Interruption of the Calvin cycle inhibits the repair of Photosystem II from photodamage

In photosynthetic organisms, impairment of the activities of enzymes in the Calvin cycle enhances the extent of photoinactivation of Photosystem II (PSII). We investigated the molecular mechanism responsible for this phenomenon in the unicellular green alga Chlamydomonas reinhardtii. When the Calvin cycle was interrupted by glycolaldehyde, which is known to inhibit phosphoribulokinase, the extent of photoinactivation of PSII was enhanced. The effect of glycolaldehyde was very similar to that of chloramphenicol, which inhibits protein synthesis de novo in chloroplasts. The interruption of the Calvin cycle by the introduction of a missense mutation into the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) also enhanced the extent of photoinactivation of PSII. In such mutant 10-6C cells, neither glycolaldehyde nor chloramphenicol has any additional effect on photoinactivation. When wild-type cells were incubated under weak light after photodamage to PSII, the activity of PSII recovered gradually and reached a level close to the initial level. However, recovery was inhibited in wild-type cells by glycolaldehyde and was also inhibited in 10-6C cells. Radioactive labelling and Northern blotting demonstrated that the interruption of the Calvin cycle suppressed the synthesis de novo of chloroplast proteins, such as the D1 and D2 proteins, but did not affect the levels of psbA and psbD mRNAs. Our results suggest that the photoinactivation of PSII that is associated with the interruption of the Calvin cycle is attributable primarily to the inhibition of the protein synthesis-dependent repair of PSII at the level of translation in chloroplasts.     

Glycerate-3-phosphate, produced by CO2 fixation in the Calvin cycle, is critical for the synthesis of the D1 protein of photosystem II

We demonstrated recently that, in intact cells of Chlamydomonas reinhardtii, interruption of CO2 fixation via the Calvin cycle inhibits the synthesis of proteins in photosystem II (PSII), in particular, synthesis of the D1 protein, during the repair of PSII after photodamage. In the present study, we investigated the mechanism responsible for this phenomenon using intact chloroplasts isolated from spinach leaves. When CO2 fixation was inhibited by exogenous glycolaldehyde, which inhibits the phosphoribulokinase that synthesizes ribulose-1,5-bisphosphate, the synthesis de novo of the D1 protein was inhibited. However, when glycerate-3-phosphate (3-PGA), which is a product of CO2 fixation in the Calvin cycle, was supplied exogenously, the inhibitory effect of glycolaldehyde was abolished. A reduced supply of CO2 also suppressed the synthesis of the D1 protein, and this inhibitory effect was also abolished by exogenous 3-PGA. These findings suggest that the supply of 3-PGA, generated by CO2 fixation, is important for the synthesis of the D1 Protein. It is likely that 3-PGA accepts electrons from NADPH and decreases the level of reactive oxygen species, which inhibit the synthesis of proteins, such as the D1 protein.     

Glycerate-3-phosphate, produced by CO2 fixation in the Calvin cycle, is critical for the synthesis of the D1 protein of photosystem II

We demonstrated recently that, in intact cells of Chlamydomonas reinhardtii, interruption of CO₂ fixation via the Calvin cycle inhibits the synthesis of proteins in photosystem II (PSII), in particular, synthesis of the D1 protein, during the repair of PSII after photodamage. In the present study, we investigated the mechanism responsible for this phenomenon using intact chloroplasts isolated from spinach leaves. When CO₂ fixation was inhibited by exogenous glycolaldehyde, which inhibits the phosphoribulokinase that synthesizes ribulose-1,5-bisphosphate, the synthesis de novo of the D1 protein was inhibited. However, when glycerate-3-phosphate (3-PGA), which is a product of CO₂ fixation in the Calvin cycle, was supplied exogenously, the inhibitory effect of glycolaldehyde was abolished. A reduced supply of CO₂ also suppressed the synthesis of the D1 protein, and this inhibitory effect was also abolished by exogenous 3-PGA. These findings suggest that the supply of 3-PGA, generated by CO₂ fixation, is important for the synthesis of the D1 Protein. It is likely that 3-PGA accepts electrons from NADPH and decreases the level of reactive oxygen species, which inhibit the synthesis of proteins, such as the D1 protein.     

Inhibition of Calvin–Benson cycle suppresses the repair of photosystem II in Symbiodinium: implications for coral bleaching

Inhibition of Calvin–Benson cycle (CBC) activity by thermal stress has been hypothesized to cause photoinhibition of photosystem II (PSII) in zooxanthellae of reef-building corals and consequently lead to bleaching. This study tests whether the interruption of CBC by glycolaldehyde (GA) leads to photoinhibition and subsequent coral bleaching in Stylophora pistillata. When S. pistillata was incubated with GA, the O₂ evolution rate declined in a dose-dependent manner and the extent of photoinhibition, reflected by a decreased maximum quantum yield of PSII (F _v/F _m), was enhanced. The effect of GA on photoinhibition was similar to that of chloramphenicol (CAP), an inhibitor of protein synthesis in chloroplasts. When S. pistillata was incubated in weak light following a high-light-induced photoinhibitory treatment, the recovery of PSII from photoinhibition was suppressed in a similar manner to both GA- and CAP-treated samples. After incubation in moderate light at 26°C, S. pistillata showed a bleaching response only in presence of GA. These results suggest that coral bleaching-like responses are caused by interruption of the CBC activity in S. pistillata and are associated with accelerated photoinhibition through suppression of the protein synthesis-dependent repair of PSII but not to an increase in photodamage to PSII.     

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.     

Environmental stress inhibits the synthesis de novo of proteins involved in the photodamage-repair cycle of Photosystem II in Synechocystis sp. PCC 6803

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 μE 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 μE 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.     

PsbT polypeptide is required for efficient repair of photodamaged photosystem II reaction center.

PsbT is a small chloroplast-encoded hydrophobic polypeptide associated with the photosystem II (PSII) core complex. A psbT-deficient mutant (Delta psbT) of the green alga Chlamydomonas reinhardtii grows photoautotrophically, whereas its growth is significantly impaired in strong light. To understand the photosensitivity of Delta psbT, we have studied the effect of strong illumination on PSII activity and proteins. It is shown that the level of PSII activity and proteins is reduced in the Delta psbT more significantly than in wild type under strong light. When recovery of the photodamaged PSII is inhibited by a chloroplast protein synthesis inhibitor, the light-induced inactivation and degradation of PSII occur similarly in wild-type and mutant cells. On the contrary, the recovery of PSII activity after partial photoinactivation is remarkably delayed in the Delta psbT cells, suggesting that PsbT is required for efficient recovery of the photodamaged PSII complex. These results therefore present the first evidence for involvement of this small PSII polypeptide in the recovery process. Partial disintegration of the purified PSII core complex and localization of PSII proteins in the resulting PSII subcore complexes have revealed that PsbT is associated with D1/D2 heterodimer. A possible role of PsbT in the recovery process is discussed.     

Increased reliance upon photosystem II repair following acclimation to high-light by coral-dinoflagellate symbioses

Changing light environments force photoautotroph cells, including coral symbionts, to acclimate to maintain photosynthesis. Photosystem II (PSII) is subjected to photoinactivation at a rate proportional to the incident light, and cells must adjust their rates of protein repair to counter this photoinactivation. We examined PSII function in the coral symbiont Symbiodinium to determine the effect of photoacclimation on their capacity for PSII repair. Colonies of the coral Stylophora pistillata were collected from moderate light environments on the Lizard Island reef (Queensland, Australia) and transported to a local field station, where they were assigned to lower or higher light regimes and allowed to acclimate for 2 weeks. Following this photoacclimation period, the low-light acclimated corals showed greater symbiont density, higher chlorophyll per symbiont cell, and higher photosystem II protein than high-light acclimated corals did. Subsequently, we treated the corals with lincomycin, an inhibitor of chloroplastic protein synthesis, and exposed them to a high-light treatment to separate the effect of de novo protein synthesis in PSII repair from intrinsic susceptibility to photoinactivation. Low-light acclimated corals showed a sharp initial drop in PSII function but inhibition of PSII repair provoked only a modest additional drop in PSII function, compared to uninhibited corals. In high-light acclimated corals inhibition of PSII repair provoked a larger drop in PSII function, compared to uninhibited high-light corals. The greater lincomycin effects in the corals pre-acclimated to high-light show that high-light leads to an increased reliance on the PSII repair cycle.     

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.     

Cold transiently activates calcium-permeable channels in Arabidopsis mesophyll cells

Oxygenation of ribulose-1,5-bisphosphate catalyzed by Rubisco produces glycolate-2-P. The photorespiratory pathway, which consists of photorespiratory carbon and nitrogen cycles, metabolizes glycolate-2-P to the Calvin cycle intermediate glycerate-3-P and is proposed to be important for avoiding photoinhibition of photosystem II (PSII), especially in C3 plants. We show here that mutants of Arabidopsis (Arabidopsis thaliana) with impairment of ferredoxin-dependent glutamate synthase, serine hydroxymethyltransferase, glutamate/malate transporter, and glycerate kinase had accelerated photoinhibition of PSII by suppression of the repair of photodamaged PSII and not acceleration of the photodamage to PSII. We found that suppression of the repair process was attributable to inhibition of the synthesis of the D1 protein at the level of translation. Our results suggest that the photorespiratory pathway helps avoid inhibition of the synthesis of the D1 protein, which is important for the repair of photodamaged PSII upon interruption of the Calvin cycle.     

Assembly of the D1 precursor in monomeric photosystem II reaction center precomplexes precedes chlorophyll a-triggered accumulation of reaction center II in barley etioplasts

Assembly of plastid-encoded chlorophyll binding proteins of photosystem II (PSII) was studied in etiolated barley seedlings and isolated etioplasts and either the absence or presence of de novo chlorophyll synthesis. De novo assembly of reaction center complexes in etioplasts was characterized by immunological analysis of protein complexes solubilized from inner etioplast membranes and separated in sucrose density gradients. Previously characterized membrane protein complexes from chloroplasts were utilized as molecular mass standards for sucrose density gradient separation analysis. In etiolated seedlings, induction of chlorophyll a synthesis resulted in the accumulation of D1 in a dimeric PSII reaction center (RCII) complex. In isolated etioplasts, de novo chlorophyll a synthesis directed accumulation of D1 precursor in a monomeric RCII precomplex that also included D2 and cytochrome b(559). Chlorophyll a synthesis that was chemically prolonged in darkness neither increased the yield of RCII monomers nor directed assembly of RCII dimers in etioplasts. We therefore conclude that in etioplasts, assembly of the D1 precursor in monomeric RCII precomplexes precedes chlorophyll a-triggered accumulation of reaction center monomers.     

Genetic Enhancement of the Ability to Tolerate Photoinhibition by Introduction of Unsaturated Bonds into Membrane Glycerolipids

Strong light leads to damage to photosynthetic machinery, particularly at low temperatures, and the main site of the damage is the D1 protein of the photosystem II (PSII) complex. Here we describe that transformation of Synechococcus sp. PCC 7942 with the desA gene for a [delta]12 desaturase increased unsaturation of membrane lipids and enhanced tolerance to strong light. To our knowledge, this is the first report of the successful genetic enhancement of tolerance to strong light. Analysis of the light-induced inactivation and of the subsequent recovery of the activity of the PSII complex revealed that the recovery process was markedly accelerated by the genetic transformation. Labeling experiments with [35S]L-methionine also revealed that the synthesis of the D1 protein de novo at low temperature, which was a prerequisite for the restoration of the PSII complex, was much faster in the transformed cells than in the wild-type cells. These findings demonstrate that the ability of membrane lipids to desaturate fatty acids is important for the photosynthetic organisms to tolerate strong light, by accelerating the synthesis of the D1 protein de novo.     

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.     

Resistance to photoinhibition of photosystem II and catalase and antioxidative protection in high mountain plants

In leaves of three alpine high mountain plants, Homogyne alpina, Ranunculus glacialis and Soldanella alpina, both photosystem II (PSII) and the enzyme catalase appeared to he highly resistant to photoinactivation under natural field conditions. While the Dl protein of PSII and catalase have a rapid turnover in light and require continuous new protein synthesis in non-adapted plants, little apparent photoinactivation of PSII or catalase was induced in the alpine plants by translation inhibitors or at low temperature, suggesting that turnover of the Dl protein and catalase was slow in these leaves. In vitro PSII was rapidly inactivated in light in isolated thylakoids from H. alpina and R. glacialis. In isolated intact chloroplasts from R. glacialis, photoinactivation of PSII was slower than in thylakoids. Partially purified catalase from R. glacialis and S. alpina was as sensitive to photoinactivation in vitro as catalases from other sources. Catalase from H. alpina had, however, a 10-fold higher stability in light. The levels of xanthophyll cycle carotenoids, of the antioxidants ascorbate and glulathione, and of the activities of catalase, superoxide dismutase and glutathione reductase were very high in S. alpina, intermediate in H. alpina, but very low in R. glacialis. However, isolated chloroplasts from all three alpine species contained much higher concentrations of ascorbate and glutathione than chloroplasts from lowland plants.     

Effects of the lack of phosphatidylglycerol on the donor side of photosystem II

Our previous studies with the pgsA mutant of the cyanobacterium Synechocystis sp. PCC6803 (hereafter termed pgsA mutant), which is defective for the biosynthesis of phosphatidylglycerol (PG), revealed an important role for PG in the electron acceptor side of photosystem II (PSII), especially in the electron transport between plastoquinones Q(A) and Q(B). This study now shows that PG also plays an important role in the electron donor side of PSII, namely, the oxygen-evolving system. Analyses of purified PSII complexes indicated that PSII from PG-depleted pgsA mutant cells sustained only approximately 50% of the oxygen-evolving activity compared to wild-type cells. Dissociation of the extrinsic proteins PsbO, PsbV, and PsbU, which are required for stabilization of the manganese (Mn) cluster, followed by the release of a Mn atom, was observed in PSII of the PG-depleted mutant cells. The released PsbO rebound to PSII when PG was added back to the PG-depleted mutant cells, even when de novo protein synthesis was inhibited. Changes in photosynthetic activity of the PG-depleted pgsA mutant cells induced by heat treatment or dark incubation resembled those of DeltapsbO, DeltapsbV, and DeltapsbU mutant cells. These results suggest that PG plays an important role in binding extrinsic proteins required for sustaining a functional Mn cluster on the donor side of PSII.     

Photoprotection of residual functional photosystem II units that survive illumination in the absence of repair, and their critical role in subsequent recovery

Photosystem II (PSII) complexes, which split water into oxygen, protons and electrons in photosynthesis, require light but are also inactivated by it. Recovery of PSII from photoinactivation requires de novo protein synthesis. PSII in capsicum leaf segments were photoinactivated in the absence of chloroplast-encoded protein synthesis. At large photon exposures and despite the absence of repair, a residual fraction of PSII remained functional, being ca 0.08–0.2 depending on the ease of gas exchange in the tissue. This study revealed that the residual functional PSII was photoprotected by both (1) reaction-center quenching of excitation energy by photoinactivated PSII even when little or no PSII activity was permitted, and (2) antenna quenching, which was dependent on a trans-thylakoid pH gradient sustained mainly by linear electron transport and facilitated by the residual functional PSII complexes themselves. Significantly, little or no contribution to photoprotection of PSII was observed from cyclic electron flow around PSI. Further, the small residual functional PSII population was critical for recovery of the photoinactivated PSII complexes. Thus, photoinactivated and residual functional PSII complexes in leaves play a mutually beneficial role in each other's ultimate survival.