Photooxidative Damage Research Articles

Loss of inner-envelope K+/H+ exchangers impairs plastid rRNA maturation and gene expression.

The inner-envelope K+ EFFLUX ANTIPORTERS (KEA) 1 and 2 are critical for chloroplast development, ion homeostasis, and photosynthesis. However, the mechanisms by which changes in ion flux across the envelope affect organelle biogenesis remained elusive. Chloroplast development requires intricate coordination between the nuclear genome and the plastome. Many mutants compromised in plastid gene expression (PGE) display a virescent phenotype, that is delayed greening. The phenotypic appearance of Arabidopsis thaliana kea1 kea2 double mutants fulfills this criterion, yet a link to PGE has not been explored. Here, we show that a simultaneous loss of KEA1 and KEA2 results in maturation defects of the plastid ribosomal RNAs. This may be caused by secondary structure changes of rRNA transcripts and concomitant reduced binding of RNA-processing proteins, which we documented in the presence of skewed ion homeostasis in kea1 kea2. Consequently, protein synthesis and steady-state levels of plastome-encoded proteins remain low in mutants. Disturbance in PGE and other signs of plastid malfunction activate GENOMES UNCOUPLED 1-dependent retrograde signaling in kea1 kea2, resulting in a dramatic downregulation of GOLDEN2-LIKE transcription factors to halt expression of photosynthesis-associated nuclear-encoded genes (PhANGs). PhANG suppression delays the development of fully photosynthesizing kea1 kea2 chloroplasts, probably to avoid progressing photo-oxidative damage. Overall, our results reveal that KEA1/KEA2 function impacts plastid development via effects on RNA-metabolism and PGE.

High light-induced photoinhibition is not limiting seedling establishment at abrupt treeline ecotones in New Zealand.

Seedlings of New Zealand's treeline-forming Fuscospora cliffortioides (Hook.f.) perform poorly beyond the established canopy, limiting treeline advance. To test the long-standing assumption that photoinhibition impairs regeneration in the subalpine belt of New Zealand's Southern Alps, we assessed photosystem II (PSII) performance of seedling-sized individuals and microclimate variation. We performed diurnal, non-invasive chlorophyll-a-fluorescence measurements on exposed and canopy-sheltered individuals at two sites in New Zealand's Southern Alps during summer and winter. Diurnal recordings of the effective (ΦPSII) and optimal (Fv/Fm) photosynthetic quantum yield were supplemented with light response curves and micro-temperature recordings. ΦPSII returned to near-optimal values around 0.8 after 30min of shading, which rules out accumulative or long-term photoinhibition effects. The maximum electron transport rate derived from rapid light curves was significantly higher (+12%) in exposed compared with canopy-shaded individuals. Summer temperature fluctuated widely on the scree (-0.5 to 60.5°C) and near seedlings (-2 to 26.5°C). Our results revealed a remarkable level of light adaptation and contradict previous studies hinting at high light-induced photoinhibition as a treeline-limiting factor in the Southern Alps. By linking low ΦPSII on winter mornings, and large, sudden temperature drops in summer, we suspect that cold-induced photoinhibition might occur but the rapid recovery of ΦPSII seen across a wide temperature range makes lethal photo-oxidative damage rather unlikely. Given the demonstrably low summer frost tolerance of F. cliffortioides, cold-related damage resulting from frost events during the growing season or embolism induced by frost drought may offer more plausible explanations for the poor seedling establishment. Duration and frequency of these events could diminish with global warming, which may promote treeline advance.

High Carotenoid Mutants of Chlorella vulgaris Show Enhanced Biomass Yield under High Irradiance.

Microalgae represent a carbon-neutral source of bulk biomass, for extraction of high-value compounds and production of renewable fuels. Due to their high metabolic activity and reproduction rates, species of the genus Chlorella are highly productive when cultivated in photobioreactors. However, wild-type strains show biological limitations making algal bioproducts expensive compared to those extracted from other feedstocks. Such constraints include inhomogeneous light distribution due to high optical density of the culture, and photoinhibition of the surface-exposed cells. Thus, the domestication of algal strains for industry makes it increasingly important to select traits aimed at enhancing light-use efficiency while withstanding excess light stress. Carotenoids have a crucial role in protecting against photooxidative damage and, thus, represent a promising target for algal domestication. We applied chemical mutagenesis to Chlorella vulgaris and selected for enhanced tolerance to the carotenoid biosynthesis inhibitor norflurazon. The NFR (norflurazon-resistant) strains showed an increased carotenoid pool size and enhanced tolerance towards photooxidative stress. Growth under excess light revealed an improved carbon assimilation rate of NFR strains with respect to WT. We conclude that domestication of Chlorella vulgaris, by optimizing both carotenoid/chlorophyll ratio and resistance to photooxidative stress, boosted light-to-biomass conversion efficiency under high light conditions typical of photobioreactors. Comparison with strains previously reported for enhanced tolerance to singlet oxygen, reveals that ROS resistance in Chlorella is promoted by at least two independent mechanisms, only one of which is carotenoid-dependent.

The Lutein and Zeaxanthin in Pregnancy (L-ZIP) study\u2014carotenoid supplementation during pregnancy: ocular and systemic effects\u2014study protocol for a randomized controlled trial

BackgroundLutein (L), zeaxanthin (Z), and meso-zeaxanthin (MZ), collectively called the macular pigment (MP), are dietary carotenoids that preferentially localize in the macula of the human eye. MP protects the macula from photo-oxidative damage and enhances visual function. Inadequate maternal intake of carotenoids, coupled with the placental transfer of maternal carotenoids to support fetal brain and retina development, potentially put mothers at risk of depletion systemically and in their ocular tissues. Presently, maternal carotenoid status throughout pregnancy remains poorly characterized, and no prospective randomized controlled trial of L and Z supplementation via prenatal vitamins has assessed maternal and infants’ systemic and ocular carotenoid status during pregnancy. We hypothesize that prenatal maternal carotenoid supplementation will counteract maternal carotenoid depletion during pregnancy and will improve biomarkers of carotenoid status of both mothers and infants.MethodsLutein and Zeaxanthin in Pregnancy (L-ZIP) is a phase 2, single-center, prospective, double-masked, randomized active-controlled clinical trial conducted at the John A. Moran Eye Center, University of Utah, Salt Lake City, UT, USA. Participants consume a daily standard prenatal multivitamin with no added carotenoids and are randomized (1:1 allocation) to receive either a capsule containing 10 mg L and 2 mg Z in safflower oil (Carotenoid group) or a capsule containing only safflower oil with no added carotenoids (Control group) for a period of 6 to 8 months. Skin, serum, and ocular carotenoids are measured at every study visit (i.e., within the first trimester [baseline], second trimester, third trimester, and 0–2 weeks postpartum). Skin carotenoid assessment is by resonance Raman spectroscopy (RRS); serum carotenoid status is quantified using high-performance liquid chromatography (HPLC); and MP is measured with the dual-wavelength autofluorescence. Infants’ MP and foveal anatomy are assessed using RetCam retinal camera and Bioptigen SD-OCT, respectively. The primary outcomes are changes in maternal systemic and ocular carotenoid status during pregnancy.DiscussionL-ZIP is the first prospective RCT to investigate maternal carotenoid status throughout pregnancy and to determine whether prenatal maternal carotenoid supplementation will offset maternal carotenoid depletion and improve biomarkers of maternal and infant’s carotenoid status. Findings from L-ZIP will strengthen recommendations regarding prenatal carotenoid supplementation and consequently inform policy decisions.Trial registrationClinicalTrials.gov NCT03750968. Registered on November 23, 2018

Pre treatment with Bacillus subtilis mitigates drought induced photo-oxidative damages in okra by modulating antioxidant system and photochemical activity.

The online version contains supplementary material available at 10.1007/s12298-021-00982-8.

The molecular pH-response mechanism of the plant light-stress sensor PsbS

Plants need to protect themselves from excess light, which causes photo-oxidative damage and lowers the efficiency of photosynthesis. Photosystem II subunit S (PsbS) is a pH sensor protein that plays a crucial role in plant photoprotection by detecting thylakoid lumen acidification in excess light conditions via two lumen-faced glutamates. However, how PsbS is activated under low-pH conditions is unknown. To reveal the molecular response of PsbS to low pH, here we perform an NMR, FTIR and 2DIR spectroscopic analysis of Physcomitrella patens PsbS and of the E176Q mutant in which an active glutamate has been replaced. The PsbS response mechanism at low pH involves the concerted action of repositioning of a short amphipathic helix containing E176 facing the lumen and folding of the luminal loop fragment adjacent to E71 to a 310-helix, providing clear evidence of a conformational pH switch. We propose that this concerted mechanism is a shared motif of proteins of the light-harvesting family that may control thylakoid inter-protein interactions driving photoregulatory responses.

Dissection of the Mechanisms of Growth Inhibition Resulting from Loss of the PII Protein in the Cyanobacterium Synechococcus elongatus PCC 7942.

In cyanobacteria, the PII protein (the glnB gene product) regulates a number of proteins involved in nitrogen assimilation including PipX, the coactivator of the global nitrogen regulator protein NtcA. In Synechococcus elongatus PCC 7942, construction of a PII-less mutant retaining the wild-type pipX gene is difficult because of the toxicity of uncontrolled action of PipX and the other defect(s) resulting from the loss of PIIper se, but the nature of the PipX toxicity and the PipX-independent defect(s) remains unclear. Characterization of a PipX-less glnB mutant (PD4) in this study showed that the loss of PII increases the sensitivity of PSII to ammonium. Ammonium was shown to stimulate the formation of reactive oxygen species in the mutant cells. The ammonium-sensitive growth phenotype of PD4 was rescued by the addition of an antioxidant α-tocopherol, confirming that photo-oxidative damage was the major cause of the growth defect. A targeted PII mutant retaining wild-type pipX was successfully constructed from the wild-type S. elongatus strain (SPc) in the presence of α-tocopherol. The resulting mutant (PD1X) showed an unusual chlorophyll fluorescence profile, indicating extremely slow reduction and re-oxidation of QA, which was not observed in mutants defective in both glnB and pipX. These results showed that the aberrant action of uncontrolled PipX resulted in an impairment of the electron transport reactions in both the reducing and oxidizing sides of QA.

Local adaptation optimizes photoprotection strategies in a Neotropical legume tree under drought stress.

Photoprotection is a plant functional mechanism to prevent photooxidative damage by excess light. This is most important when carbon assimilation is limited by drought, and as such, it entails a trade-off between carbon assimilation vs stress avoidance. The ecological adaptation of plants to local water availability can lead to different photoprotective strategies. To test this, we used different provenances of Caesalpinia spinosa (Mol.) Kuntze (commonly known as 'tara') along a precipitation gradient. Tara is a Neotropical legume tree with high ecological and commercial value, found in dry tropical forests, which are increasingly threatened by climate change. Morphological and physiological responses of tara provenances were analysed under three different treatments of drought and leaflet immobilization, i.e., light stress, in a common garden greenhouse experiment. Tara quickly responded to drought by reducing stomatal conductance, evapotranspiration, photochemical efficiency, carbon assimilation and growth, while increasing structural and chemical photoprotection (leaflet angle and pigments for thermal dissipation). Leaflet closure was an efficient photoprotection strategy with overall physiological benefits for seedlings as it diminished the evaporative demand and avoided photodamage, but also entailed costs by reducing net carbon assimilation opportunities. These responses depended on seed origin, with seedlings from the most xeric locations showing the highest dehydration tolerance, suggesting local adaptation and highlighting the value of different strategies under distinct environments. This plasticity in its response to environmental stress allows tara to thrive in locations with contrasting water availability. Our findings increase the understanding of the factors controlling the functional ecology of tara in response to drought, which can be leveraged to improve forecasts of changes in its distribution range, and for planning restoration projects with this keystone tree species.

The Leafless Orchid Cymbidium macrorhizon Performs Photosynthesis in the Pericarp during the Fruiting Season.

Photosynthesis with highly photoreactive chlorophyll (Chl) provides energy for plant growth but with simultaneous risk of photooxidative damage and photoprotection costs. Although the leafless orchid Cymbidium macrorhizon mostly depends on mycorrhizal fungi for carbon, it accumulates Chl particularly during fruiting and may not be fully mycoheterotrophic. In fact, stable isotopic analysis suggested that the fruiting C. macrorhizon specimens obtain a significant proportion of its carbon demands through photosynthesis. However, actual photosynthetic characteristics of this leafless orchid are unknown. To reveal the functionality of photosynthetic electron transport in C. macrorhizon, we compared its photosynthetic properties with those of its relative mixotrophic orchid Cymbidium goeringii and the model plant Arabidopsis thaliana. Compared with C. goeringii and A. thaliana, maximum photochemical efficiency of PSII was substantially low in C. macrorhizon. Chl fluorescence induction kinetics revealed that the electron transport capacity of PSII was limited in C. macrorhizon. Chl fluorescence analysis at 77 K suggested partial energetic disconnection of the light-harvesting antenna from the PSII reaction center in C. macrorhizon. Despite its low PSII photochemical efficiency, C. macrorhizon showed photosynthetic electron transport activity both in the field and under laboratory conditions. Cymbidium macrorhizon developed strong nonphotochemical quenching in response to increased light intensity as did C. goeringii, suggesting the functionality of photoprotective systems in this orchid. Moreover, C. macrorhizon fruit developed stomata on the pericarp and showed net O2-evolving activity. Our data demonstrate that C. macrorhizon can perform photosynthetic electron transport in the pericarp, although its contribution to net carbon acquisition may be limited.

Downregulation of miR-17-92 Cluster by PERK Fine-Tunes Unfolded Protein Response Mediated Apoptosis.

An important event in the unfolded protein response (UPR) is activation of the endoplasmic reticulum (ER) kinase PERK. The PERK signalling branch initially mediates a prosurvival response, which progresses to a proapoptotic response upon prolonged ER stress. However, the molecular mechanisms of PERK-mediated cell death are not well understood. Here we show that expression of the primary miR-17-92 transcript and mature miRNAs belonging to the miR-17-92 cluster are decreased during UPR. We found that miR-17-92 promoter reporter activity was reduced during UPR in a PERK-dependent manner. Furthermore, we show that activity of the miR-17-92 promoter is repressed by ectopic expression of ATF4 and NRF2. Promoter deletion analysis mapped the region responding to UPR-mediated repression to a site in the proximal region of the miR-17-92 promoter. Hypericin-mediated photo-oxidative ER damage reduced the expression of miRNAs belonging to the miR-17-92 cluster in wild-type but not in PERK-deficient cells. Importantly, ER stress-induced apoptosis was inhibited upon miR-17-92 overexpression in SH-SY5Y and H9c2 cells. Our results reveal a novel function for ATF4 and NRF2, where repression of the miR-17-92 cluster plays an important role in ER stress-mediated apoptosis. Mechanistic details are provided for the potentiation of cell death via sustained PERK signalling mediated repression of the miR-17-92 cluster.

PROTECTIVE ROLES OF D1 PROTEIN TURNOVER AND THE XANTHOPHYLL CYCLE IN TOMATO (SOLANUM LYCOPERSICUM) UNDER SUB-HIGH TEMPERATURE AND HIGH LIGHT STRESS

<List> <ListItem><ItemContent> • D1 turnover plays a more important role than xanthophyll cycle in photoprotection under sub-high temperature and high light (HH) conditions in tomato. </ItemContent></ListItem> <ListItem><ItemContent> • D1 protein turnover and xanthophyll cycle avoided the accumulation of excess energy and photooxidative damage of photosystem through maintaining photosynthetic pigments content and inducing changes in NPQ core components. </ItemContent></ListItem> <ListItem><ItemContent> • D1 protein turnover and xanthophyll cycle can also avoided accumulation of ROS and membrane lipid peroxidation by maintaining the activity of the ROS scavenging system. </ItemContent></ListItem></List> D1 protein turnover and the xanthophyll cycle (XC) are important photoprotective mechanisms in plants that operate under adverse conditions. Here, streptomycin sulfate (SM) and dithiothreitol (DTT) were used in tomato plants as inhibitors of D1 protein turnover and XC to elucidate their photoprotective impacts under sub-high temperature and high light conditions (HH, 35°C, 1000 µmol·m^-²·s^-¹). SM and DTT treatments significantly reduced the net photosynthetic rate, apparent quantum efficiency, maximum photochemical efficiency, and potential activity of photosystem II, leading to photoinhibition and a decline in plant biomass under HH. The increase in reactive oxygen species levels resulted in thylakoid membrane lipid peroxidation. In addition, there were increased non-photochemical quenching and decreased chlorophyll pigments in SM and DTT application, causing an inhibition of D1 protein production at both transcriptional and translational levels. Overall, inhibition of D1 turnover caused greater photoinhibition than XC inhibition. Additionally, the recovery levels of most photosynthesis indicators in DTT-treated plants were higher than in SM-treated plants. These findings support the view that D1 turnover has a more important role than XC in photoprotection in tomato under HH conditions.

Distinct photo-oxidation-induced cell death pathways lead to selective killing of human breast cancer cells

Lack of effective treatments for aggressive breast cancer is still a major global health problem. We have previously reported that photodynamic therapy using methylene blue as photosensitizer (MB-PDT) massively kills metastatic human breast cancer, marginally affecting healthy cells. In this study, we aimed to unveil the molecular mechanisms behind MB-PDT effectiveness and specificity towards tumor cells. Through lipidomics and biochemical approaches, we demonstrated that MB-PDT efficiency and specificity rely on polyunsaturated fatty acid-enriched membranes and on the better capacity to deal with photo-oxidative damage displayed by non-tumorigenic cells. We found out that, in tumorigenic cells, lysosome membrane permeabilization is accompanied by ferroptosis and/or necroptosis. Our results also pointed at a cross-talk between lysosome-dependent cell death (LDCD) and necroptosis induction after photo-oxidation, and contributed to broaden the understanding of MB-PDT-induced mechanisms and specificity in breast cancer cells. Therefore, we demonstrated that efficient approaches could be designed on the basis of lipid composition and metabolic features for hard-to-treat cancers. The results further reinforce MB-PDT as a therapeutic strategy for highly aggressive human breast cancer cells.

Influence of salinity stress on PSII in barley (Hordeum vulgare L.) genotypes, probed by chlorophyll-a fluorescence

ObjectivesChlorophyll-a fluorescence is an efficient tool to determine the photosynthetic capacity of plants and the health status of plants under normal or stress conditions including salinity stress. This study was aimed to elucidate changes in the efficiency of photosystem II (PSII) in barley genotypes differing in degree of salt tolerance, which can be used for phenotyping in the breeding program for developing salt-tolerant cultivars. MethodologyTwelve barley (Hordeum vulgare L.) genotypes were subjected to salt stress and salt stress reduced the growth of all barley genotypes, which is associated with a decline in chlorophyll and K+ contents (roots and leaves) and increase in Na+. Of the 12 barley genotypes, one salt-tolerant (B-10008) and one salt-sensitive barley genotype (B-14011) was selected to further investigate the structural stability of PSII using fast chlorophyll a kinetic analysis under salinity stress. ResultsThe shape of OJIP transients changed due to salt stress in both salt-sensitive and salt-tolerant barley genotypes indicating a disturbance in structural stability at various points of PSII. The detailed analysis of JIP-test parameters suggested that salt stress caused photoinhibition of PSII due to enhanced inactive reaction centers, reduced absorption flux (ABS/RC), low transfer of electrons per reaction center (ETO/RC) and enhanced accumulation of QA¯ (VJ) thus reducing primary photochemistry (TRO/RC, ɸPO). These changes in PSII resulted in the reduction of the maximum quantum yield of PSII (Fv/Fm) and performance index (PIABS). Moreover, salinity stress enhanced dissipation energy flux per reaction center (DIO/RC) as a protective measure to save PSII from photooxidative damage in thylakoid membrane. ConclusionThe appearance of positive K and L-bands supported the idea that salt stress dissociated the light-harvesting complex from core proteins of PSII, damaged oxygen-evolving complex and reduced the structural stability of PSII by disturbing the electron transfer between acceptor and donor sides of PSII especially in salt sensitive genotype (B-14011). Moreover, such an adverse effect of salt stress on PSII was less in salt-tolerant barley genotype (B-10008). Thus, some JIP-test parameters can be used as potential phenotype marker for screening salt-tolerant genotypes.

Phagocytosis by the Retinal Pigment Epithelium: Recognition, Resolution, Recycling.

Tissue-resident phagocytes are responsible for the routine binding, engulfment, and resolution of their meals. Such populations of cells express appropriate surface receptors that are tailored to recognize the phagocytic targets of their niche and initiate the actin polymerization that drives internalization. Tissue-resident phagocytes also harbor enzymes and transporters along the endocytic pathway that orchestrate the resolution of ingested macromolecules from the phagolysosome. Solutes fluxed from the endocytic pathway and into the cytosol can then be reutilized by the phagocyte or exported for their use by neighboring cells. Such a fundamental metabolic coupling between resident phagocytes and the tissue in which they reside is well-emphasized in the case of retinal pigment epithelial (RPE) cells; specialized phagocytes that are responsible for the turnover of photoreceptor outer segments (POS). Photoreceptors are prone to photo-oxidative damage and their long-term health depends enormously on the disposal of aged portions of the outer segment. The phagocytosis of the POS by the RPE is the sole means of this turnover and clearance. RPE are themselves mitotically quiescent and therefore must resolve the ingested material to prevent their toxic accumulation in the lysosome that otherwise leads to retinal disorders. Here we describe the sequence of events underlying the healthy turnover of photoreceptors by the RPE with an emphasis on the signaling that ensures the phagocytosis of the distal POS and on the transport of solutes from the phagosome that supersedes its resolution. While other systems may utilize different receptors and transporters, the biophysical and metabolic manifestations of such events are expected to apply to all tissue-resident phagocytes that perform regular phagocytic programs.

Genetic engineering of potato (Solanum tuberosum L.) for enhanced α-tocopherols and abiotic stress tolerance.

Vitamin E (α-tocopherol) is a lipid-soluble essential vitamin recognized for improvement in degenerative health conditions, abating cancer risk, and coronary heart diseases in humans. While in plants, it acts as a free radical scavenger that protects cells against oxidative and photooxidative damages. The daily consumption of potato makes it a key target for biofortification with vitamins for eliminating vitamin deficiency in large populations. Vitamin E biosynthetic pathway genes have been overexpressed in plants via genetic engineering to enhance the α-tocopherol content. Major genes involved in the vitamin E biosynthesis in plants viz. the homogentisate-phytyltransferase (At-HPT) and γ-tocopherol-methyltransferase (At-γ-TMT), isolated from Arabidopsis were constitutively overexpressed in potato (Solanum tuberosum L.). The molecular analyses of independent transgenic lines revealed a stable integration of both the genes in the plant genome. The transgenic potato exhibited significantly improved vitamin E contents up to 173-258% in comparison to the untransformed control plants. Transgenic tissues also exhibited increased cellular antioxidant enzymes, proline, osmolyte, and glutathione content that are directly correlated with the ability of the plant to withstand abiotic stresses imposed by salt (NaCl) and heavy metal (CdCl2 ). Therefore, the current strategy of increasing the vitamin E content in potato with enhanced tolerance to abiotic stresses might greatly aid efforts to engineer crops for human health benefits and greater yield under adverse environmental conditions.

Characterization of UVA-Induced Alterations to Transfer RNA Sequences.

Ultraviolet radiation (UVR) adversely affects the integrity of DNA, RNA, and their nucleoside modifications. By employing liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based RNA modification mapping approaches, we identified the transfer RNA (tRNA) regions most vulnerable to photooxidation. Photooxidative damage to the anticodon and variable loop regions was consistently observed in both modified and unmodified sequences of tRNA upon UVA (λ 370 nm) exposure. The extent of oxidative damage measured in terms of oxidized guanosine, however, was higher in unmodified RNA compared to its modified version, suggesting an auxiliary role for nucleoside modifications. The type of oxidation product formed in the anticodon stem–loop region varied with the modification type, status, and whether the tRNA was inside or outside the cell during exposure. Oligonucleotide-based characterization of tRNA following UVA exposure also revealed the presence of novel photoproducts and stable intermediates not observed by nucleoside analysis alone. This approach provides sequence-specific information revealing potential hotspots for UVA-induced damage in tRNAs.

Inhibition of microRNA-155 Protects Retinal Function Through Attenuation of Inflammation in Retinal Degeneration

Although extensively investigated in inflammatory conditions, the role of pro-inflammatory microRNAs (miRNAs), miR-155 and miR-146a, has not been well-studied in retinal degenerative diseases. We therefore aimed to explore the role and regulation of these miRNA in the degenerating retina, with a focus on miR-155. C57BL/6J mice were subjected to photo-oxidative damage for up to 5 days to induce focal retinal degeneration. MiR-155 expression was quantified by qRT-PCR in whole retina, serum, and small-medium extracellular vesicles (s-mEVs), and a PrimeFlow™ assay was used to identify localisation of miR-155 in retinal cells. Constitutive miR-155 knockout (KO) mice and miR-155 and miR-146a inhibitors were utilised to determine the role of these miRNA in the degenerating retina. Electroretinography was employed as a measure of retinal function, while histological quantification of TUNEL+ and IBA1+ positive cells was used to quantify photoreceptor cell death and infiltrating immune cells, respectively. Upregulation of miR-155 was detected in retinal tissue, serum and s-mEVs in response to photo-oxidative damage, localising to the nucleus of a subset of retinal ganglion cells and glial cells and in the cytoplasm of photoreceptors. Inhibition of miR-155 showed increased function from negative controls and a less pathological pattern of IBA1+ cell localisation and morphology at 5 days photo-oxidative damage. While neither dim-reared nor damaged miR-155 KO animals showed retinal histological difference from controls, following photo-oxidative damage, miR-155 KO mice showed increased a-wave relative to controls. We therefore consider miR-155 to be associated with the inflammatory response of the retina in response to photoreceptor-specific degeneration.

The water soluble Zn(II) and Mg(II) phthalocyanines: Synthesis, photochemical, DNA photodamage and PDT effects against A549 cells

In this study, the phthalonitrile compound 4-(thieno[3,2-b]pyridin-7-yloxy] phthalonitrile (3), it’s peripherally tetra substituted zinc(II) (4) and magnesium(II) (5) phthalocyanines and their water-soluble derivatives (ZnPcQ) and (MgPcQ) were synthesized. The compounds were characterized with a combination of various spectroscopic techniques. The singlet oxygen quantum yields of ZnPcQ and MgPcQ were determined as 0.59 and 0.36. Their DNA nuclease, photodamage and oxidative photodamage experiments were investigated using agarose gel electrophoresis on supercoiled plasmid pBR322 DNA. The PDT effects of the compounds were investigated using MTT assay toward A549 cells. ZnPcQ and MgPcQ did not have damage effects on supercoiled plasmid pBR322 DNA without irradiation whereas ZnPcQ showed significant photodamage and oxidative photodamage effects upon irradiation. The photodamage mechanism experiments showed that singlet oxygen had a crucial role in the photodamage pathway. MgPcQ did not show considerable cytotoxic and phototoxic effects on A549 cell for 24 h. On the other hand, the cell viability of A549 was 69 ± 2.70 % at 10 μM without irradiation in the presence of ZnPcQ. A549 cells treated with 5 and 10 μM of ZnPcQ upon irradiation, A549 presented 75 ± 5.90 % and 44 ± 7.0 % of viabilities, respectively. These results suggested that ZnPcQ is a promising candidate for PDT and worthy of further research.

Barley Plants Overexpressing Ferrochelatases (HvFC1 and HvFC2) Show Improved Photosynthetic Rates and Have Reduced Photo-Oxidative Damage under Drought Stress than Non-Transgenic Controls

We investigated the roles of two Ferrochelatases (FCs), which encode the terminal enzyme for heme biosynthesis, in drought and oxidative stress tolerance in model cereal plant barley (Hordeum vulgare). Three independent transgenic lines ectopically overexpressing either barley FC1 or FC2 were selected and evaluated under well-watered, drought, and oxidative stress conditions. Both HvFC1 and HvFC2 overexpressing transgenics showed delayed wilting and maintained higher photosynthetic performance relative to controls, after exposure to soil dehydration. In each case, HvFC overexpression significantly upregulated the nuclear genes associated with detoxification of reactive oxygen species (ROS) upon drought stress. Overexpression of HvFCs, also suppressed photo-oxidative damage induced by the deregulated tetrapyrrole biosynthesis mutant tigrinad12. Previous studies suggest that only FC1 is implicated in stress defense responses, however, our study demonstrated that both FC1 and FC2 affect drought stress tolerance. As FC-derived free heme was proposed as a chloroplast-to-nuclear signal, heme could act as an important signal, stimulating drought responsive nuclear gene expression. This study also highlighted tetrapyrrole biosynthetic enzymes as potential targets for engineering improved crop performance, both in well-watered and water-limited environments.

Soybean (Glycine max) PHYTOCHROME-INTERACTION FACTOR 1 induced skotomorphogenesis and de-etiolation in Arabidopsis

Skotomorphogenesis occurs after germination and before excavation in plants. It inhibits excessive absorbed energy in cells and can prevent the lethal photooxidative damage caused by transitioning from skotomorphogenesis to photomorphogenesis for light energy utilization. To investigate the mechanisms underlying photoreactions in soybean [Glycine max (L.) Merr.], we identified and isolated soybean phytochrome-interaction factor 1 (GmPIF1). A yeast two-hybrid (Y2H) assay showed that GmPIF1 interacted with photoactive PHYTOCHROME A (PHYA) and B (PHYB) in both soybean and Arabidopsis (GmPHYA, GmPHYB, AtPHYA, and AtPHYB). To analyze its function, we ectopically over-expressed GmPIF1 in wild type and pif1 mutant Arabidopsis. In etiolated seedlings, GmPIF1 caused hypocotyl elongation, cotyledon closed, apical hooks folded, and less accumulation of protochlorophyllide. In Y2H, GmPIF1 interacted with AtHDA15 that inhibited chlorophyll synthesis under dark conditions. After transition from darkness to white light, GmPIF1 promotes the reduction of photobleaching and induced de-etiolation. Moreover, GmPIF1 inhibited PHYA- and PHYB-mediated seed germination. Our findings increase our understanding of the regulatory network of light response in soybean and provide useful gene resources for soybean breeding in programs and genetics engineering.