 6803. Based on these results and the analysis of Arabidopsis mutants, a general model for PSII repair is suggested in which FtsH complexes alone are able to degrade damaged D1.     

The family of Deg proteases in cyanobacteria and chloroplasts of higher plants

The family of Deg proteases is present in nearly all organisms from bacteria to higher plants. This family consists of ATP-independent serine endopeptidases with a catalytic domain of trypsin type and up to three PDZ domains, involved in protein–protein interactions. Sixteen deg genes (originally named deg P1–16) were found in Arabidopsis thaliana , and the chloroplast location was predicted or experimentally proven for seven proteins. The cyanobacterium Synechocystis sp. PCC6803 contains three Deg homologues, HtrA (DegP), HhoA (DegQ) and HhoB (DegS), but their number can vary between one and six in other photosynthetic Prokaryota. Interestingly, all of these proteases are evolutionarily more closely related within one species than proteases with the same names present in other organisms. This means that Deg proteases from A. thaliana are not necessarily the closest relatives of cyanobacterial DegP. Therefore, we propose to change the misleading original name 'DegP' to 'Deg' for A. thaliana enzymes. Here, we summarize the expression, location and functions of Deg proteases from cyanobacteria and chloroplasts of higher plants, with special emphasis on their role in the photosystem II (PSII) repair cycle under light stress conditions.     

The exposed N-terminal tail of the D1 subunit is required for rapid D1 degradation during photosystem II repair in Synechocystis sp PCC 6803

The selective replacement of photodamaged D1 protein within the multisubunit photosystem II (PSII) complex is an important photoprotective mechanism in chloroplasts and cyanobacteria. FtsH proteases are involved at an early stage of D1 degradation, but it remains unclear how the damaged D1 subunit is recognized, degraded, and replaced. To test the role of the N-terminal region of D1 in PSII biogenesis and repair, we have constructed mutants of the cyanobacterium Synechocystis sp PCC 6803 that are truncated at the exposed N terminus. Removal of 5 or 10 residues blocked D1 synthesis, as assessed in radiolabeling experiments, whereas removal of 20 residues restored the ability to assemble oxygen-evolving dimeric PSII complexes but inhibited PSII repair at the level of D1 degradation. Overall, our results identify an important physiological role for the exposed N-terminal tail of D1 at an early step in selective D1 degradation. This finding has important implications for the recognition of damaged D1 and its synchronized replacement by a newly synthesized subunit.     

Protection by α-tocopherol of the repair of photosystem II during photoinhibition in Synechocystis sp. PCC 6803

α-Tocopherol is a lipophilic antioxidant that is an efficient scavenger of singlet oxygen. We investigated the role of α-tocopherol in the protection of photosystem II (PSII) from photoinhibition using a mutant of the cyanobacterium Synechocystis sp. PCC 6803 that is deficient in the biosynthesis of α-tocopherol. The activity of PSII in mutant cells was more sensitive to inactivation by strong light than that in wild-type cells, indicating that lack of α-tocopherol enhances the extent of photoinhibition. However, the rate of photodamage to PSII, as measured in the presence of chloramphenicol, which blocks the repair of PSII, did not differ between the two lines of cells. By contrast, the repair of PSII from photodamage was suppressed in mutant cells. Addition of α-tocopherol to cultures of mutant cells returned the extent of photoinhibition to that in wild-type cells, without any effect on photodamage. The synthesis de novo of various proteins, including the D1 protein that plays a central role in the repair of PSII, was suppressed in mutant cells under strong light. These observations suggest that α-tocopherol promotes the repair of photodamaged PSII by protecting the synthesis de novo of the proteins that are required for recovery from inhibition by singlet oxygen.     

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.     

A change in the sensitivity of elongation factor G to oxidation protects photosystem II from photoinhibition in Synechocystis sp. PCC 6803

The repair of photosystem II (PSII) after photodamage is particularly sensitive to oxidative stress and inhibition of such repair is associated with the oxidation of specific cysteine residues in elongation factor G (EF-G), a key translation factor, in the cyanobacterium Synechocystis sp. PCC 6803. Expression of mutated EF-G with a target cysteine residue replaced by serine in Synechocystis resulted in the protection of PSII from photoinhibition. This protection was attributable to the enhanced repair of PSII via acceleration of the synthesis of the D1 protein, which might have been due to reduced sensitivity of protein synthesis to oxidative stress.     

The FtsH protease slr0228 is important for quality control of photosystem II in the thylakoid membrane of Synechocystis sp. PCC 6803

The cyanobacterium Synechocystis sp. PCC 6803 contains four members of the FtsH protease family. One of these, FtsH (slr0228), has been implicated recently in the repair of photodamaged photosystem II (PSII) complexes. We have demonstrated here, using a combination of blue native PAGE, radiolabeling, and immunoblotting, that FtsH (slr0228) is required for selective replacement of the D1 reaction center subunit in both wild type PSII complexes and in PSII subcomplexes lacking the PSII chlorophyll a-binding subunit CP43. To test whether FtsH (slr0228) has a more general role in protein quality control in vivo, we have studied the synthesis and degradation of PSII subunits in wild type and in defined insertion and missense mutants incapable of proper assembly of the PSII holoenzyme. We discovered that, when the gene encoding FtsH (slr0228) was disrupted in these strains, the overall level of assembly intermediates and unassembled PSII proteins markedly increased. Pulse-chase experiments showed that this was due to reduced rates of degradation in vivo. Importantly, analysis of epitope-tagged and green fluorescent protein-tagged strains revealed that slr0228 was present in the thylakoid and not the cytoplasmic membrane. Overall, our results show that FtsH (slr0228) plays an important role in controlling the removal of PSII subunits from the thylakoid membrane and is not restricted to selective D1 turnover.     

Systematic analysis of the relation of electron transport and ATP synthesis to the photodamage and repair of photosystem II in Synechocystis

The photosynthetic machinery and, in particular, the photosystem II (PSII) complex are susceptible to strong light, and the effects of strong light are referred to as photodamage or photoinhibition. In living organisms, photodamaged PSII is rapidly repaired and, as a result, the extent of photoinhibition represents a balance between rates of photodamage and the repair of PSII. In this study, we examined the roles of electron transport and ATP synthesis in these two processes by monitoring them separately and systematically in the cyanobacterium Synechocystis sp. PCC 6803. We found that the rate of photodamage, which was proportional to light intensity, was unaffected by inhibition of the electron transport in PSII, by acceleration of electron transport in PSI, and by inhibition of ATP synthesis. By contrast, the rate of repair was reduced upon inhibition of the synthesis of ATP either via PSI or PSII. Northern blotting and radiolabeling analysis with [(35)S]Met revealed that synthesis of the D1 protein was enhanced by the synthesis of ATP. Our observations suggest that ATP synthesis might regulate the repair of PSII, in particular, at the level of translation of the psbA genes for the precursor to the D1 protein, whereas neither electron transport nor the synthesis of ATP affects the extent of photodamage.     

Digalactosyldiacylglycerol is required for stabilization of the oxygen-evolving complex in photosystem II

The galactolipid digalactosyldiacylglycerol (DGDG) is present in the thylakoid membranes of oxygenic photosynthetic organisms such as higher plants and cyanobacteria. Recent x-ray crystallographic analysis of protein-cofactor supercomplexes in thylakoid membranes revealed that DGDG molecules are present in the photosystem II (PSII) complex (four molecules per monomer), suggesting that DGDG molecules play important roles in folding and assembly of subunits in the PSII complex. However, the specific role of DGDG in PSII has not been fully clarified. In this study, we identified the dgdA gene (slr1508, a ycf82 homolog) of Synechocystis sp. PCC6803 that presumably encodes a DGDG synthase involved in the biosynthesis of DGDG by comparison of genomic sequence data. Disruption of the dgdA gene resulted in a mutant defective in DGDG synthesis. Despite the lack of DGDG, the mutant cells grew as rapidly as the wild-type cells, indicating that DGDG is not essential for growth in Synechocystis. However, we found that oxygen-evolving activity of PSII was significantly decreased in the mutant. Analyses of the PSII complex purified from the mutant cells indicated that the extrinsic proteins PsbU, PsbV, and PsbO, which stabilize the oxygen-evolving complex, were substantially dissociated from the PSII complex. In addition, we found that heat susceptibility but not dark-induced inactivation of oxygen-evolving activity was notably increased in the mutant cells in comparison to the wild-type cells, suggesting that the PsbU subunit is dissociated from the PSII complex even in vivo. These results demonstrate that DGDG plays important roles in PSII through the binding of extrinsic proteins required for stabilization of the oxygen-evolving complex.     

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 .     

Degradation of PsbO by the Deg Protease HhoA Is Thioredoxin Dependent

The widely distributed members of the Deg/HtrA protease family play an important role in the proteolysis of misfolded and damaged proteins. Here we show that the Deg protease rHhoA is able to degrade PsbO, the extrinsic protein of the Photosystem II (PSII) oxygen-evolving complex in Synechocystis sp. PCC 6803 and in spinach. PsbO is known to be stable in its oxidized form, but after reduction by thioredoxin it became a substrate for recombinant HhoA (rHhoA). rHhoA cleaved reduced eukaryotic (specifically, spinach) PsbO at defined sites and created distinct PsbO fragments that were not further degraded. As for the corresponding prokaryotic substrate (reduced PsbO of Synechocystis sp. PCC 6803), no PsbO fragments were observed. Assembly to PSII protected PsbO from degradation. For Synechocystis sp. PCC 6803, our results show that HhoA, HhoB, and HtrA are localized in the periplasma and/or at the thylakoid membrane. In agreement with the idea that PsbO could be a physiological substrate for Deg proteases, part of the cellular fraction of the three Deg proteases of Synechocystis sp. PCC 6803 (HhoA, HhoB, and HtrA) was detected in the PSII-enriched membrane fraction.     

Role of FtsH2 in the repair of Photosystem II in mutants of the cyanobacterium Synechocystis PCC 6803 with impaired assembly or stability of the CaMn4 cluster

The FtsH2 protease, encoded by the slr0228 gene, plays a key role in the selective degradation of photodamaged D1 protein during the repair of Photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. To test whether additional proteases might be involved in D1 degradation during high rates of photodamage, we have studied the synthesis and degradation of the D1 protein in ΔPsbO and ΔPsbV mutants, in which the CaMn₄ cluster catalyzing oxygen evolution is less stable, and in the D1 processing mutants, D1-S345P and ΔCtpA, which are unable to assemble a functional cluster. All four mutants exhibited a dramatically increased rate of D1 degradation in high light compared to the wild-type. Additional inactivation of the ftsH2 gene slowed the rate of D1 degradation dramatically and increased the level of PSII complexes. We conclude that FtsH2 plays a major role in the degradation of both precursor and mature forms of D1 following donor-side photoinhibition. However, this conclusion concerned only D1 assembled into larger complexes containing at least D2 and CP47. In the ΔpsbEFLJ deletion mutant blocked at an early stage in PSII assembly, unassembled D1 protein was efficiently degraded in the absence of FtsH2 pointing to the involvement of other protease(s). Significantly, the ΔPsbO mutant displayed unusually low levels of cellular chlorophyll at extremely low-light intensities. The possibilities that PSII repair may limit the availability of chlorophyll for the biogenesis of other chlorophyll-binding proteins and that PsbO might have a regulatory role in PSII repair are discussed.     

Membrane lipid unsaturation modulates processing of the photosystem II reaction-center protein D1 at low temperatures.

The role of membrane lipid unsaturation in the restoration of photosystem II (PSII) function and in the synthesis of the D1 protein at different temperatures after photoinhibition was studied in wild-type cells and a mutant of Synechocystis sp. PCC 6803 with genetically inactivated desaturase genes. We show that posttranslational carboxyl-terminal processing of the precursor form of the D1 protein is an extremely sensitive reaction in the PSII repair cycle and is readily affected by low temperatures. Furthermore, the threshold temperature at which perturbations in D1-protein processing start to emerge is specifically dependent on the extent of thylakoid membrane lipid unsaturation, as indicated by comparison of wild-type cells with the mutant defective in desaturation of 18:1 fatty acids of thylakoid membranes. When the temperature was decreased from 33 degrees C (growth temperature) to 18 degrees C, the inability of the fatty acid mutant to recover from photoinhibition was accompanied by a failure to process the newly synthesized D1 protein, which accumulated in considerable amounts as an unprocessed precursor D1 protein. Precursor D1 integrated into PSII monomer and dimer complexes even at low temperatures, but no activation of oxygen evolution occurred in these complexes in mutant cells defective in fatty acid unsaturation.     

Synechocystis 6803 mutants expressing distinct forms of the Photosystem II D1 protein from Synechococcus 7942: relationship between the psbA coding region and sensitivity to visible and UV-B radiation

Synechocystis PCC 6803 mutants expressing either the "low light" (D1:1) or the "high light" (D1:2) form of the Photosystem II (PSII) D1 protein from Synechococcus PCC 7942 were constructed and characterized with respect to properties of PSII and sensitivity to visible and UV-B radiation. The AI and AIII mutants (containing only the D1:1 and D1:2 forms, respectively) exhibited very similar PSII characteristics as the control strain and they differed only in the accelerated decay kinetics of flash-induced variable fluorescence measured in the presence of DCMU. However, the mutants showed increased sensitivity to photodamage induced by visible and UV-B radiation, with higher loss of PSII activity in the AI than in the AIII strain. Thus, the difference between strains containing D1:1 and D1:2 found previously in Synechococcus 7942 is maintained after transfer of corresponding psbA genes into Synechocystis 6803 and is directly related to the coding region of these genes. The higher light sensitivity of the AI mutant is caused partly by the higher rate of photodamage and partly by the less efficient PSII repair.     

Requirement of carotene isomerization for the assembly of photosystem II in Synechocystis sp. PCC 6803

Carotene isomerase mutant (crtH mutant) cells of Synechocystis sp. PCC 6803 can accumulate beta-carotene under light conditions. However, the mutant cells grown under a light-activated heterotrophic growth condition contained detectable levels of neither beta-carotene nor D1 protein of the photosystem (PS) II reaction center, and no oxygen-evolving activity of PSII was detected. beta-Carotene and D1 protein appeared and a high level of PSII activity was detected after the cells were transferred to a continuous light condition. The PSI activities of thylakoid membranes from mutant cells were almost the same as those of thylakoid membranes from wild-type cells, both before and after transfer to the continuous light condition. These results suggest that beta-carotene is required for the assembly of PSII but not for that of PSI.     

Singlet oxygen inhibits the repair of photosystem II by suppressing the translation elongation of the D1 protein in Synechocystis sp. PCC 6803

Singlet oxygen, generated during photosynthesis, is a strong oxidant that can, potentially, damage various molecules of biological importance. We investigated the effects in vivo of singlet oxygen on the photodamage to photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. Increases in intracellular concentrations of singlet oxygen, caused by the presence of photosensitizers, such as rose bengal and ethyl eosin, stimulated the apparent photodamage to PSII. However, actual photodamage to PSII, as assessed in the presence of chloramphenicol, was unaffected by the production of singlet oxygen. These observations suggest that singlet oxygen produced by added photosensitizers acts by inhibiting the repair of photodamaged PSII. Labeling of proteins in vivo revealed that singlet oxygen inhibited the synthesis of proteins de novo and, in particular, the synthesis of the D1 protein. Northern blotting analysis indicated that the accumulation of psbA mRNAs, which encode the D1 protein, was unaffected by the production of singlet oxygen. Subcellular localization of polysomes with bound psbA mRNAs suggested that the primary target of singlet oxygen might be the elongation step of translation.