Photooxidative Damage Research Articles

Biosynthesis of Ascorbic Acid as a Glucose-Induced Photoprotective Process in the Extremophilic Red Alga Galdieria partita.

The extremophilic red alga Galdieria partita is a facultative heterotroph that occupies mostly low-light microhabitats. However, the exceptional detection of abundant populations of G. partita in sunlight-exposed soil raises the possibility that exogenous organic carbon sources protect cells from photo-oxidative damage. The present study aimed to identify the photoprotective process activated by exogenous glucose under photo-oxidative stress. We demonstrated that exogenous glucose mitigated the photo-oxidative damage of cells exposed to 300 μmol photons m–2 s–1 photosynthetic active radiation. Photosynthesis carbon assimilation scarcely contributed to the cell growth in the presence of glucose, but the photosynthetic apparatus was nevertheless maintained and protected by glucose in a concentration-dependent manner. Supplementation of glucose increased expression of the L-gulonolactone oxidase gene essential for ascorbic acid biosynthesis, whereas no enhanced expression of the genes involved in carotenoid or tocopherol biosynthesis was observed. Under the photo-oxidative stress condition, the ascorbic acid content was strongly enhanced by exogenous glucose. We propose that the biosynthesis of ascorbic acid is one of the major photoprotective processes induced by exogenous glucose. The elucidation of how ascorbic acid is involved in scavenging reactive oxygen species provides key insights into the photoprotective mechanism in red algae.

An extreme heatwave enhanced the xanthophyll de-epoxidation state in leaves of Eucalyptus trees grown in the field.

Heatwaves are becoming more frequent with climate warming and can impact tree growth and reproduction. Eucalyptus parramattensis can cope with an extreme heatwave in the field via transpiratory cooling and enhanced leaf thermal tolerance that protected foliar tissues from photo-inhibition and photo-oxidation during natural midday irradiance. Here, we explored whether changes in foliar carotenoids and/or the xanthophyll cycle state can facilitate leaf acclimation to long-term warming and/or an extreme heatwave event. We found that leaves had similar carotenoid levels when grown for one year under ambient and experimental long-term warming (+ 3°C) conditions in whole tree chambers. Exposure to a 4-day heatwave (> 43°C) significantly altered the xanthophyll de-epoxidation state of carotenoids revealing one mechanism by which trees could minimise foliar photo-oxidative damage. The levels of zeaxanthin were significantly higher in both young and old leaves during the heatwave, revealing that violaxanthin de-epoxidation and perhaps de novo zeaxanthin synthesis contributed to enhancement of the xanthophyll cycle state. In a future climate of long-term warming and increased heatwave events, leaves of E. parramattensis will be able to utilise biochemical strategies to alter the xanthophyll cycle state and cope with extreme temperatures under natural solar irradiation.

Two dominant boreal conifers use contrasting mechanisms to reactivate photosynthesis in the spring

Boreal forests are dominated by evergreen conifers that show strongly regulated seasonal photosynthetic activity. Understanding the mechanisms behind seasonal modulation of photosynthesis is crucial for predicting how these forests will respond to changes in seasonal patterns and how this will affect their role in the terrestrial carbon cycle. We demonstrate that the two co-occurring dominant boreal conifers, Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies), use contrasting mechanisms to reactivate photosynthesis in the spring. Scots pine downregulates its capacity for CO2 assimilation during winter and activates alternative electron sinks through accumulation of PGR5 and PGRL1 during early spring until the capacity for CO2 assimilation is recovered. In contrast, Norway spruce lacks this ability to actively switch between different electron sinks over the year and as a consequence suffers severe photooxidative damage during the critical spring period.

Photoacclimation and Photoprotection of Juvenile Sporophytes of Macrocystis pyrifera (Laminariales, Phaeophyceae) Under High-light Conditions During Short-term Shallow-water Cultivation1.

This study was designed to understand better if and how juvenile sporophytes of Macrocystis pyrifera can photoacclimate to high-light conditions when transplanted from 10 to 3 meters over 7d. Acclimation of adult sporophytes to light regimes in the bathymetric gradient has been extensively documented. It primarily depends on photoacclimation and translocation of resources among blades. Among other physiological differences, juvenile sporophytes of M. pyrifera lack the structural complexity shown by adults. As such, juveniles may primarily depend on their photoacclimation capacities to maintain productivity and even avoid mortality under changing light regimes. However, little is known about how these mechanisms operate in young individuals. The capacity of sporophytes to photoacclimate was assessed by examining changes in their photosynthetic performance, pigment content, and bio-optical properties of the blade. Sporophytes nutritional status and oxidative damage were also determined. Results showed that juvenile sporophytes transplanted to shallow water were able to regulate light harvesting by reducing pigment concentration, and thus, absorptance and photosynthetic efficiency. Also, shallow-water sporophytes notably enhanced the dissipation of light energy as heat (NPQ) as a photoprotective mechanism. Generally, these adjustments allowed sporophytes to manage the absorption and utilization of light energy, hence reducing the potential for photo-oxidative damage. Furthermore, no substantial changes were found in the internal reserves (i.e., soluble carbohydrates and nitrogen) of these sporophytes. To our knowledge, these results are the first to provide robust evidence of photoprotective and photoacclimation strategies in juveniles of M. pyrifera, allowing them to restrict or avoid photodamage during shallow-water cultivation.

Cyclic electron flow modulate the linear electron flow and reactive oxygen species in tomato leaves under high temperature

The cyclic electron flow (CEF) around photosystem I (PSI) plays a crucial role in photosynthesis and also functions in plant tolerance of abiotic environmental stress. However, the role of PGR5/PGRL1- and NDH-dependent CEF in tomato under hightemperature (HT) is poorly understood. Here, we assessed the photoprotective effect of these pathways in tomato leaves under HT by using antimycin A (AA) and rotenone (R), which are chemical inhibitors of PGR5/PGRL1- and NDH-dependent CEF, respectively. The results showed that AA treatment caused significantly greater inhibition of CEF under HT compared to R treatment. Moreover, AA treatment caused a greater decrease in maximal photochemistry efficiency (Fv/Fm) and increased damage to the donor and acceptor side of photosystem II (PSII); however, the limitation of the acceptor side in PSI [Y(NA)] was significantly increased. In addition, thylakoid membrane integrity was compromised and reactive oxygen species, proton gradient (ΔpH), antioxidant enzyme activity, and the expression of photosystem core subunit genes were significantly decreased under AA treatment. These findings indicate that PGR5/PGRL1-dependent CEF protects PSII and PSI from photooxidative damage through the formation of ΔpH while maintaining thylakoid membrane integrity and normal gene expression levels of core photosystem components. This study demonstrates that PGR5/PGRL1-dependent CEF plays a major role in HT response in tomato.

Transcriptomic and metabolic analysis of an astaxanthin-hyperproducing Haematococcus pluvialis mutant obtained by low-temperature plasma (LTP) mutagenesis under high light irradiation

Haematococcus pluvialis can accumulate sufficient levels of astaxanthin under various stress conditions. High light irradiation is essential for astaxanthin accumulation in Haematococcus pluvialis. However, a lack of genomic information limits the understanding of its physiological metabolism for high astaxanthin production. In this work, we investigated the wild-type (WT) strain and mutant strain (named as M3 which was obtained in our previous work) under high light irradiation and compared the difference in astaxanthin biosynthesis. We collected and analyzed the data of de novo transcriptome information of Haematococcus pluvialis at 24 h under high light stress. Our transcriptomic results indicated that M3 strain had higher utilization efficiency of CO2 to provide the precursors of carotenoid and fatty acid biosynthesis by increasing the expression levels of phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), malate dehydrogenase (ME), ribulose bisphosphate carboxylase/oxygenase (Rubisco) activase (RCA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) while decreasing the expression levels of fructose-1, 6-bisphosphatase (FBP). The analysis of pigments, chlorophyll fluorescence and the quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that M3 strain maintained higher photosynthetic activity by regulating chlororespiration pathway and elevating non-photosynthetic pigment (lutein, β-carotene, and astaxanthin) content to alleviate photooxidative damage. Moreover, the M3 strain showed higher fatty acid content than the WT strain. Overall, combinative analysis of de novo transcriptomic and physiological data provided information necessary for not only a better understanding of the difference in astaxanthin biosynthesis between WT and M3 strains but also the feasibility of genetic engineering of Haematococcus pluvialis in the future.

Formulation and Evaluation of Sunscreen Gels Containing Mangiferin Isolated from Phaleria macrocarpa Fruits

Objectives: Ultraviolet (UV)-mediated photoreaction and photo-oxidation damage the skin, which can be prevented by using sunscreens as photo protective agents. Mangiferin is a major constituent of Phaleria macrocarpa fruits that has both sunscreen and antioxidant activity. This study aimed to formulate and evaluate sunscreen gel made from mangiferin isolated from P. marcocarpha fruits. Methods: Sunscreen gels were formulated using three different concentrations of mangiferin (1.25%, 2.5%, and 5%) and their physicochemical parameters (color, odor, homogeneity, spread ability, pH, and accelerated stability) were tested. The in vitro Sun Protection Factor (SPF) of the gels was determined using UV spectrophotometry. Sensory evaluation (hedonic test) was performed with a panel of 32 untrained panelists. Skin irritation test was conducted on 20 female volunteers using a skin patch. Results: The three mangiferin sunscreen gels showed high absorbance at wavelengths of 290–360 nm. The SPF was 11.2, 38.6, and and 88.53 at a mangiferin concentration of 1.25%, 2.5%, and 5%, respectively. The gels’ pH was in the proper range (5.8–6.0), and they showed good spread ability, no phase separation, and acceptable consistency. They were found to be stable during a two-month stability study. A gel containing 2.5% concentration of mangiferin is the most preferred formula. In addition, they did not irritate the skin. Conclusion: Gels formulation containing mangiferin at concentrations of 1.25, 2.5, and 5% are effective as sunscreens. The gel meets the requirements on physicochemical parameters and is stable for two months storages at temperatures 8°C, 25°C and 40°C.

Differences in isoprenoid-mediated energy dissipation pathways between coastal and interior Douglas-fir seedlings in response to drought.

Plants have evolved energy dissipation pathways to reduce photooxidative damage under drought when photosynthesis is hampered. Non-volatile and volatile isoprenoids are involved in non-photochemical quenching of excess light energy and scavenging of reactive oxygen species. A better understanding of trees' ability to cope with and withstand drought stress will contribute to mitigate the negative effects of prolonged drought periods expected under future climate conditions. Therefore we investigated if Douglas-fir (Pseudotsuga menziesii(Mirb.)) provenances from habitats with contrasting water availability reveal intraspecific variation in isoprenoid-mediated energy dissipation pathways. In a controlled drought experiment with 1-year-old seedlings of an interior and a coastal Douglas-fir provenance, we assessed the photosynthetic capacity, pool sizes of non-volatile isoprenoids associated with the photosynthetic apparatus, as well as pool sizes and emission of volatile isoprenoids. We observed variation in the amount and composition of non-volatile and volatile isoprenoids among provenances, which could be linked to variation in photosynthetic capacity under drought. The coastal provenance exhibited an enhanced biosynthesis and emission of volatile isoprenoids, which is likely sustained by generally higher assimilation rates under drought. In contrast, the interior provenance showed an enhanced photoprotection of the photosynthetic apparatus by generally higher amounts of non-volatile isoprenoids and increased amounts of xanthophyll cycle pigments under drought. Our results demonstrate that there is intraspecific variation in isoprenoid-mediated energy dissipation pathways among Douglas-fir provenances, which may be important traits when selecting provenances suitable to grow under future climate conditions.

Preparation and Evaluation of Sunscreen Nanoemulsions with Synergistic Efficacy on SPF by Combination of Soybean Oil, Avobenzone, and Octyl Methoxycinnamate.

BACKGROUND:Soybean oil contains vitamin E and acts as a natural sunscreen which can absorb Ultra Violet (UV) B light and has antioxidant properties to reduce the photooxidative damage that results from UV-induced Reactive Oxygen Species production. The UV blocking from most natural oils is insufficient to obtain a high UV protection. The strategies for preparations of sunscreen products with high SPF can be done by nanoemulsion formulation and Ultra Violet filter combinations of Soybean Oil, Avobenzone and Octyl methoxycinnamate.AIM:The purpose of this study was to prepare and in vitro efficacy evaluation of sunscreen nanoemulsion containing Soybean oil, Avobenzone and Octyl methoxycinnamate.METHODS:The sunscreen nanoemulsions were prepared by the high energy emulsification method. The formulation uses a combination of 3% Avobenzone, 7.5% Octyl methoxycinnamate, with different ratio of Soybean oil and Liquid Paraffin. The nanoemulsion was evaluated for droplet sizes by using particle size analyzer, physical stability in room temperature (25 ± 2°C during experiment for 12 weeks of storage, physical stability (cycling test), phase separation by centrifugation at 3750 rpm for 5 hours, pH, viscosity, and Sun Protection Factor (SPF) value by UV spectrophotometric. The SPF value of sunscreen nanoemulsion was compared to sunscreen nanoemulsion without Soybean Oil and sunscreen emulsion. Particle morphology observation of nanoemulsion by using Transmission Electron Microscope.RESULTS:The sunscreen nanoemulsion formulation containing a combination of 3% Avobenzone, 7.5% Octyl methoxycinnamate with a ratio of 2.73% Soybean Oil and 0.27% Paraffin Oil resulted in the smallest average droplet size of 68.47 nm. The sunscreen nanoemulsion without Soybean Oil had an average droplet size of 384.07 nm. The globules size was increased during the experiment for 12 weeks of storage at room temperature, but there was no phase separation after centrifugation. The formulation of sunscreen emulsion, phase separation was formed after centrifugation. The nanoemulsion had a pH value of 7.23 ± 0.06 and a viscosity value of 133.33 ± 7.22 cP. The sunscreen nanoemulsion containing a combination of 3% Avobenzone, 7.5% Octyl methoxycinnamate 2.73%, Soybean Oil, 2.73% and 0.27% Liquid Paraffin had SPF value (21.57 ± 1.21) higher than sunscreen nanoemulsion without Soybean Oil (16.52 ± 0.98) and sunscreen emulsion (15.10 ± 0.22). The TEM analysis of globules morphology showed that the sunscreen nanoemulsion formed a spherical globule.CONCLUSION:The sunscreen nanoemulsion containing a combination of 3% Avobenzone, 7.5% Octyl Methoxycinnamate, 2.73% Soybean Oil and 0.27% Liquid Paraffin showed synergistic sunscreen efficacy on SPF. This sunscreen nanoemulsion is more stable than sunscreen emulsion formulation during the experiment for 12 weeks at room temperature.

Leaf gas exchange, photochemical responses and oxidative damages in assai (Euterpe oleracea Mart.) seedlings subjected to high temperature stress

In this work, we aimed to examine the magnitude of changes in leaf gas exchange, chlorophyll fluorescence, and antioxidant responses in assai (Euterpe oleracea Mart.) seedlings subjected for 14 days to different temperatures (28 °C, control treatment, and 36 and 40 °C, high-temperature stress). Exposure to 36 °C decreased stomatal conductance (gs), photochemical quenching (qL), and non- photochemical quenching (NPQ) by 27, 53, and 20%, respectively. Further, it increased the photosynthetically active radiation (PAR) fraction that was absorbed by photosystem (PS)-II-antenna (but neither used in photochemistry nor dissipated thermally) by 14%. Moreover, incubation of seedlings at 36 °C induced a 53% increase in ascorbate peroxidase (APX) activity and a 16% decrease in hydrogen peroxide (H2O2); however, there were neither photooxidative damages to the PS nor lipid peroxidation. In contrast, seedlings incubated at 40 °C exhibited significantly decreased net CO2 assimilation rate (93%), gs (83%), transpiration (75%), and water use efficiency (71%). The maximum efficiency of PS II photochemistry did not differ compared to control plants, but the actual quantum yield of PS II electron transport, qL, and NPQ were decreased by 74, 60 and 19%, respectively. At 40 °C, there was increased photorespiration and elevated superoxide dismutase and APX activities, but the seedlings exhibited increased lipid peroxidation and general chlorosis in the youngest leaves. Taken together, our findings support that assai seedlings were tolerant to 36 °C but very sensitive to 40 °C.

A New Light on Photosystem II Maintenance in Oxygenic Photosynthesis.

Life on earth is sustained by oxygenic photosynthesis, a process that converts solar energy, carbon dioxide, and water into chemical energy and biomass. Sunlight is essential for growth and productivity of photosynthetic organisms. However, exposure to an excessive amount of light adversely affects fitness due to photooxidative damage to the photosynthetic machinery, primarily to the reaction center of the oxygen-evolving photosystem II (PSII). Photosynthetic organisms have evolved diverse photoprotective and adaptive strategies to avoid, alleviate, and repair PSII damage caused by high-irradiance or fluctuating light. Rapid and harmless dissipation of excess absorbed light within antenna as heat, which is measured by chlorophyll fluorescence as non-photochemical quenching (NPQ), constitutes one of the most efficient protective strategies. In parallel, an elaborate repair system represents another efficient strategy to maintain PSII reaction centers in active states. This article reviews both the reaction center-based strategy for robust repair of photodamaged PSII and the antenna-based strategy for swift control of PSII light-harvesting (NPQ). We discuss evolutionarily and mechanistically diverse strategies used by photosynthetic organisms to maintain PSII function for growth and productivity under static high-irradiance light or fluctuating light environments. Knowledge of mechanisms underlying PSII maintenance would facilitate bioengineering photosynthesis to enhance agricultural productivity and sustainability to feed a growing world population amidst climate change.

A thylakoid membrane-bound and redox-active rubredoxin (RBD1) functions in de novo assembly and repair of photosystem II

Photosystem II (PSII) undergoes frequent photooxidative damage that, if not repaired, impairs photosynthetic activity and growth. How photosynthetic organisms protect vulnerable PSII intermediate complexes during de novo assembly and repair remains poorly understood. Here, we report the genetic and biochemical characterization of chloroplast-located rubredoxin 1 (RBD1), a PSII assembly factor containing a redox-active rubredoxin domain and a single C-terminal transmembrane α-helix (TMH) domain. RBD1 is an integral thylakoid membrane protein that is enriched in stroma lamellae fractions with the rubredoxin domain exposed on the stromal side. RBD1 also interacts with PSII intermediate complexes containing cytochrome b559 Complementation of the Chlamydomonas reinhardtii (hereafter Chlamydomonas) RBD1-deficient 2pac mutant with constructs encoding RBD1 protein truncations and site-directed mutations demonstrated that the TMH domain is essential for de novo PSII assembly, whereas the rubredoxin domain is involved in PSII repair. The rubredoxin domain exhibits a redox midpoint potential of +114 mV and is proficient in 1-electron transfers to a surrogate cytochrome c in vitro. Reduction of oxidized RBD1 is NADPH dependent and can be mediated by ferredoxin-NADP+ reductase (FNR) in vitro. We propose that RBD1 participates, together with the cytochrome b559, in the protection of PSII intermediate complexes from photooxidative damage during de novo assembly and repair. This role of RBD1 is consistent with its evolutionary conservation among photosynthetic organisms and the fact that it is essential in photosynthetic eukaryotes.

Oxidative post-translational modification of EXECUTER1 is required for singlet oxygen sensing in plastids

Environmental information perceived by chloroplasts can be translated into retrograde signals that alter the expression of nuclear genes. Singlet oxygen (1O2) generated by photosystem II (PSII) can cause photo-oxidative damage of PSII but has also been implicated in retrograde signaling. We previously reported that a nuclear-encoded chloroplast FtsH2 metalloprotease coordinates 1O2-triggered retrograde signaling by promoting the degradation of the EXECUTER1 (EX1) protein, a putative 1O2 sensor. Here, we show that a 1O2-mediated oxidative post-translational modification of EX1 is essential for initiating 1O2-derived signaling. Specifically, the Trp643 residue in DUF3506 domain of EX1 is prone to oxidation by 1O2. Both the substitution of Trp643 with 1O2-insensitive amino acids and the deletion of the DUF3506 domain abolish the EX1-mediated 1O2 signaling. We thus provide mechanistic insight into how EX1 senses 1O2 via Trp643 located in the DUF3506 domain.

Sensing β-carotene oxidation in photosystem II to master plant stress tolerance.

Stressful environmental conditions lead to the production of reactive oxygen species in the chloroplasts, due to limited photosynthesis and enhanced excitation pressure on the photosystems. Among these reactive species, singlet oxygen (1 O2 ), which is generated at the level of the PSII reaction center, is very reactive, readily oxidizing macromolecules in its immediate surroundings, and it has been identified as the principal cause of photooxidative damage in plant leaves. The two β-carotene molecules present in the PSII reaction center are prime targets of 1 O2 oxidation, leading to the formation of various oxidized derivatives. Plants have evolved sensing mechanisms for those PSII-generated metabolites, which regulate gene expression, putting in place defense mechanisms and alleviating the effects of PSII-damaging conditions. A new picture is thus emerging which places PSII as a sensor and transducer in plant stress resilience through its capacity to generate signaling metabolites under excess light energy. This review summarizes new advances in the characterization of the apocarotenoids involved in the PSII-mediated stress response and of the pathways elicited by these molecules, among which is the xenobiotic detoxification.

Lutein and zeaxanthin isomers may attenuate photo-oxidative retinal damage via modulation of G protein-coupled receptors and growth factors in rats

BackgroundRetina photoreceptor cells are specially adapted for functioning over comprehensive ambient light conditions. Lutein and Zeaxanthin isomers (L/Zi) can protect photoreceptor cells against excessive light degeneration. Efficacy of L/Zi has been assessed on some G protein-coupled receptors (GPCRs), transcription and neurotrophic factors in the retina of rats exposed to incremental intense light emitting diode (LED) illumination conditions. MethodsForty-two male rats (age: 8 weeks) were randomly assigned to six treatment groups, 7 rats each. The rats with a 3x2 factorial design were kept under 3 intense light conditions (12hL/12hD, 16hL/8hD, 24hL/0hD) and received two levels of L/Zi (0 or 100 mg/kg BW) for two months. Increased nuclear factor-kappa B (NF-κB), glial fibrillary acid protein (GFAP), and decreased Rhodopsin (Rho), Rod arrestin (Sag), G Protein Subunit Alpha Transducin1 (Gnat1), neural cell adhesion molecule (NCAM), growth-associated protein-43 (GAP43), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and heme oxygenase 1 (HO-1) were observed in 24 h light intensity adaptation followed by 16 h IL and 8 h D. ResultsL/Zi administration significantly improved antioxidant capacity and retinal Rho, Rod-arrestin (Sag), Gnat1, NCAM, GAP43, BDNF, NGF, IGF1, Nrf2, and HO-1 levels. However, the levels of NF-κB and GFAP levels were decreased by administration of L/Zi. ConclusionsAccording to these results, L/Zi may be assumed as an adjunct therapy to prevent early photoreceptor cell degeneration and neutralize free radicals derived from oxidative stress.

3-Hydroxykynurenine bound to eye lens proteins induces oxidative modifications in crystalline proteins through a type I photosensitizing mechanism

Photosensitized reactions mediated by endogenous chromophores have been associated with the etiology of age-related cataract disease. Endogenous chromophores such as 3-hydroxykynurenine (3OHKN) can be found in both free form, and bound to crystallin proteins. However, their efficiency in generating photo-induced oxidative modifications on eye lens proteins is not completely understood. In this work, the efficiency and photodynamic activity of 3OHKN bound to both lysine (3OHKN-Lys) and bovine lens proteins (3OHKN-BLP) was assessed and compared with the photosensitizing activity of the major chromophore arising from glucose degradation (GDC). The photosensitizing activity of 3OHKN-Lys, 3OHKN-BLP and GDC was characterized by measurement of singlet oxygen quantum yields, O2 consumption, SDS-PAGE and amino acid analysis of the photo-oxidized proteins.Singlet oxygen quantum yields under 20% O2 atmosphere were 0.02, 0.01, and 0.27 for 3OHKN-Lys, 3OHKN-BLP and GDC, respectively. O2 consumption by photosensitized reactions was more efficient for 3OHKN-BLP, with the extent of O2 consumption being ∼28% higher than for 3OHKN-Lys and GDC under both 5 and 20% O2. SDS-PAGE showed that protein crosslinking is dependent on the O2 concentration, and more extensive at 5 than 20% O2. GDC and 3OHKN-Lys were the most efficient crosslinkers at 20 and 5% O2, respectively. Amino acid analysis of the irradiated proteins showed consumption of Trp, His, Tyr and Phe, and formation of kynurenine (from Trp), methionine sulfoxide (from Met) and DOPA (from Tyr). Kynurenine formation was dependent on the O2 concentration with higher amounts detected at 5 than 20% O2 for 3OHKN-BLP and 3OHKN-Lys, with 3OHKN-BLP the most efficient sensitizer. Our results suggest that 3OHKN-BLP can elicit photo-oxidative damage mainly by a type I photosensitizing mechanism, with this likely to be the most prevalent pathway at the low physiologic O2 concentrations in the eye lens.

A vitamin-C-derived DNA modification catalysed by an algal TET homologue.

Methylation of cytosine to 5-methylcytosine (5mC) is a prevalent DNA modification found in many organisms. Sequential oxidation of 5mC by ten-eleven translocation (TET) dioxygenases results in a cascade of additional epigenetic marks and promotes demethylation of DNA in mammals1,2. However, the enzymatic activity and function of TET homologues in other eukaryotes remains largely unexplored. Here we show that the green alga Chlamydomonas reinhardtii contains a 5mC-modifying enzyme (CMD1) that is a TET homologue and catalyses the conjugation of a glyceryl moiety to the methyl group of 5mC through a carbon-carbon bond, resulting in two stereoisomeric nucleobase products. The catalytic activity of CMD1 requires Fe(II) and the integrity of its binding motif His-X-Asp, which is conserved in Fe-dependent dioxygenases3. However, unlike previously described TET enzymes, which use 2-oxoglutarate as a co-substrate4, CMD1 uses L-ascorbic acid (vitamin C) as an essential co-substrate. Vitamin C donates the glyceryl moiety to 5mC with concurrent formation of glyoxylic acid and CO2. The vitamin-C-derived DNA modification is present in the genome of wild-type C. reinhardtii but at a substantially lower level in a CMD1 mutant strain. The fitness of CMD1 mutant cells during exposure to high light levels is reduced. LHCSR3, a gene that is critical for the protection of C. reinhardtii from photo-oxidative damage under high light conditions, is hypermethylated and downregulated in CMD1 mutant cells compared to wild-type cells, causing a reduced capacity for photoprotective non-photochemical quenching. Our study thus identifies a eukaryotic DNA base modification that is catalysed by a divergent TET homologue and unexpectedly derived from vitamin C, and describes its role as a potential epigenetic mark that may counteract DNA methylation in the regulation of photosynthesis.

Suppression of Light-Induced Retinal Degeneration by Quercetin via the AP-1 Pathway in Rats.

We examined the cytoprotective effect of quercetin via activator protein (AP-1) and the heat shock protein 70 (Hsp70) pathway against light-induced retinal degeneration in rats. Quercetin was administered intraperitoneally to Sprague-Dawley rats for seven days before light exposure to intense white fluorescent light (3000 lux) for 24 h. Light-induced retinal damage was determined by the number of rows of photoreceptor cell nuclei, the microstructures of the rod outer segments and retinal pigment epithelium, and terminal deoxynucleotidyl transferase (TdT)-mediated 2′-Deoxyuridine-5′-triphosphate (dUTP) nick end labeling. To elucidate the cytoprotective mechanism of quercetin, expression levels were measured in the rat retinas of 8-hydroxy-deoxyguanosine (8-OHdG), a marker of oxidative stress; Hsp70; and transcription factor AP-1 transcription activity. Pretreatment with quercetin inhibited light-induced photoreceptor cellular apoptosis and subsequent retinal degeneration in rats. 8-OHdG and Hsp70 protein expressions were up-regulated markedly by light exposure and suppressed by quercetin pretreatment. The results of an electrophoretic mobility shift assay showed that AP-1-binding activity was activated by light exposure, and binding of c-Fos and c-Jun, but not JunB, mediated the binding activity. Intraperitoneal administration of quercetin decreases photooxidative damage in the retina and mediates cytoprotection against light-induced photoreceptor cell degeneration in rats. Suppression of the heterodimeric combination of c-Jun and c-Fos proteins at the AP-1 binding site is highly involved in quercetin-mediated cytoprotection.

Photoprotection and growth under different lights of Arabidopsis single and double mutants for energy dissipation (npq4) and state transitions (pph1).

Arabidopsis single and double mutants for energy dissipation (npq4) and state transitions (pph1, blocked in State II) show enhanced growth and flowers + siliques production under controlled low-light conditions. Non-photochemical quenching (NPQ) is a short-term regulation important to maintain efficient photosynthesis and to avoid photooxidative damages by dissipation of excess energy. Full activation of NPQ in plants requires the protonation of the PsbS protein, which is the sensor of the low lumenal pH triggering the thermal dissipation. State transitions are a second important photosynthetic regulation to respond to changes in light quality and unbalanced excitation of photosystems. State transitions allow energy redistribution between PSI and PSII through the reversible exchange of LHCII antenna complexes between photosystems thanks to the opposite action of the STN7 kinase and PPH1 phosphatase: phosphorylation of LHCII promotes its mobilization from PSII to PSI, while dephosphorylation has the opposite effect. In this work, we produced the pph1/npq4 double mutant and characterized some photosynthetic, growth and reproduction properties in comparison with wild-type and single-mutant plants in high- and low-light conditions. Results indicate that in high light, the pph1 mutant maintains good photoprotection ability, while npq4 plants show more susceptibility to photodamages. The pph1/npq4 double mutant showed a resistance to high-light stress similar to that of the single npq4 mutant. In low-light condition, the single mutants showed a significant increase of growth and flowering compared to wild-type plants and this effect was further enhanced in the pph1/npq4 double mutant. Results suggest that photosynthetic optimisation to improve crop growth and productivity might be possible, at least under controlled low-light conditions, by modifying NPQ and regulationof state transitions.

Efficacy of novel selective NLRP3 inhibitors in human and murine retinal pigment epithelial cells.

NLRP3 inflammasome activation in the retinal pigment epithelium (RPE) is observed in atrophic age-related macular degeneration (AMD), and pharmacological NLRP3 inhibition may provide a therapeutic strategy to halt disease progression. We tested selective NLRP3 inhibitors (IFM-514, IFM-632, and CRID3) for their efficacy in human and murine RPE cells. Inflammasome activation was induced in primary human RPE cells and ARPE-19 cells following priming with IL-1α by different stimuli, including lysosomal membrane permeabilization by leucyl-leucine methyl ester (Leu-Leu-OMe), oxidative damage induced by hydrogen peroxide, lipofuscin-mediated photooxidative damage induced by incubation with 4-hydroxynonenal-modified photoreceptor outer segments and subsequent blue light irradiation, and P2X7 receptor activation by benzoylbenzoyl-ATP. Independent of the applied activation mechanism, treatment with the NLRP3 inhibitors IFM-632, IFM-514, and CRID3 resulted in a significant suppression of inflammasome activation as assessed by IL-1β and LDH release. Likewise, inflammasome activation in blue light-irradiated Abca4-/- mouse and Leu-Leu-OMe-treated wild-type mouse RPE/choroid/sclera eye cups was significantly reduced by treatment with the NLRP3 inhibitors. These results indicate that the investigated selective NLRP3 inhibitors are effective in human and murine RPE cells, thus representing promising agents for the future evaluation of inflammasome inhibition as a therapeutic strategy in atrophic AMD. KEY MESSAGES: • NLRP3 inhibitors suppress inflammasome activation in human RPE cells independent of trigger. • Light-induced inflammasome activation in Abca4-/- mouse eye cups is reduced by NLRP3 inhibitors. • Novel selective NLRP3 inhibitors are effective in human and murine RPE cells. • Promising compounds for pharmaceutical intervention in atrophic AMD.