Damage to photosystem II by lipid peroxidation products.

BACKGROUND: Photosystem II proteins of higher plant chloroplasts are prone to oxidative stress, and most prominently the reaction center-binding D1 protein is damaged under abiotic stress. The reactive oxygen species produced under these stress conditions have been suggested to be responsible for the protein injury. SCOPE OF REVIEW: Recently, it has been shown that the primary and secondary products of non-enzymatic and enzymatic lipid peroxidation have a capability to modify photosystem II proteins. Here, we give an overview showing how lipid peroxidation products formed under light stress and heat stress in the thylakoid membranes cause oxidative modification of proteins in higher plant photosystem II. MAJOR CONCLUSIONS: Damage to photosystem II proteins by lipid peroxidation products represents a new mechanism underlying photoinhibition and heat inactivation. GENERAL SIGNIFICANCE: Complete characterization of photosystem II protein damage is of crucial importance because avoidance of the damage makes plants to survive under various abiotic stresses. Further physiological significance of photosystem II protein oxidation by lipid peroxidation product should have a potential relevance to plant acclimation because the oxidized proteins might serve as signaling molecules.

Quality Control of Photosystem II: The Mechanisms for Avoidance and Tolerance of Light and Heat Stresses are Closely Linked to Membrane Fluidity of the Thylakoids.

When oxygenic photosynthetic organisms are exposed to excessive light and/or heat, Photosystem II is damaged and electron transport is blocked. In these events, reactive oxygen species, endogenous radicals and lipid peroxidation products generated by photochemical reaction and/or heat cause the damage. Regarding light stress, plants first dissipate excessive light energy captured by light-harvesting chlorophyll protein complexes as heat to avoid the hazards, but once light stress is unavoidable, they tolerate the stress by concentrating damage in a particular protein in photosystem II, i.e., the reaction-center binding D1 protein of Photosystem II. The damaged D1 is removed by specific proteases and replaced with a new copy produced through de novo synthesis (reversible photoinhibition). When light intensity becomes extremely high, irreversible aggregation of D1 occurs and thereby D1 turnover is prevented. Once the aggregated products accumulate in Photosystem II complexes, removal of them by proteases is difficult, and irreversible inhibition of Photosystem II takes place (irreversible photoinhibition). Important is that various aspects of both the reversible and irreversible photoinhibition are highly dependent on the membrane fluidity of the thylakoids. Heat stress-induced inactivation of photosystem II is an irreversible process, which may be also affected by the fluidity of the thylakoid membranes. Here I describe why the membrane fluidity is a key to regulate the avoidance and tolerance of Photosystem II on environmental stresses.

Born in 1949 in postwar Japan.

In this article, I would like to look back at my life as a researcher of photosynthesis. I was born in 1949, and grew up and was educated in postwar Japan in the 1950s and 1960s. I have studied photosynthesis, in particular Photosystem II, after research experiences in the USA and UK. My study of Photosystem II has continued over 43 years until now. Through the present retrospection, I would like to suggest that all photosynthesis researchers, including the members of the "49ers", many other established scientists, and young students as well, should not simply stay in the lab working hard on their studies and writing papers; but should also do something for the public. People want to learn from us about many critical social issues such as the environment, food, energy and, most importantly, peace. I believe that our knowledge must form an important basis for people to take action to create a peaceful and harmonious human society.

Quality control of photosystem II: direct imaging of the changes in the thylakoid structure and distribution of FtsH proteases in spinach chloroplasts under light stress.

Under light stress, the reaction center-binding protein D1 of PSII is photo-oxidatively damaged and removed from PSII complexes by proteases located in the chloroplast. A protease considered to be responsible for degradation of the damaged D1 protein is the metalloprotease FtsH. We showed previously that the active hexameric FtsH protease is abundant at the grana margin and the grana end membranes, and this homo-complex removes the photodamaged D1 protein in the grana. Here, we showed a change in the distribution of FtsH in spinach thylakoids during excessive illumination by transmission electron microscopy (TEM) and immunogold labeling of FtsH. The change in distribution of the protease was accompanied by structural changes to the thylakoids, which we detected using spinach leaves by TEM after chemical fixation of the samples. Quantitative analyses showed several characteristic changes in the structure of the thylakoids, including shrinkage of the grana, outward bending of the marginal portions of the thylakoids and an increase in the height of the grana stacks under excessive illumination. The increase in the height of the grana stacks may include swelling of the thylakoids and an increase in the partition gaps between the thylakoids. These data strongly suggest that excessive illumination induces partial unstacking of the thylakoids, which enables FtsH to access easily the photodamaged D1 protein. Finally three-dimensional tomography of the grana was recorded to observe the effect of light stress on the overall structure of the thylakoids.

Quality control of Photosystem II: the molecular basis for the action of FtsH protease and the dynamics of the thylakoid membranes.

The reaction center-binding D1 protein of Photosystem II is damaged by excessive light, which leads to photoinhibition of Photosystem II. The damaged D1 protein is removed immediately by specific proteases, and a metalloprotease FtsH located in the thylakoid membranes is involved in the proteolytic process. According to recent studies on the distribution and organization of the protein complexes/supercomplexes in the thylakoid membranes, the grana of higher plant chloroplasts are crowded with Photosystem II complexes and light-harvesting complexes. For the repair of the photodamaged D1 protein, the majority of the active hexameric FtsH proteases should be localized in close proximity to the Photosystem II complexes. The unstacking of the grana may increase the area of the grana margin and facilitate easier access of the FtsH proteases to the damaged D1 protein. These results suggest that the structural changes of the thylakoid membranes by light stress increase the mobility of the membrane proteins and support the quality control of Photosystem II.

Quality control of PSII: behavior of PSII in the highly crowded grana thylakoids under excessive light.

The grana thylakoids of higher plant chloroplasts are crowded with PSII and the associated light-harvesting complexes (LHCIIs). They constitute supercomplexes, and often form semi-crystalline arrays in the grana. The crowded condition of the grana may be necessary for efficient trapping of excitation energy by LHCII under weak light, but it might hinder proper movement of LHCII necessary for reversible aggregation of LHCII in the energy-dependent quenching of Chl fluorescence under moderate high light. When the thylakoids are illuminated with extreme high light, the reaction center-binding D1 protein of PSII is photodamaged, and the damaged protein migrates to the grana margins for degradation and subsequent repair. In both moderate and extreme high-light conditions, fluidity of the thylakoid membrane is crucial. In this review, we first provide an overview of photoprotective processes, then discuss changes in membrane fluidity and mobility of the protein complexes in the grana under excessive light, which are closely associated with photoprotection of PSII. We hypothesize that reversible aggregation of LHCII, which is necessary to avoid light stress under moderate high light, and swift turnover of the photodamaged D1 protein under extreme high light are threatened by irreversible protein aggregation induced by reactive oxygen species in photochemical reactions.

Quality control of Photosystem II: reversible and irreversible protein aggregation decides the fate of Photosystem II under excessive illumination.

In response to excessive light, the thylakoid membranes of higher plant chloroplasts show dynamic changes including the degradation and reassembly of proteins, a change in the distribution of proteins, and large-scale structural changes such as unstacking of the grana. Here, we examined the aggregation of light-harvesting chlorophyll-protein complexes and Photosystem II core subunits of spinach thylakoid membranes under light stress with 77K chlorophyll fluorescence; aggregation of these proteins was found to proceed with increasing light intensity. Measurement of changes in the fluidity of thylakoid membranes with fluorescence polarization of diphenylhexatriene showed that membrane fluidity increased at a light intensity of 500-1,000 µmol photons m(-) (2) s(-) (1), and decreased at very high light intensity (1,500 µmol photons m(-) (2) s(-) (1)). The aggregation of light-harvesting complexes at moderately high light intensity is known to be reversible, while that of Photosystem II core subunits at extremely high light intensity is irreversible. It is likely that the reversibility of protein aggregation is closely related to membrane fluidity: increases in fluidity should stimulate reversible protein aggregation, whereas irreversible protein aggregation might decrease membrane fluidity. When spinach leaves were pre-illuminated with moderately high light intensity, the qE component of non-photochemical quenching and the optimum quantum yield of Photosystem II increased, indicating that Photosystem II/light-harvesting complexes rearranged in the thylakoid membranes to optimize Photosystem II activity. Transmission electron microscopy revealed that the thylakoids underwent partial unstacking under these light stress conditions. Thus, protein aggregation is involved in thylakoid dynamics and regulates photochemical reactions, thereby deciding the fate of Photosystem II.

Quality control of photosystem II: lipid peroxidation accelerates photoinhibition under excessive illumination.

Environmental stresses lower the efficiency of photosynthesis and sometimes cause irreversible damage to plant functions. When spinach thylakoids and Photosystem II membranes were illuminated with excessive visible light (100-1,000 µmol photons m(-1) s(-1)) for 10 min at either 20°C or 30°C, the optimum quantum yield of Photosystem II decreased as the light intensity and temperature increased. Reactive oxygen species and endogenous cationic radicals produced through a photochemical reaction at and/or near the reaction center have been implicated in the damage to the D1 protein. Here we present evidence that lipid peroxidation induced by the illumination is involved in the damage to the D1 protein and the subunits of the light-harvesting complex of Photosystem II. This is reasoned from the results that considerable lipid peroxidation occurred in the thylakoids in the light, and that lipoxygenase externally added in the dark induced inhibition of Photosystem II activity in the thylakoids, production of singlet oxygen, which was monitored by electron paramagnetic resonance spin trapping, and damage to the D1 protein, in parallel with lipid peroxidation. Modification of the subunits of the light-harvesting complex of Photosystem II by malondialdehyde as well as oxidation of the subunits was also observed. We suggest that mainly singlet oxygen formed through lipid peroxidation under light stress participates in damaging the Photosystem II subunits.

Quality control of Photosystem II: where and how does the degradation of the D1 protein by FtsH proteases start under light stress?--Facts and hypotheses.

Degradation of the reaction center-binding D1 protein of Photosystem II is central in photoinhibition of Photosystem II. In higher plant chloroplasts, Photosystem II complexes are abundant in the grana. It has been suggested that the Photosystem II complexes containing photodamaged D1 protein migrate for their repair from the grana to the non-appressed stroma thylakoids, where the photodamaged D1 protein is degraded by a specific protease(s) such as filamentation temperature sensitive H (FtsH) protease. There are several possible ways to activate the FtsH proteases. As FtsH is a membrane-bound ATP-dependent metalloprotease, it requires ATP and zinc as essential part of its catalytic mechanism. It is also suggested that a membrane protein(s) associated with FtsH is required for modulation of the FtsH activity. Here, we propose several possible mechanisms for activation of the proteases, which depend on oligomerization of the monomer subunits. In relation to the oligomerization of FtsH subunits, we also suggest unique distribution of active FtsH hexamers on the thylakoids: hexamers of the FtsH proteases are localized near the Photosystem II complexes at the grana. Degradation of the D1 protein probably takes place in the grana rather than in the stroma thylakoids to circumvent long-distance migration of both the Photosystem II complexes containing the photodamaged D1 protein and the proteases.

Isolation of photosystem II-enriched membranes and the oxygen-evolving complex subunit proteins from higher plants.

We describe methods to isolate highly active oxygen-evolving photosystem II (PSII) membranes and core complexes from higher plants, and to purify subunits of the oxygen-evolving complex (OEC). The membrane samples used as the material for various in vitro studies of PSII are prepared by solubilizing thylakoid membranes with the nonionic detergent Triton X-100, and the core complexes are prepared by further solubilization of the PSII membranes with n-dodecyl-ß-D-maltoside (ß-DDM). The OEC subunit proteins are dissociated from the PSII-enriched membranes by alkaline or salt treatment, and are then purified by ion-exchange chromatography using an automated high performance liquid chromatography system.

Assay of photoinhibition and heat inhibition of photosystem II in higher plants.

When thylakoids of higher plant chloroplasts are exposed to excessive light or moderate heat stress, photosystem II reaction center-binding protein D1 is damaged. The photodamage of the D1 protein is caused by reactive oxygen species, mostly singlet oxygen, and also by endogenous cationic radicals generated by the photochemical reactions of photosystem II. Moreover, it was shown recently that the damage to the D1 protein by moderate heat stress is due to reactive oxygen species produced by lipid peroxidation near photosystem II. To maintain photosystem II activity, the oxidatively damaged D1 protein must be replaced by a newly synthesized copy, and thus degradation and removal of the photo- or heat-damaged D1 protein are essential for maintaining the viability of photosystem II. In this chapter, we describe the methods for assaying photoinhibition and heat inhibition of photosystem II in higher plant materials.

Quality control of photosystem II: FtsH hexamers are localized near photosystem II at grana for the swift repair of damage.

The reaction center-binding D1 protein of Photosystem II is oxidatively damaged by excessive visible light or moderate heat stress. The metalloprotease FtsH has been suggested as responsible for the degradation of the D1 protein. We have analyzed the distribution and subunit structures of FtsH in spinach thylakoids and various membrane fractions derived from the thylakoids using clear native polyacrylamide gel electrophoresis and Western blot analysis. FtsH was found not only in the stroma thylakoids but also in the Photosystem II-enriched grana membranes. Monomeric, dimeric, and hexameric FtsH proteases were present as major subunit structures in thylakoids, whereas only hexameric FtsH proteases were detected in Triton X-100-solubilized Photosystem II membranes. Importantly, among the membrane fractions examined, hexameric FtsH proteases were most abundant in the Photosystem II membranes. In accordance with this finding, D1 degradation took place in the Photosystem II membranes under light stress. Sucrose density gradient centrifugation analysis of thylakoids and the Photosystem II membranes solubilized with n-dodecyl-ß-d-maltoside and a chemical cross-linking study of thylakoids showed localization of FtsH near the Photosystem II light-harvesting chlorophyll-protein supercomplexes in the grana. These results suggest that part of the FtsH hexamers are juxtapositioned to PSII complexes in the grana in darkness, carrying out immediate degradation of the photodamaged D1 protein under light stress.

Study on the effects of chloride depletion on photosystem II using different chloride depletion methods.

Chloride is an indispensable factor for the functioning of oxygen evolving complex (OEC) and has protective and activating effects on photosystem II. In this study we have investigated mainly by EPR, the properties of chloride-sufficient, chloride-deficient and chloride-depleted thylakoid membranes and photosystem II enriched membranes from spinach. The results on the effects of different chloride depletion methods on the structural and functional aspects of photosystem II showed that chloride-depletion by treating PS II membranes with high pH is a relatively harsh way causing a significant and irreparable damage to the PS II donor side. Damage to the acceptor side of PS II was recovered almost fully in chloride-deficient as well as chloride-depleted PS II membranes.

Quality control of photosystem II: Thylakoid unstacking is necessary to avoid further damage to the D1 protein and to facilitate D1 degradation under light stress in spinach thylakoids.

Photosystem II is vulnerable to light damage. The reaction center-binding D1 protein is impaired during excessive illumination and is degraded and removed from photosystem II. Using isolated spinach thylakoids, we investigated the relationship between light-induced unstacking of thylakoids and damage to the D1 protein. Under light stress, thylakoids were expected to become unstacked so that the photodamaged photosystem II complexes in the grana and the proteases could move on the thylakoids for repair. Excessive light induced irreversible unstacking of thylakoids. By comparing the effects of light stress on stacked and unstacked thylakoids, photoinhibition of photosystem II was found to be more prominent in stacked thylakoids than in unstacked thylakoids. In accordance with this finding, EPR spin trapping measurements demonstrated higher production of hydroxyl radicals in stacked thylakoids than in unstacked thylakoids. We propose that unstacking of thylakoids has a crucial role in avoiding further damage to the D1 protein and facilitating degradation of the photodamaged D1 protein under light stress.

Quality control of photosystem II: impact of light and heat stresses.

Photosystem II is vulnerable to various abiotic stresses such as strong visible light and heat. Under both stresses, the damage seems to be triggered by reactive oxygen species, and the most critical damage occurs in the reaction center-binding D1 protein. Recent progress has been made in identifying the protease involved in the degradation of the photo- or heat-damaged D1 protein, the ATP-dependent metalloprotease FtsH. Another important result has been the discovery that the damaged D1 protein aggregates with nearby polypeptides such as the D2 protein and the antenna chlorophyll-binding protein CP43. The degradation and aggregation of the D1 protein occur simultaneously, but the relationship between the two is not known. We suggest that phosphorylation and dephosphorylation of the D1 protein, as well as the binding of the extrinsic PsbO protein to Photosystem II, play regulatory roles in directing the damaged D1 protein to the two alternative pathways.

In vivo quality control of photosystem II in cyanobacteria Synechocystis sp. PCC 6803: D1 protein degradation and repair under the influence of light, heat and darkness.

The cells of Synechocystis sp. PCC 6803 were subjected under photoinhibitory irradiation (600 micromolm(-2)s(-1)) at various temperatures (20-40 degrees C) to study in vivo quality control of photosystem II (PSII). The protease biogenesis and its consequences on photosynthetic efficiency (chlorophyll fluorescence ratio Fv/Fm) of the PSII, D1 degradation and repair were monitored during illumination and darkness. The loss in Fv/Fm value and degradation of D1 protein occurred not only under high light exposure, but also continued when the cells were subjected under dark restoration process after high light exposure. No loss in Fv/Fm value or D1 degradation occurred during recovery under growth/low light (30 micromol m(-2) s(-1)). Further, it helped the resynthesis of new D1 protein, essential to sustain quality control of PSII. In vivo triggering of D1 protein required high light exposure to switch-on the protease biosynthesis to maintain protease pool which induced temperature-dependent enzymatic proteolysis of photodamaged D1 protein during photoinhition and dark incubation. Our findings suggested the involvement and overexpression of a membrane-bound FtsH protease during high light exposure which caused degradation of D1 protein, strictly regulated by high temperature (30-40 degrees C). However, lower temperature (20 degrees C) prevented further loss of photoinhibited PSII efficiency in vivo and also retarded temperature-dependent proteolytic process of D1 degradation.

Quality control of photosystem II: reactive oxygen species are responsible for the damage to photosystem II under moderate heat stress.

Moderate heat stress (40 degrees C for 30 min) on spinach thylakoid membranes induced cleavage of the reaction center-binding D1 protein of photosystem II, aggregation of the D1 protein with the neighboring polypeptides D2 and CP43, and release of three extrinsic proteins, PsbO, -P, and -Q. These heat-induced events were suppressed under anaerobic conditions or by the addition of sodium ascorbate, a general scavenger of reactive oxygen species. In accordance with this, singlet oxygen and hydroxyl radicals were detected in spinach photosystem II membranes incubated at 40 degrees C for 30 min with electron paramagnetic resonance spin-trapping spectroscopy. The moderate heat stress also induced significant lipid peroxidation under aerobic conditions. We suggest that the reactive oxygen species are generated by heat-induced inactivation of a water-oxidizing manganese complex and through lipid peroxidation. Although occurring in the dark, the damages caused by the moderate heat stress to photosystem II are quite similar to those induced by excessive illumination where reactive oxygen species are involved.

Quality control of Photosystem II: cleavage and aggregation of heat-damaged D1 protein in spinach thylakoids.

Moderate heat stress (40 degrees C, 30 min) on spinach thylakoids induced cleavage of the D1 protein, producing an N-terminal 23-kDa fragment, a C-terminal 9-kDa fragment, and aggregation of the D1 protein. A homologue of Arabidopsis FtsH2 protease, which is responsible for degradation of the damaged D1 protein, was abundant in the stroma thylakoids. Two processes occurred in the thylakoids in response to heat stress: dephosphorylation of the D1 protein in the stroma thylakoids, and aggregation of the phosphorylated D1 protein in the grana. Heat stress also induced the release of the extrinsic PsbO, P and Q proteins from Photosystem II, which affected D1 degradation and aggregation significantly. The cleavage and aggregation of the D1 protein appear to be two alternative processes influenced by protein phosphorylation/dephosphorylation, distribution of FtsH, and intactness of the thylakoids.

Quality control of photosystem II. Cleavage of reaction center D1 protein in spinach thylakoids by FtsH protease under moderate heat stress.

When spinach thylakoids were subjected to moderate heat stress (40 degrees C for 30 min), oxygen evolution was inhibited, and cleavage of the reaction center-binding protein D1 of photosystem II took place, producing 23-kDa N-terminal fragments. The D1 cleavage was greatly facilitated by the addition of 0.15 mM ZnCl2 and 1 mM ATP and was completely inhibited by 1 mM EDTA, indicating the participation of an ATP-dependent metalloprotease(s) in the D1 cleavage. Herbicides 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, bromoxynil, and ioxynil, all of which bind to the Q(B) site, inhibited the D1 cleavage, suggesting that the DE-loop of the D1 protein is the heat-sensitive cleavage site. We solubilized the protease by treating the thylakoids with 2 M KSCN and detected a protease activity in the supernatant by gelatin activity gel electrophoresis in the 70-80-kDa region. The antibodies against tobacco FtsH and Arabidopsis FtsH2 reacted with a 70-80-kDa band of the KSCN-solubilized fraction, which suggests the presence of FtsH in the fraction. In accordance with this finding, we identified the homolog to Arabidopsis FtsH8 in the 70-80-kDa region by matrix-assisted laser desorption ionization time-of-flight mass analysis of the thylakoids. The KSCN-solubilized fraction was successively reconstituted with thylakoids to show heat-induced cleavage of the D1 protein and production of the D1 fragment. These results strongly suggest that an FtsH protease(s) is involved in the primary cleavage of the D1 protein under moderate heat stress.