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.     

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.     

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.     

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.     

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.     

Quality control of Photosystem II: an FtsH protease plays an essential role in the turnover of the reaction center D1 protein in Synechocystis PCC 6803 under heat stress as well as light stress conditions.

The role of an AAA protease FtsH (slr0228) in the turnover of the D1 protein was studied under moderate heat stress conditions using wild-type cells of the cyanobacterium Synechocystis PCC 6803 and the mutant cells lacking a homologue of FtsH (slr0228). When the growth temperature of the wild-type was shifted from 30 degrees C to 40 degrees C, growth and oxygen-evolving activity were partially inhibited. Under the same heat stress, growth of the mutant was inhibited more significantly (63% inhibition after 5 days heat stress, compared with 26% inhibition with the wild-type cells) and the oxygen-evolving activity was also impaired in parallel. With heat stress at 42 degrees C, the level of the D1 protein of wild type cells was decreased, whereas that in mutant cells was not. The responses of cyanobacterial cells to heat stress observed here are quite similar to those to light stress that were reported previously. From these results, we suggest that the FtsH protease (slr0228) is responsible for both the heat-induced and light-induced degradation of the D1 protein. Notably, the amount of FtsH increased when the wild-type cells were exposed to heat stress or light stress, indicating that the up-regulation of the FtsH protease in the thylakoids is crucial for the cyanobacterial cells to cope with these abiotic stresses.     

Parallel expression of alternate forms of psbA2 gene provides evidence for the existence of a targeted D1 repair mechanism in Synechocystis sp. PCC 6803

The D1 protein of Photosystem II (PSII) is recognized as the main target of photoinhibitory damage and exhibits a high turnover rate due to its degradation and replacement during the PSII repair cycle. Damaged D1 is replaced by newly synthesized D1 and, although reasonable, there is no direct evidence for selective replacement of damaged D1. Instead, it remains possible that increased turnover of D1 subunits occurs in a non-selective manner due for example, to a general up-regulation of proteolytic activity triggered during damaging environmental conditions, such as high light. To determine if D1 degradation is targeted to damaged D1 or generalized to all D1, we developed a genetic system involving simultaneous dual expression of wild type and mutant versions of D1 protein. Dual D1 strains (nS345P:eWT and nD170A:eWT) expressed a wild type (WT) D1 from ectopic and a damage prone mutant (D1-S345P, D1-D170A) from native locus on the chromosome. Characterization of strains showed that all dual D1 strains restore WT like phenotype with high PSII activity. Higher PSII activity indicates increased population of PSII reaction centers with WT D1. Analysis of steady state levels of D1 in nS345P:eWT by immunoblot showed an accumulation of WT D1 only. But, in vivo pulse labeling confirmed the synthesis of both S345P (exists as iD1) and WT D1 in the dual strain. Expression of nS345P:eWT in FtsH2 knockout background showed accumulation of both iD1 and D1 proteins. This demonstrates that dual D1 strains express both forms of D1, yet only damage prone PSII complexes are selected for repair providing evidence that the D1 degradation process is targeted towards damaged PSII complexes. Since the N-terminus has been previously shown to be important for the degradation of damaged D1, the possibility that the highly conserved cysteine 18 residue situated in the N-terminal domain of D1 is involved in the targeted repair process was tested by examining site directed mutants of this and the other cysteines of the D1 protein. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

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.     

Amino acid composition of leaf,grain and bracts of japonica rice (<Emphasis Type="Italic">Oryza Sativa</Emphasis> ssp. <Emphasis Type="Italic">japonica</Emphasis>) and its response to nitrogen fertilization

Heat stress is one of the main abiotic stresses that limit plant growth. The effects of high temperature on oxidative damage, PSII activity and D1 protein turnover were studied in three wheat varieties with different heat susceptibility (CS, YN949 and AK58). The results showed that heat stress induced lower lipid peroxidation in AK58 and YN949 than CS, which was related to different changes of SOD, CAT, POD and H₂O₂. Similarly, AK58 and YN949 performed better PSII photochemical efficiency (F_v/F_m, ΦPSII and ETR) under high temperature, which was attributed to rapid synthesis and degradation of D1 protein. Moreover, higher expression of D1 protein turnover-related genes (PsbA, STN8, PBCP, Deg1, Deg2, Deg5, Deg8, FtsH1/5 and FtsH2/8) and SOD activity in AK58 and YN949 under normal conditions also established a basis for acclimatizing high temperatures, thereby alleviating PSII photoinhibition and reducing oxidative damage when exposed to heat stress.     

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.     

Recent advances in understanding the assembly and repair of photosystem II

Background

Photosystem II (PSII) is the light-driven water:plastoquinone oxidoreductase of oxygenic photosynthesis and is found in the thylakoid membrane of chloroplasts and cyanobacteria. Considerable attention is focused on how PSII is assembled in vivo and how it is repaired following irreversible damage by visible light (so-called photoinhibition). Understanding these processes might lead to the development of plants with improved growth characteristics especially under conditions of abiotic stress.

Scope

Here we summarize recent results on the assembly and repair of PSII in cyanobacteria, which are excellent model organisms to study higher plant photosynthesis.

Conclusions

Assembly of PSII is highly co-ordinated and proceeds through a number of distinct assembly intermediates. Associated with these assembly complexes are proteins that are not found in the final functional PSII complex. Structural information and possible functions are beginning to emerge for several of these ‘assembly’ factors, notably Ycf48/Hcf136, Psb27 and Psb28. A number of other auxiliary proteins have been identified that appear to have evolved since the divergence of chloroplasts and cyanobacteria. The repair of PSII involves partial disassembly of the damaged complex, the selective replacement of the damaged sub-unit (predominantly the D1 sub-unit) by a newly synthesized copy, and reassembly. It is likely that chlorophyll released during the repair process is temporarily stored by small CAB-like proteins (SCPs). A model is proposed in which damaged D1 is removed in Synechocystis sp. PCC 6803 by a hetero-oligomeric complex composed of two different types of FtsH sub-unit (FtsH2 and FtsH3), with degradation proceeding from the N-terminus of D1 in a highly processive reaction. It is postulated that a similar mechanism of D1 degradation also operates in chloroplasts. Deg proteases are not required for D1 degradation in Synechocystis 6803 but members of this protease family might play a supplementary role in D1 degradation in chloroplasts under extreme conditions.     

Assembling and maintaining the Photosystem II complex in chloroplasts and cyanobacteria

Plants, algae and cyanobacteria grow because of their ability to use sunlight to extract electrons from water. This vital reaction is catalysed by the Photosystem II (PSII) complex, a large multi-subunit pigment-protein complex embedded in the thylakoid membrane. Recent results show that assembly of PSII occurs in a step-wise fashion in defined regions of the membrane system, involves conserved auxiliary factors and is closely coupled to chlorophyll biosynthesis. PSII is also repaired following damage by light. FtsH proteases play an important role in selectively removing damaged proteins from the complex, both in chloroplasts and cyanobacteria, whilst undamaged subunits and pigments are recycled. The chloroplastic Deg proteases play a supplementary role in PSII repair.     

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.     

Sub‐cellular location of FtsH proteases in the cyanobacterium Synechocystis sp. PCC 6803 suggests localised PSII repair zones in the thylakoid membranes

In cyanobacteria and chloroplasts, exposure to HL damages the photosynthetic apparatus, especially the D1 subunit of Photosystem II. To avoid chronic photoinhibition, a PSII repair cycle operates to replace damaged PSII subunits with newly synthesised versions. To determine the sub‐cellular location of this process, we examined the localisation of FtsH metalloproteases, some of which are directly involved in degrading damaged D1. We generated transformants of the cyanobacterium Synechocystis sp. PCC6803 expressing GFP‐tagged versions of its four FtsH proteases. The ftsH2–gfp strain was functional for PSII repair under our conditions. Confocal microscopy shows that FtsH1 is mainly in the cytoplasmic membrane, while the remaining FtsH proteins are in patches either in the thylakoid or at the interface between the thylakoid and cytoplasmic membranes. HL exposure which increases the activity of the Photosystem II repair cycle led to no detectable changes in FtsH distribution, with the FtsH2 protease involved in D1 degradation retaining its patchy distribution in the thylakoid membrane. We discuss the possibility that the FtsH2–GFP patches represent Photosystem II ‘repair zones’ within the thylakoid membranes, and the possible advantages of such functionally specialised membrane zones. Anti‐GFP affinity pull‐downs provide the first indication of the composition of the putative repair zones.     

Involvement of an FtsH homologue in the assembly of functional photosystem I in the cyanobacterium Synechocystis sp. PCC 6803

The Synechocystis sp. PCC 6803 genome encodes four putative homologues of the AAA protease FtsH, two of which (slr0228 and sll1463) have been subjected to insertional mutagenesis in this study. Disruption of sll1463 had no discernible effect but disruption of slr0228 caused a 60% reduction in the abundance of functional photosystem I, without affecting the cellular content of photosystem II or phycobilisomes. Fluorescence and immunoblotting analyses show reductions in PS I polypeptides and possible structural alterations in the residual PS I, indicating an important role for slr0228 in PS I biogenesis.     

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.     

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.     

Repair of UV-B induced damage of Photosystem II via de novo synthesis of the D1 and D2 reaction centre subunits in Synechocystis sp. PCC 6803

The repair of ultraviolet-B radiation induced damage to the structure and function of Photosystem II was studied in the cyanobacterium Synechocystis sp. PCC 6803. UV-B irradiation of intact Synechocystis cells results in the loss of steady-state oxygen evolution, an effect accompanied by a parallel loss of both D1 and D2 protein subunits of the Photosystem II reaction centre. Transfer of the UV-irradiated cells to normal growth conditions under visible light results in partial recovery of the inhibited oxygen evolving activity and restoration of the lost D1 and D2 proteins. The extent of recovery decreases with increasing degree of damage: after 50% inhibition, the original activity is completely restored within 2 hours. In contrast, after 90–95% inhibition less than half of the original activity is regained during a 4 hour recovery period. The translation inhibitor lincomycin completely blocks the recovery process if added after the UV-B treatment, and accelerates the kinetics of activity loss if added before the onset of UV-B irradiation. Substantial retardation of recovery and acceleration of activity loss is also observed if the very low intensity short wavelength contribution (<290 nm) is not filtered out from the UV-B light source. It is concluded that in intact cells UV-B induced damage of the Photosystem II complex can be repaired. This process is the first example of simultaneous D1 and D2 protein repair in Photosystem II, and considered to function as an important defence mechanism against detrimental UV-B effects in oxygenic photosynthetic organisms. De novo synthesis of the D1 and D2 reaction centre subunits is a key step of the repair process, which itself can also be inhibited by ultraviolet light, especially by the short wavelength UV-C components, or by high doses of UV-B.