Tocopherol as singlet oxygen scavenger in photosystem II

Singlet oxygen is formed in the photosystem II reaction center in the quench of P680 triplets, and the yield is dependent on light intensity and the reduction level of plastoquinone. Singlet oxygen in PS II triggers the degradation of the D1 protein. We investigated the participation of tocopherol as a singlet oxygen scavenger in this system. For this purpose, we inhibited tocopherol biosynthesis at the level of the HPP-dioxygenase in the alga Chlamydomonas reinhardtii under conditions in which plastoquinone did not limit the photosynthesis rate. In the presence of the inhibitor and in high light for 2 h, photosynthesis in vivo and photosystem II was inactivated, the D1 protein was degraded, and the tocopherol pool was depleted and fell below its turnover rate/h. The inhibited system could be fully resuscitated upon the addition of a chemical singlet oxygen quencher (diphenylamine), and partly by synthetic cell wall permeable short chain alpha- and gamma-tocopherol derivatives. We conclude that under conditions of photoinhibition and extensive D1 protein turnover tocopherol has a protective function as a singlet oxygen scavenger.     

Singlet oxygen scavenging activity of tocopherol and plastochromanol in Arabidopsis thaliana: relevance to photooxidative stress

In the present study, singlet oxygen (¹O₂) scavenging activity of tocopherol and plastochromanol was examined in tocopherol cyclase‐deficient mutant (vte1) of Arabidopsis thaliana lacking both tocopherol and plastochromanol. It is demonstrated here that suppression of tocopherol and plastochromanol synthesis in chloroplasts isolated from vte1 Arabidopsis plants enhanced ¹O₂ formation under high light illumination as monitored by electron paramagnetic resonance spin‐trapping spectroscopy. The exposure of vte1 Arabidopsis plants to high light resulted in the formation of secondary lipid peroxidation product malondialdehyde as determined by high‐pressure liquid chromatography. Furthermore, it is shown here that the imaging of ultra‐weak photon emission known to reflect oxidation of lipids was unambiguously higher in vte1 Arabidopsis plants. Our results indicate that tocopherol and plastochromanol act as efficient ¹O₂ scavengers and protect effectively lipids against photooxidative damage in Arabidopsis plants.     

New prenyllipid metabolites identified in Arabidopsis during photo‐oxidative stress

In the present study, we have identified new prenyllipid metabolites formed during high light stress in Arabidopsis thaliana, whose origin and function remained unknown so far. It was found that plastoquinone‐C accumulates mainly in the reduced form under high light conditions, as well as during short‐term excess light illumination both in the wild‐type and tocopherol biosynthetic vte1 mutant, suggesting that plastoquinone‐C, a singlet oxygen‐derived prenyllipid, is reduced in chloroplasts by photosystem II or enzymatically, outside thylakoids. Plastoquinone‐B, a fatty acid ester of plastoquinone‐C, was identified for the first time in Arabidopsis in high light grown wild‐type plants and during short‐time, excess light illumination of the wild‐type plants and the vte1 mutant. The gene expression analysis showed that vte2 gene is most pronouncedly up‐regulated among the prenyllipid biosynthetic genes under high light and induction of its expression is mainly caused by an increased level of singlet oxygen, as was demonstrated in experiments with D₂O‐treated plants under excess light conditions.     

Tocopherol biosynthesis: chemistry, regulation and effects of environmental factors

Tocopherols are lipophilic antioxidants and together with tocotrienols belong to the vitamin-E family. The four forms of tocopherols (??-, ??-, ??- and ??-tocopherols) consist of a polar chromanol ring and lipophilic prenyl chain with differences in the position and number of methyl groups. The biosynthesis of tocopherols takes place mainly in plastids of higher plants from precursors derived from two metabolic pathways: homogentisic acid, an intermediate of degradation of aromatic amino acids, and phytyldiphosphate, which arises from methylerythritol phosphate pathway. The regulation of tocopherol biosynthesis in photosynthetic organisms occurs, at least partially, at the level of key enzymes as such including p-hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27), homogentisate phytyltransferase (HPT, EC 2.5.1.-), tocopherol cyclase (TC, EC 5.4.99.-), and two methyltransferases. Tocopherol biosynthesis changes during plant development and in response toward different stresses induced by high-intensity light, drought, high salinity, heavy metals, and chilling. It is supposed that scavenging of lipid peroxy radicals and quenching of singlet oxygen are the main functions of tocopherols in photosynthetic organisms. The antioxidant action of tocopherols is related to the formation of tocopherol quinone and its following recycling or degradation. However, until now, the mechanisms of tocopherol degradation in plants have not been established in detail. This review focuses on mechanisms of tocopherols biosynthesis and its regulation in photosynthetic organisms. In addition, available information on tocopherol degradation is summarized.     

Singlet oxygen production in photosystem II and related protection mechanism

High-light illumination of photosynthetic organisms stimulates the production of singlet oxygen by photosystem II (PSII) and causes photo-oxidative stress. In the PSII reaction centre, singlet oxygen is generated by the interaction of molecular oxygen with the excited triplet state of chlorophyll (Chl). The triplet Chl is formed via charge recombination of the light-induced charge pair. Changes in the midpoint potential of the primary electron donor P(680) of the primary acceptor pheophytin or of the quinone acceptor Q(A), modulate the pathway of charge recombination in PSII and influence the yield of singlet oxygen formation. The involvement of singlet oxygen in the process of photoinhibition is discussed. Singlet oxygen is efficiently quenched by beta-carotene, tocopherol or plastoquinone. If not quenched, it can trigger the up-regulation of genes, which are involved in the molecular defence response of photosynthetic organisms against photo-oxidative stress.     

Tocopherol quinone content of green algae and higher plants revised by a new high-sensitive fluorescence detection method using HPLC--effects of high light stress and senescence

A rapid, sensitive fluorescence method was applied here for detection of oxidized tocopherol quinones in total plant tissue extracts using HPLC, employing a post-column reduction of these compounds by a Zn column. Using this method, we were able to detect both alpha- and gamma-tocopherol quinones in Chamydomonas reinhardii with a very high degree of sensitivity. The levels of both compounds increased under high light stress in the presence of pyrazolate in parallel to a decrease in the content of the corresponding tocopherols. The formation of tocopherol quinones from tocopherols was apparently due to their oxidation by singlet oxygen, which is formed in photosystem II under high light stress. alpha-Tocopherol quinone was also detected in a variety of higher plants of different age, and its level was found to increase during senescence in leaves grown under natural conditions. In contrast to alpha-tocopherol quinone, gamma-tocopherol quinone was not found in the higher plant species investigated with the exception of young runner bean leaves, where the levels of both compounds increased dramatically during cold and light stress. Taking advantage of native fluorescence of the reduced alpha-tocopherol quinone (alpha-tocopherol quinol), it can be detected in plant tissue extracts with a high sensitivity. In young runner bean leaves, alpha-tocopherol quinol was found at a level similar to alpha-tocopherol.     

A reporter system for the individual detection of hydrogen peroxide and singlet oxygen: its use for the assay of reactive oxygen species produced in vivo

A reporter system for the assay of reactive oxygen species (ROS) was developed in Chlamydomonas reinhardtii, a plant model organism well suited for the application of inhibitors and generators of various types of ROS. This system employs various HSP70A promoter segments fused to a Renilla reniformis luciferase gene as a reporter. Transformants with the complete HSP70A promoter were inducible by both hydrogen peroxide and singlet oxygen. Constructs that lacked upstream heat-shock elements (HSEs) were inducible by hydrogen peroxide, indicating that this induction does not require such HSEs. Rather, downstream elements located between positions -81 to -149 with respect to the translation start site appear to be involved. In contrast, upstream sequences are essential for the response to singlet oxygen. Thus, activation by singlet oxygen appears to require promoter elements that are different from those used by hydrogen peroxide. ROS generated endogenously by treatment of the alga with metronidazole, protoporphyrin IX, dinoterb or high light intensities were detected by this reporter system, and distinguished as production of hydrogen peroxide (metronidazole) and singlet oxygen (protoporphyrin IX, dinoterb, high light). This system thus makes it possible to test whether, under varying environmental conditions including the application of abiotic stress, hydrogen peroxide or singlet oxygen or both are produced.     

Hydroxy‐plastochromanol and plastoquinone‐C as singlet oxygen products during photo‐oxidative stress in Arabidopsis

In the present study, we have shown that hydroxy‐plastochromanol and plastoquinone‐C, the hydroxy derivatives of plastochromanol and plastoquinone‐9, respectively, are specifically formed from the parent compounds upon action of singlet oxygen and can be regarded as stable, specific, natural products of singlet oxygen action during photo‐oxidative stress in vivo. The presented data indicate that plastoquinone‐C formation dominates mainly during relatively short periods of high light stress where efficient production of singlet oxygen takes place, whereas hydroxy‐plastochromanol is rather formed under conditions of long‐term, less pronounced generation of singlet oxygen. An interesting observation was that hydroxy‐plastochromanol is formed even at very low light conditions (5–10 μmol photons m^?2 s^?1), indicating that singlet oxygen is generated not only during high light stress but also its formation by photosystem II is inseparably connected with the functioning of this photosystem even at the lowest light intensities.     

Characterization of singlet oxygen-accumulating mutants isolated in a screen for altered oxidative stress response in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Background  

When photosynthetic organisms are exposed to harsh environmental conditions such as high light intensities or cold stress, the production of reactive oxygen species like singlet oxygen is stimulated in the chloroplast. In Chlamydomonas reinhardtii singlet oxygen was shown to act as a specific signal inducing the expression of the nuclear glutathione peroxidase gene GPXH/GPX5 during high light stress, but little is known about the cellular mechanisms involved in this response. To investigate components affecting singlet oxygen signaling in C. reinhardtii, a mutant screen was performed.     

Acclimation to singlet oxygen stress in Chlamydomonas reinhardtii

In an aerobic environment, responding to oxidative cues is critical for physiological adaptation (acclimation) to changing environmental conditions. The unicellular alga Chlamydomonas reinhardtii was tested for the ability to acclimate to specific forms of oxidative stress. Acclimation was defined as the ability of a sublethal pretreatment with a reactive oxygen species to activate defense responses that subsequently enhance survival of that stress. C. reinhardtii exhibited a strong acclimation response to rose bengal, a photosensitizing dye that produces singlet oxygen. This acclimation was dependent upon photosensitization and occurred only when pretreatment was administered in the light. Shifting cells from low light to high light also enhanced resistance to singlet oxygen, suggesting an overlap in high-light and singlet oxygen response pathways. Microarray analysis of RNA levels indicated that a relatively small number of genes respond to sublethal levels of singlet oxygen. Constitutive overexpression of either of two such genes, a glutathione peroxidase gene and a glutathione S-transferase gene, was sufficient to enhance singlet oxygen resistance. Escherichia coli and Saccharomyces cerevisiae exhibit well-defined responses to reactive oxygen but did not acclimate to singlet oxygen, possibly reflecting the relative importance of singlet oxygen stress for photosynthetic organisms.     

The Cyanobacterial Photoactive Orange Carotenoid Protein Is an Excellent Singlet Oxygen Quencher

Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the photosynthetic reaction centers under high-light conditions. The photoactive orange carotenoid protein (OCP) is essential in this mechanism as a light sensor and energy quencher. When OCP is photoactivated by strong blue-green light, it is able to dissipate excess energy as heat by interacting with phycobilisomes. As a consequence, charge separation and recombination leading to the formation of singlet oxygen diminishes. Here, we demonstrate that OCP has another essential role. We observed that OCP also protects Synechocystis cells from strong orange-red light, a condition in which OCP is not photoactivated. We first showed that this photoprotection is related to a decrease of singlet oxygen concentration due to OCP action. Then, we demonstrated that, in vitro, OCP is a very good singlet oxygen quencher. By contrast, another carotenoid protein having a high similarity with the N-terminal domain of OCP is not more efficient as a singlet oxygen quencher than a protein without carotenoid. Although OCP is a soluble protein, it is able to quench the singlet oxygen generated in the thylakoid membranes. Thus, OCP has dual and complementary photoprotective functions as an energy quencher and a singlet oxygen quencher.     

Singlet oxygen and non-photochemical quenching contribute to oxidation of the plastoquinone-pool under high light stress in Arabidopsis

The redox state of plastoquinone-pool in chloroplasts is crucial for driving many responses to variable environment, from short-term effects to those at the gene expression level. In the present studies, we showed for the first time that the plastoquinone-pool undergoes relatively fast oxidation during high light stress of low light-grown Arabidopsis plants. This oxidation was not caused by photoinhibition of photosystem II, but mainly by singlet oxygen generated in photosystem II and non-photochemical quenching in light harvesting complex antenna of the photosystem, as revealed in experiments with a singlet oxygen scavenger and with Arabidopsis npq4 mutant. The latter mechanism suppresses the influx of electrons to the plastoquinone-pool preventing its excessive reduction. The obtained results are of crucial importance in light of the function of the redox state of the plastoquinone-pool in triggering many high light-stimulated physiological responses of plants.     

Effects of phospholipids on the antioxidant activity of α-tocopherol in the singlet oxygen oxidation of canola oil

This study evaluated the effects of phosphatidylcholine (PC) or phosphatidylethanolamine (PE) on the antioxidative activity of α-tocopherol during oxidation of canola oil by singlet oxygen at 10°C for seven hours. Singlet oxygen was produced by chlorophyll b (4 ppm) under 1,700 lux. The oxidation of oil was evaluated by headspace oxygen consumption by gas chromatography and peroxide values (POVs). Concentrations of PC, PE, chlorophyll, and α-tocopherol were determined by HPLC. PC and PE protected chlorophyll from degradation, but they accelerated the degradation of α-tocopherol under singlet oxygen. Contents of PC and PE did not change for seven hours under singlet oxygen. α-Tocopherol significantly lowered POV and headspace oxygen consumption of canola oil under singlet oxygen, and its antioxidant activity was increased by the co-presence of PC and PE. PC and PE increased chemical quenching of singlet oxygen by tocopherol in decreasing the oil oxidation.     

Effect of free-radical and oxygen scavengers on photochemically generated oxygen toxicity and on the aerotolerance of Campylobacter jejuni

Exposure of a nutrient agar medium to the combined action of fluorescent light and air produced toxic factors in the medium which affected the growth of Campylobacter jejuni. Sodium dithionite (5-10 mM), a powerful reducing agent, and catalase were effective in counteracting the injurious action of light and air. Among the quenchers of singlet oxygen tested, only histidine had a beneficial effect on the recovery of C. jejuni in the photo-oxidized medium, while the addition of superoxide dismutase, a hydroxyl radical scavenger such as cysteamine, or the free radical antioxidants tocopherol and butylated hydroxy toluene, did not increase the recovery rate of photochemically injured cells. Histidine (40 mM) and dithionite (5-10 mM) also assisted recovery of C. jejuni inoculated on nutrient agar stored in air in the dark. Cysteamine and dithionite were toxic to Campylobacter when added at concentrations of greater than or equal to 10 mM and greater than or equal to 20 mM, respectively. A high inoculum of C. jejuni could not be recovered in unsupplemented nutrient agar incubated in air but was recovered in atmospheres containing 17 or 21% oxygen plus 10% carbon dioxide. The addition of dithionite, catalase or histidine resulted some colony formation on nutrient agar incubated in air. Among the scavengers tested, only dithionite was consistently able to maintain the viability of C. jejuni on nutrient agar stored in air for longer than 4 weeks. In view of the ability of catalase, dithionite and histidine to enhance the aerotolerance of C. jejuni, it is concluded that various oxygen species might be involved in the toxicity of high levels of oxygen.     

Function of beta-carotene and tocopherol in photosystem II

New and known structural and functional insights in the role of beta-carotene and of alpha-tocopherol in photosytem II are reviewed. A concept is presented connecting the failure of P680 triplet quenching by beta-carotene with the formation of singlet oxygen and its scavenging in the turnover of the D1 protein and by tocopherol in the maintenance of PS II structure and function.     

Effect of free-radical and oxygen scavengers on photochemically generated oxygen toxicity and on the aerotolerance of Campylobacter jejuni

Exposure of a nutrient agar medium to the combined action of fluorescent light and air produced toxic factors in the medium which affected the growth of Campylobacter jejuni . Sodium dithionite (5–10 mM), a powerful reducing agent, and catalase were effective in counteracting the injurious action of light and air. Among the quenchers of singlet oxygen tested, only histidine had a beneficial effect on the recovery of C. jejuni in the photo-oxidized medium, while the addition of superoxide dismutase, a hydroxyl radical scavenger such as cysteamine, or the free radical antioxidants tocopherol and butylated hydroxy toluene, did not increase the recovery rate of photochemically injured cells. Histidine (40 mM) and dithionite (5–10 mM) also assisted recovery of C. jejuni inoculated on nutrient agar stored in air in the dark. Cysteamine and dithionite were toxic to Campylobacter when added at concentrations of ≥10 mM and ≥ 20 mM, respectively. A high inoculum of C. jejuni could not be recovered in unsupplemented nutrient agar incubated in air but was recovered in atmospheres containing 17 or 21% oxygen plus 10% carbon dioxide. The addition of dithionite, catalase or histidine resulted in some colony formation on nutrient agar incubated in air. Among the scavengers tested, only dithionite was consistently able to maintain the viability of C. jejuni on nutrient agar stored in air for longer than 4 weeks. In view of the ability of catalase, dithionite and histidine to enhance the aerotolerance of C. jejuni , it is concluded that various oxygen species might be involved in the toxicity of high levels of oxygen.     

Imaging the production of singlet oxygen in vivo using a new fluorescent sensor, Singlet Oxygen Sensor Green

Singlet oxygen is known to be produced by cells in response to photo-oxidative stresses and wounding. Due to singlet oxygen being highly reactive, it is thought to have a very short half-life in biological systems and, consequently, it is difficult to detect. A new commercially available reagent (singlet oxygen sensor green, SOSG), which is highly selective for singlet oxygen, was applied to a range of biological systems that are known to generate singlet oxygen. Induction of singlet oxygen production by the addition of myoglobin to liposome preparations demonstrated that the singlet oxygen-induced increases in SOSG fluorescence closely followed the increase in the concentration of conjugated dienes, which is stoichiometrically related to singlet oxygen production. Applications of photo-oxidative stresses to diatom species and leaves, which are known to result in the production of singlet oxygen, produced large increases in SOSG fluorescence, as did the addition of 3-(3',4'-dichlorophenyl)1,1-dimethylurea (DCMU) to these systems, which inhibits electron transport in photosystem II and stimulates singlet oxygen production. The conditional fluorescent (flu) mutant of Arabidopsis produces singlet oxygen when exposed to light after a dark period, and this coincided with a large increase in SOSG fluorescence. Wounding of leaves was followed by an increase in SOSG fluorescence, even in the dark. It is concluded that SOSG is a useful in vivo probe for the detection of singlet oxygen.     

Role of singlet oxygen in chloroplast to nucleus retrograde signaling in Chlamydomonas reinhardtii

High light illumination of photosynthetic organisms stimulates the production of singlet oxygen by photosystem II and causes photooxidative stress. In Chlamydomonas reinhardtii, singlet oxygen also induces the expression of the nuclear-encoded glutathione peroxidase homologous gene GPXH. We provide evidence that singlet oxygen stimulates GPXH expression by activating a signaling mechanism outside the thylakoid membrane. Singlet oxygen from photosystem II could be detected with specific probes in the aqueous phase of isolated thylakoid suspensions and the cytoplasm of high light stressed cells. This indicates that singlet oxygen can stimulate a response farther from its production site than generally believed.     

The effect of carotenoids on the concentration of singlet oxygen in lipid membranes

An effect of β-carotene and its polar derivative, zeaxanthin, on a concentration of singlet oxygen in lipid membranes was studied in a model system. The carotenoids were incorporated into the membranes of small unilamellar liposomes at a concentration of 0.15 mol% with respect to lipid. Singlet oxygen was generated in a liposome suspension via photosensitization of toluidine blue, and its concentration in a membrane was detected with application of a specific fluorescence probe (singlet oxygen sensor green reagent) located in the lipid bilayer. The results show the carotenoid-dependent decrease in the concentration of singlet oxygen in the membranes formed with unsaturated lipids (egg yolk phosphatidylcholine and digalactosyldiacylglycerol) but not in the case of the membranes formed with a saturated lipid (dimyristoylphosphatidylcholine). The effect of carotenoids was about twice as high as in the case of cholesterol present in liposomes at the same concentration. The results suggest that carotenoids protect membranes formed with unsaturated lipids against singlet oxygen through combined activity of different mechanisms: modification of structural properties of the lipid bilayers, physical quenching of singlet oxygen and chemical reactions leading to the pigment oxidation. The latter conclusion is based on the analysis of the absorption spectra of liposomes before and after light exposure. An importance of the different modes of protection by carotenoids against single oxygen toxicity towards biomembranes is discussed.