Oxidation of elongation factor G inhibits the synthesis of the D1 protein of photosystem II

Oxidative stress inhibits the repair of photodamaged photosystem II (PSII). This inhibition is due initially to the suppression, by reactive oxygen species (ROS), of the synthesis de novo of proteins that are required for the repair of PSII, such as the D1 protein, at the level of translational elongation. To investigate in vitro the mechanisms whereby ROS inhibit translational elongation, we developed a translation system in vitro from the cyanobacterium Synechocystis sp. PCC 6803. The synthesis of the D1 protein in vitro was inhibited by exogenous H2O2. However, the addition of reduced forms of elongation factor G (EF-G), which is known to be particularly sensitive to oxidation, was able to reverse the inhibition of translation. By contrast, the oxidized forms of EF-G failed to restore translational activity. Furthermore, the overexpression of EF-G of Synechocystis in another cyanobacterium Synechococcus sp. PCC 7942 increased the tolerance of cells to H2O2 in terms of protein synthesis. These observations suggest that EF-G might be the primary target, within the translational machinery, of inhibition by ROS.     

Genomic investigation of the system for selenocysteine incorporation in the bacterial domain

The Photosystem II complex (PSII) is susceptible to inactivation by strong light, and the inactivation caused by strong light is referred to as photoinactivation or photoinhibition. In photosynthetic organisms, photoinactivated PSII is rapidly repaired and the extent of photoinactivation reflects the balance between the light-induced damage (photodamage) to PSII and the repair of PSII. In this study, we examined these two processes separately and quantitatively under stress conditions in the cyanobacterium Synechocystis sp. PCC 6803. The rate of photodamage was proportional to light intensity over a range of light intensities from 0 to 2000 microE m(-2) s(-1), and this relationship was not affected by environmental factors, such as salt stress, oxidative stress due to H2O2, and low temperature. The rate of repair also depended on light intensity. It was high under weak light and reached a maximum of 0.1 min(-1) at 300 microE m(-2) s(-1). By contrast to the rate of photodamage, the rate of repair was significantly reduced by the above-mentioned environmental factors. Pulse-labeling experiments with radiolabeled methionine revealed that these environmental factors inhibited the synthesis de novo of proteins. Such proteins included the D1 protein which plays an important role in the photodamage-repair cycle. These observations suggest that the repair of PSII under environmental stress might be the critical step that determines the outcome of the photodamage-repair cycle.     

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.     

Involvement of the HtrA family of proteases in the protection of the cyanobacterium Synechocystis PCC 6803 from light stress and in the repair of photosystem II

Photosystem II (PSII) is prone to irreversible light-induced damage, with the D1 polypeptide a major target. Repair processes operate in the cell to replace a damaged D1 subunit within the complex with a newly synthesized copy. As yet, the molecular details of PSII repair are relatively obscure despite the critical importance of this process for maintaining PSII activity and cell viability. We are using the cyanobacterium Synechocystis sp. PCC 6803 to identify the various proteases and chaperones involved in D1 turnover in vivo. Two families of proteases are being studied: the FtsH family (four members) of Zn(2+)-activated nucleotide-dependent proteases; and the HtrA (or DegP) family (three members) of serine-type proteases. In this paper, we report the results of our studies on a triple mutant in which all three copies of the htrA gene family have been inactivated. Growth of the mutant on agar plates was inhibited at high light intensities, especially in the presence of glucose. Oxygen evolution measurements indicated that, under conditions of high light, the rate of synthesis of functional PSII was less in the mutant than in the wild-type. Immunoblotting experiments conducted on cells blocked in protein synthesis further indicated that degradation of D1 was slowed in the mutant. Overall, our observations indicate that the HtrA family of proteases are involved in the resistance of Synechocystis 6803 to light stress and play a part, either directly or indirectly, in the repair of PSII in vivo.     

How do environmental stresses accelerate photoinhibition?

Environmental stress enhances the extent of photoinhibition, a process that is determined by the balance between the rate of photodamage to photosystem II (PSII) and the rate of its repair. Recent investigations suggest that exposure to environmental stresses, such as salt, cold, moderate heat and oxidative stress, do not affect photodamage but inhibit the repair of PSII through suppression of the synthesis of PSII proteins. In particular, production of D1 protein is downregulated at the translation step by the direct inactivation of the translation machinery and/or by primarily interrupting the fixation of CO2. The latter results in the creation of reactive oxygen species (ROS), which in turn block the synthesis of PSII proteins in chloroplasts.     

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.     

Revised scheme for the mechanism of photoinhibition and its application to enhance the abiotic stress tolerance of the photosynthetic machinery

When photosynthetic organisms are exposed to abiotic stress, their photosynthetic activity is significantly depressed. In particular, photosystem II (PSII) in the photosynthetic machinery is readily inactivated under strong light and this phenomenon is referred to as photoinhibition of PSII. Other types of abiotic stress act synergistically with light stress to accelerate photoinhibition. Recent studies of photoinhibition have revealed that light stress damages PSII directly, whereas other abiotic stresses act exclusively to inhibit the repair of PSII after light-induced damage (photodamage). Such inhibition of repair is associated with suppression, by reactive oxygen species (ROS), of the synthesis of proteins de novo and, in particular, of the D1 protein, and also with the reduced efficiency of repair under stress conditions. Gene-technological improvements in the tolerance of photosynthetic organisms to various abiotic stresses have been achieved via protection of the repair system from ROS and, also, by enhancing the efficiency of repair via facilitation of the turnover of the D1 protein in PSII. In this review, we summarize the current status of research on photoinhibition as it relates to the effects of abiotic stress and we discuss successful strategies that enhance the activity of the repair machinery. In addition, we propose several potential methods for activating the repair system by gene-technological methods.     

Very strong UV-A light temporally separates the photoinhibition of photosystem II into light-induced inactivation and repair

When organisms that perform oxygenic photosynthesis are exposed to strong visible or UV light, inactivation of photosystem II (PSII) occurs. However, such organisms are able rapidly to repair the photoinactivated PSII. The phenomenon of photoinactivation and repair is known as photoinhibition. Under normal laboratory conditions, the rate of repair is similar to or faster than the rate of photoinactivation, preventing the detailed analysis of photoinactivation and repair as separate processes. We report here that, using strong UV-A light from a laser, we were able to analyze separately the photoinactivation and repair of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803. Very strong UV-A light at 364 nm and a photon flux density of 2600 micromol photons m(-2) s(-1) inactivated the oxygen-evolving machinery and the photochemical reaction center of PSII within 1 or 2 min before the first step in the repair process, namely, the degradation of the D1 protein, occurred. During subsequent incubation of cells in weak visible light, the activity of PSII recovered fully within 30 min and this process depended on protein synthesis. During subsequent incubation of cells in darkness for 60 min, the D1 protein of the photoinactivated PSII was degraded. Further incubation in weak visible light resulted in the rapid restoration of the activity of PSII. These observations suggest that very strong UV-A light is a useful tool for the analysis of the repair of PSII after photoinactivation.     

Regulation of Translation by the Redox State of Elongation Factor G in the Cyanobacterium Synechocystis sp. PCC 6803

Elongation factor G (EF-G), a key protein in translational elongation, was identified as a primary target of inactivation by reactive oxygen species within the translational machinery of the cyanobacterium Synechocystis sp. PCC 6803 (Kojima, K., Oshita, M., Nanjo, Y., Kasai, K., Tozawa, Y., Hayashi, H., and Nishiyama, Y. (2007) Mol. Microbiol. 65, 936–947). In the present study, we found that inactivation of EF-G (Slr1463) by H₂O₂ was attributable to the oxidation of two specific cysteine residues and formation of a disulfide bond. Substitution of these cysteine residues by serine residues protected EF-G from inactivation by H₂O₂ and allowed the EF-G to mediate translation in a translation system in vitro that had been prepared from Synechocystis. The disulfide bond in oxidized EF-G was reduced by thioredoxin, and the resultant reduced form of EF-G regained the activity to mediate translation in vitro. Western blotting analysis showed that levels of the oxidized form of EF-G increased under strong light in a mutant that lacked NADPH-thioredoxin reductase, indicating that EF-G is reduced by thioredoxin in vivo. These observations suggest that the translational machinery is regulated by the redox state of EF-G, which is oxidized by reactive oxygen species and reduced by thioredoxin, a transmitter of reducing signals generated by the photosynthetic transport of electrons.Reactive oxygen species (ROS)² are produced as inevitable by-products of the light-driven reactions of photosynthesis. The superoxide radical, hydrogen peroxide (H₂O₂), and the hydroxyl radical are produced as a result of the photosynthetic transport of electrons, whereas singlet state oxygen (singlet oxygen) is produced by the transfer of excitation energy (1). Exposure of the photosynthetic machinery to strong light promotes the production of ROS and gives rise to oxidative stress (1).Strong light rapidly inactivates photosystem II (PSII). This phenomenon is referred to as photoinhibition (2–4), and it occurs when the rate of photodamage to PSII exceeds the rate of the repair of photodamaged PSII (5). The actions of ROS in the photoinhibition of PSII have been studied extensively, and several mechanisms for photoinhibition have been proposed (5). Recent studies of the effects of ROS on photodamage and repair have revealed that ROS act primarily by inhibiting the repair of photodamaged PSII and not by damaging PSII directly (5–9). Such studies have also shown that photodamage to PSII is an exclusively light-dependent event; photodamage is initiated by disruption of the manganese cluster of the oxygen-evolving complex upon absorption of light, in particular UV and blue light, with subsequent damage to the reaction center upon absorption of visible light by chlorophylls (10–12).Inhibition of the repair of PSII has been attributed to the suppression, by ROS, of the synthesis de novo of proteins that are required for the repair of PSII, such as the D1 protein, which forms a heterodimer with the D2 protein in the reaction center, in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis) (6, 7), in Chlamydomonas (13), and in plants (14, 15). Analysis of polysomes in Synechocystis has demonstrated that ROS inhibit the synthesis de novo of proteins primarily at the elongation step of translation, suggesting that some proteins involved in translational elongation might be the targets of inactivation by ROS (6, 7).A translation system in vitro was successfully prepared from Synechocystis, and biochemical investigations using this translation system have revealed that elongation factor G (EF-G), a GTP-binding protein that catalyzes the translocation of peptidyl-tRNA (16), is a primary target of inactivation by ROS (17). EF-G is reversibly inactivated by ROS in a redox-dependent manner; it is inactive in the oxidized form and active in the reduced form (17). Moreover, it has been proposed that changes in the activity of EF-G might depend on and be regulated by the redox states of cysteine residues within EF-G (17). However, the specific cysteine residues within EF-G that might be the targets of ROS and might be responsible for redox regulation remain to be determined.In the present study, we investigated the redox state of Slr1463, the EF-G that is phylogenetically closest to chloroplast EF-G among three homologs of EF-G in Synechocystis (17). We determined that two specific cysteine residues in the EF-G of Synechocystis were targets of oxidation by ROS. The resultant disulfide bond between the two cysteine residues was efficiently reduced by thioredoxin. In addition, we observed that EF-G was reduced by thioredoxin in vivo. Our findings revealed the mechanism of the ROS-induced inactivation of EF-G and suggested a mechanism for the redox regulation of translation by electrons generated during photosynthesis.     

A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II

Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to the imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. Photodamage is initiated by the direct effects of light on the oxygen-evolving complex and, thus, photodamage to PSII is unavoidable. Studies of the effects of oxidative stress on photodamage and subsequent repair have revealed that reactive oxygen species (ROS) act primarily by inhibiting the repair of photodamaged PSII and not by damaging PSII directly. Thus, strong light has two distinct effects on PSII; it damages PSII directly and it inhibits the repair of PSII via production of ROS. Investigations of the ROS-induced inhibition of repair have demonstrated that ROS suppress the synthesis de novo of proteins and, in particular, of the D1 protein, that are required for the repair of PSII. Moreover, a primary target for inhibition by ROS appears to be the elongation step of translation. Inhibition of the repair of PSII by ROS is accelerated by the deceleration of the Calvin cycle that occurs when the availability of CO₂ is limited. In this review, we present a new paradigm for the action of ROS in photoinhibition.     

A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II

Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to the imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. Photodamage is initiated by the direct effects of light on the oxygen-evolving complex and, thus, photodamage to PSII is unavoidable. Studies of the effects of oxidative stress on photodamage and subsequent repair have revealed that reactive oxygen species (ROS) act primarily by inhibiting the repair of photodamaged PSII and not by damaging PSII directly. Thus, strong light has two distinct effects on PSII; it damages PSII directly and it inhibits the repair of PSII via production of ROS. Investigations of the ROS-induced inhibition of repair have demonstrated that ROS suppress the synthesis de novo of proteins and, in particular, of the D1 protein, that are required for the repair of PSII. Moreover, a primary target for inhibition by ROS appears to be the elongation step of translation. Inhibition of the repair of PSII by ROS is accelerated by the deceleration of the Calvin cycle that occurs when the availability of CO(2) is limited. In this review, we present a new paradigm for the action of ROS in photoinhibition.     

Recovery of photosystem II activity in photoinhibited synechocystis cells: light-dependent translation activity is required besides light-independent synthesis of the D1 protein

Irreversible photoinactivation of photosystem II (PSII) results in the degradation of the reaction center II D1 protein. In Synechocystis PCC 6714 cells, recovery of PSII activity requires illumination. The rates of photoinactivation and recovery of PSII activity in the light are similar in cells grown in minimal (MM) or glucose-containing medium (GM). Reassembly of PSII with newly synthesized proteins requires degradation of the D1 protein of the photoinactivated PSII. This process may occur in darkness in both types of cells. The degraded D1 protein is, however, only partially replaced by newly synthesized protein in MM cells in darkness while a high level of D1 protein synthesis occurs in darkness in the GM cells. The newly synthesized D1 protein in darkness appears to be assembled with other PSII proteins. However, PSII activity is not recovered in such cells. Illumination of the cells in absence but not in the presence of protein synthesis inhibitors allows recovery of PSII activity.     

Protein synthesis is the primary target of reactive oxygen species in the photoinhibition of photosystem II

Photoinhibition of photosystem II (PSII) occurs when the rate of photodamage to PSII exceeds the rate of the repair of photodamaged PSII. Recent examination of photoinhibition by separate determinations of photodamage and repair has revealed that the rate of photodamage to PSII is directly proportional to the intensity of incident light and that the repair of PSII is particularly sensitive to the inactivation by reactive oxygen species (ROS). The ROS-induced inactivation of repair is attributable to the suppression of the synthesis de novo of proteins, such as the D1 protein, that are required for the repair of PSII at the level of translational elongation. Furthermore, molecular analysis has revealed that the ROS-induced suppression of protein synthesis is associated with the specific inactivation of elongation factor G via the formation of an intramolecular disulfide bond. Impairment of various mechanisms that protect PSII against photoinhibition, including photorespiration, thermal dissipation of excitation energy, and the cyclic transport of electrons, decreases the rate of repair of PSII via the suppression of protein synthesis. In this review, we present a newly established model of the mechanism and the physiological significance of repair in the regulation of the photoinhibition of PSII.     

An intermediate membrane subfraction in cyanobacteria is involved in an assembly network for Photosystem II biogenesis

Early steps in the biogenesis of Photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803 are thought to occur in a specialized membrane fraction that is characterized by the specific accumulation of the PSII assembly factor PratA and its interaction partner pD1, the precursor of the D1 protein of PSII. Here, we report the molecular characterization of this membrane fraction, called the PratA-defined membrane (PDM), with regard to its lipid and pigment composition and its association with PSII assembly factors, including YCF48, Slr1471, Sll0933, and Pitt. We demonstrate that YCF48 and Slr1471 are present and that the chlorophyll precursor chlorophyllide a accumulates in the PDM. Analysis of PDMs from various mutant lines suggests a central role for PratA in the spatial organization of PSII biogenesis. Moreover, quantitative immunoblot analyses revealed a network of interdependences between several PSII assembly factors and chlorophyll synthesis. In addition, formation of complexes containing both YCF48 and Sll0933 was substantiated by co-immunoprecipitation experiments. The findings are integrated into a refined model for PSII biogenesis in Synechocystis 6803.     

Expression of a highly active catalase VktA in the cyanobacterium Synechococcus elongatus PCC 7942 alleviates the photoinhibition of photosystem II

The repair of photosystem II (PSII) after photodamage is particularly sensitive to reactive oxygen species—such as H₂O₂, which is abundantly produced during the photoinhibition of PSII. In the present study, we generated a transformant of the cyanobacterium Synechococcus elongatus PCC 7942 that expressed a highly active catalase, VktA, which is derived from a facultatively psychrophilic bacterium Vibrio rumoiensis, and examined the effect of expression of VktA on the photoinhibition of PSII. The activity of PSII in transformed cells declined much more slowly than in wild-type cells when cells were exposed to strong light in the presence of H₂O₂. However, the rate of photodamage to PSII, as monitored in the presence of chloramphenicol, was the same in the two lines of cells, suggesting that the repair of PSII was protected by the expression of VktA. The de novo synthesis of the D1 protein, which is required for the repair of PSII, was activated in transformed cells under the same stress conditions. Similar protection of the repair of PSII in transformed cells was also observed under strong light at a relatively low temperature. Thus, the expression of the highly active catalase mitigates photoinhibition of PSII by protecting protein synthesis against damage by H₂O₂ with subsequent enhancement of the repair of PSII.     

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.     

Dissection of photodamage at low temperature and repair in darkness suggests the existence of an intermediate form of photodamaged photosystem II

Irradiation of the photosynthetic machinery with strong light induces damage to the photosystem II complex (PSII), and this phenomenon is referred to as photodamage. In an attempt to characterize the mechanism of photodamage to PSII, we examined the events associated with photodamage by monitoring the phenomenon in Synechocystis sp. PCC 6803 at a low temperature. After the activity of PSII had been reduced to 10% of the original activity by exposure of Synechocystis cells to strong light at 10 degrees C, recovery was allowed to proceed at 34 degrees C in darkness. Under these conditions, approximately 50% of the activity of PSII was restored within 60 min. The recovery in darkness did not require protein synthesis, as demonstrated by Western blotting analysis and a radiolabeling experiment with [(35)S]methionine. We also observed a similar recovery of PSII in darkness in isolated thylakoid membranes. Our findings, together with those of other studies, suggest the presence of an intermediate form of photodamaged PSII that is generated prior to the formation of photodamaged PSII.