GSH Accumulation Research Articles

Exogenous γ-glutamic acid (GABA) induces proline and glutathione synthesis in alleviating Cd-induced photosynthetic inhibition and oxidative damage in tobacco leaves

ABSTRACT The effects of exogenous γ-glutamic acid (GABA) on photosynthetic gas exchange capacity, PSII function, proline (Pro) synthesis and ROS metabolism of tobacco leaves under cadmium (Cd) stress were studied. The results showed that both stomatal and non-stomatal factors were the important factors to limit the photosynthetic capacity of tobacco leaves under Cd stress. Pro accumulation and glutathione (GSH) content increase are the important ways for tobacco leaves to adapt to Cd stress. Exogenous GABA significantly increased the stability of oxygen-evolving complex (OEC) and the electron transfer rate of PSII in tobacco leaves under Cd stress, and increased Gs and Pn in varying degrees. Exogenous GABA also increased the expression of P5CS and P5CR genes in tobacco leaves under Cd stress, thus promoting Pro accumulation. In conclusion, 0.5 mmol·L−1 exogenous GABA can not only improve the carbon assimilation ability by increasing the Gs , but also alleviate the oxidative damage by promoting the synthesis and accumulation of Pro and GSH, and improve the stability of photosynthetic function of tobacco leaves under Cd stress.

β-aminobutyric acid induces priming defence against Botrytis cinerea in grapefruit by reducing intercellular redox status that modifies posttranslation of VvNPR1 and its interaction with VvTGA1

Either NPR1 or TGA1 serve as master redox-sensitive transcriptional regulators for the transcription of PR genes in plants. The redox modification of the two co-activators involved in BABA-induced priming resistance against Botrytis cinerea in grapes was examined in this study. The results showed that 10 mmol L−1 BABA could effectively trigger a priming defense in grapes as manifested by augmented expression levels of PR genes upon inoculation with B. cinerea. Moreover, transcriptome profiling analysis revealed that all of the sets of key genes in the enzymatic ROS scavenging system, the PPP and AsA-GSH cycle were in harmony and were transcriptionally induced in BABA-primed grapes with pathogenic infection; in addition, this enhanced expression caused the accelerated accumulation of reductive substances, namely, AsA, GSH and NADPH, resulting in reduced intercellular conditions. Under reduced conditions, the interaction of VvTGA1 and VvNPR1 in the Y2H assay implied that VvTGA1 can provide the DNA binding capacity required by VvNPR1 for activation of VvPR genes. Consequently, the transactivation of VvNPR1 by the promoters of VvPR1, VvPR2 and VvPR5 was determined via a DLR assay, and it induced the transcription of the VvPR genes. In parallel, the redox-modified reducing condition achieved with an abundant supply of reductive substances was closely associated with the translocation of NPR1 for interaction with TGA in the nucleus. Thus, the posttranslational modification and subsequent interaction of the two redox-sensitive co-activators of VvNPR1 and VvTGA1 under reduced conditions may be responsible for BABA-induced priming for effective disease resistance in grapes.

Altered mitochondrial fusion drives defensive glutathione synthesis in cells able to switch to glycolytic ATP production

Mitochondria are highly dynamic organelles. Alterations in mitochondrial dynamics are causal or are linked to numerous neurodegenerative, neuromuscular, and metabolic diseases. It is generally thought that cells with altered mitochondrial structure are prone to mitochondrial dysfunction, increased reactive oxygen species generation and widespread oxidative damage. The objective of the current study was to investigate the relationship between mitochondrial dynamics and the master cellular antioxidant, glutathione (GSH). We reveal that mouse embryonic fibroblasts (MEFs) lacking the mitochondrial fusion machinery display elevated levels of GSH, which limits oxidative damage. Moreover, targeted metabolomics and 13C isotopic labeling experiments demonstrate that cells lacking the inner membrane fusion GTPase OPA1 undergo widespread metabolic remodeling altering the balance of citric acid cycle intermediates and ultimately favoring GSH synthesis. Interestingly, the GSH precursor and antioxidant n-acetylcysteine did not increase GSH levels in OPA1 KO cells, suggesting that cysteine is not limiting for GSH production in this context. Post-mitotic neurons were unable to increase GSH production in the absence of OPA1. Finally, the ability to use glycolysis for ATP production was a requirement for GSH accumulation following OPA1 deletion. Thus, our results demonstrate a novel role for mitochondrial fusion in the regulation of GSH synthesis, and suggest that cysteine availability is not limiting for GSH synthesis in conditions of mitochondrial fragmentation. These findings provide a possible explanation for the heightened sensitivity of certain cell types to alterations in mitochondrial dynamics.

A stepwise control strategy for glutathione synthesis in Saccharomyces cerevisiae based on oxidative stress and energy metabolism.

A stepwise control strategy for enhancing glutathione (GSH) synthesis in yeast based on oxidative stress and energy metabolism was investigated. First, molasses and corn steep liquor were selected and fed as carbon source mixture at a flow rate of 1.5g/L/h and 0.4g/L/h, respectively, for increasing cell density in a 10 L fermenter. When the biomass reached 90g/L, the KMnO4 sustained-release particles, composed of 1.5% KMnO4, 3% stearic acid, 2% polyethylene glycol and 3% agar powder, were prepared and added to the fermentation broth for maintaining the oxidative stress. The results showed that the maximum GSH accumulation of the group fed KMnO4 sustained-release particles was 39.0% higher than that of KMnO4-fed group. In addition to the improved average GSH productivity and average specific production rate, the activities of GSH peroxidase, γ-glutamylcysteine synthetase and GSH reductase, enzymes taking part in GSH metabolism, were also significantly enhanced by KMnO4 sustained-release particles feeding. Finally, 6g/L sodium citrate fed as an energy adjuvant elevated the intracellular ATP level for further enhancing GSH production. Through the above stepwise strategy, the GSH accumulation reached 5.76g/L, which was 2.84-fold higher than that of the control group. The stepwise control strategy based on oxidative stress and energy metabolism significantly improved GSH accumulation in yeast.

Antioxidant responses of triangle sail mussel Hyriopsis cumingii exposed to toxic Microcystis aeruginosa and thermal stress

Harmful algal blooms (HABs) and thermal stress as climate changes become more common in global water ecosystem, especially under eutrophic habitats. Here our study examined the combined impacts of bloom forming cyanobacteria Microcystis aeruginosa and thermal stress on the antioxidant responses of the ecologically important species triangle sail mussel Hyriopsis cumingii. The differential responses of a series of enzymes, e.g. superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST), as well as signal metabolites including reactive oxygen species (ROS), malondialdehyde (MDA) and glutathione (GSH) involved in antioxidant defense mechanisms were analyzed during 14 d exposure to toxic cyanobacterium M. aeruginosa and 7 d depuration period. The activities of SOD and GPx as well as the content of ROS and MDA in H. cumingii increased, while CAT activity reduced due to M. aeruginosa exposure. Thermal stress resulted in decrease of CAT, the accumulation of GSH and the enhance of GST and SOD. Meanwhile, the interactive effects among M. aeruginosa, thermal stress and time were also observed on most parameters except for GST activity. The total amount of microcystins (MC) in sail mussels increased with concentrations of exposed M. aeruginosa, independently of the presence or absence of thermal stress. Although around 50% of MC in mussels dropped in the depuration period, most parameters showed alterations because of cyanobacteria exposure and thermal stress. Overall, these findings suggested that toxic cyanobacteria or thermal stress induces oxidative stress and severely affects the enzymes activities and intermediates level associated with antioxidant defense mechanisms in sail mussels respectively. More importantly, the toxic impacts on sail mussels could be intensified by their combination.

Enhanced Polar Auxin Transport in Tomato Polycotyledon Mutant Seems to be Related to Glutathione Levels

Glutathione-dependent root growth in Arabidopsis is linked to polar auxin transport (PAT). Arabidopsis mutants with reduced glutathione (GSH) levels also show reduced PAT. To gain an insight into the relationship between PAT and GSH level, we analyzed tomato polycotyledon mutant, pct1-2, which has enhanced PAT. Microarray analysis of gene expression in pct1-2 mutant revealed underexpression of several genes related to glutamate and glutathione metabolism. In consonance with microarray analysis, enzymatic as well as in vivo assay revealed higher glutathione levels in the early phase of pct1-2 seedling growth than WT. The inhibition of auxin transport by 2,3,5-triiodobenzoic acid (TIBA) reduced both GSH level and PIN1 expression in pct1-2 root tips. The reduction of in vivo GSH accumulation in pct1-2 root tips by buthionine sulfoximine (BSO) stimulated elongation of the short root of pct1-2 mutant akin to TIBA. The rescue of the short root phenotype of the pct1-2 mutant was restricted to TIBA and BSO. The other auxin transport inhibitors 1-N-naphthylphthalamic acid (NPA), 2-[4-(diethylamino)-2-hydroxybenzoyl] benzoic acid (BUM), 3-chloro-4-hydroxyphenylacetic acid (CHPAA), brefeldin and gravacin inhibited root elongation in both WT and pct1-2 mutant. Our results indicate a relationship between PAT and GSH levels in tomato akin to Arabidopsis. Our work also highlights that TIBA rescues the short root phenotype of the pct1-2 mutant by acting on a PAT component distinct from the site of action of other PAT inhibitors.

ATP Hydrolysis Determines Cold Tolerance by Regulating Available Energy for Glutathione Synthesis in Rice Seedling Plants

BackgroundGlutathione (GSH) is important for plants to resist abiotic stress, and a large amount of energy is required in the process. However, it is not clear how the energy status affects the accumulation of GSH in plants under cold stress.ResultsTwo rice pure lines, Zhongzao39 (ZZ39) and its recombinant inbred line 82 (RIL82) were subjected to cold stress for 48 h. Under cold stress, RIL82 suffered more damages than ZZ39 plants, in which higher increases in APX activity and GSH content were showed in the latter than the former compared with their respective controls. This indicated that GSH was mainly responsible for the different cold tolerance between these two rice plants. Interestingly, under cold stress, greater increases in contents of carbohydrate, NAD(H), NADP(H) and ATP as well as the expression levels of GSH1 and GSH2 were showed in RIL82 than ZZ39 plants. In contrast, ATPase content in RIL82 plants was adversely inhibited by cold stress while it increased significantly in ZZ39 plants. This indicated that cold stress reduced the accumulation of GSH in RIL82 plants mainly due to the inhibition on ATP hydrolysis rather than energy deficit.ConclusionWe inferred that the energy status determined by ATP hydrolysis involved in regulating the cold tolerance of plants by controlling GSH synthesis.

Gametogenesis-Related Fluctuations in Ovothiol Levels in the Mantle of Mussels from Different Estuaries: Fighting Oxidative Stress for Spawning in Polluted Waters

Reactive oxygen species present a challenge for marine organisms releasing gametes into the water. Thiol-containing molecules protect cells against oxidative stress, and ovothiol (OSH), an antioxidant-reducing mercaptohistidine, has been described as especially relevant in the oocytes of marine invertebrates. Ovothiol synthase (ovoA), in charge of the first step in OSH synthesis, was sequenced in mussels, Mytilus galloprovincialis. Transcription levels of ovoA in mantle did not significantly change along the reproductive cycle. No alterations of ovoA transcription were observed after a laboratory copper (10 µg/L) exposure or in mussels captured in a highly polluted site. Conversely, the metabolomic analysis of the hydrophilic metabolite content in mantle clearly classified mussels according to their site of origin, especially at the most advanced stages of oogenesis. Quantification of OSH-A and -B and glutathione (GSH), revealed stable levels in mantle at early gametogenesis in the unpolluted sampling site, but a strong increase in female mantle previous to spawning in the polluted site. These increased concentrations under pollution suggest that OSH-A accumulates along oogenesis, independent of gene transcription regulation. The concerted accumulation of OSH-A and GSH suggests the building of a balanced cellular redox-system to scavenge ROS produced in the oocyte before and during fertilization.

Metabolic engineering of Lactococcus lactis for high level accumulation of glutathione and S-adenosyl-L-methionine.

Glutathione (GSH) and S-adenosyl methionine (SAM) have been applied as liver-protective factors to prevent and treat many different liver damages and diseases. Due to their low stability and short half-life, oral administration of GSH or SAM might be replaced by continuous supplying through living lactic bacteria in yogurt. In this study, Lactococcus lactis was engineered via synthetic biology strategies to produce these two important molecules. The bi-functional GSH synthase gene (gshF) and SAM synthase gene (metK) were transformed into food-grade L. lactis together with an adhesion factor gene (cwaA). The highest accumulation of SAM (9.0mg/L) and GSH (17.3mg/L) was achieved after 17h cultivation of the recombinant L. lactis. Meanwhile, the autoaggregation and hydrophobicity were also improved significantly, which suggested that this engineered L. lactis might have an increased colonization-prone ability in human GI. Our studies demonstrated one potential route to self-produce and deliver the liver-healthy factors within living probiotic bacteria.

Melatonin treatment maintains nutraceutical properties of pomegranate fruits during cold storage

In this study, the mechanism activated by melatonin treatment at 100 µM for maintaining nutraceutical properties in pomegranate fruits during storage at 4 °C for 120 days was investigated. Our results showed that the higher G6PDH and 6PGDH activities in pomegranate fruits treated with melatonin may be responsible for sufficient supply of intracellular NADPH. Also, higher AA and GSH accumulation in pomegranate fruits treated with melatonin may ascribe to higher APX and GR activities coincided with lower AAO activity. In addition, pomegranate fruits treated with melatonin exhibited significantly higher PAL activity resulting in higher phenols and anthocyanins accumulation as well as higher DPPH scavenging capacity. Additionally, higher AOX gene expression in pomegranate fruits treated with melatonin may be beneficial for ROS scavenging molecules accumulation. Therefore, maintaining nutraceutical properties of pomegranate fruits treated with melatonin may ascribe to sufficient intracellular NADPH supply by promoting G6PDH and 6PGDH activities during cold storage.

Metabolic diversity conveyed by the process leading to glutathione accumulation in inactivated dry yeast: A synthetic media study.

Glutathione-rich inactivated dry yeasts (GSH-IDY) are purported to accumulate glutathione intracellularly and then released into the must. Glutathione is beneficial for wine quality, but research has highlighted that GSH-IDYs have a synergic antioxidant effect similar to that of molecular GSH. Combination of negative mode ultra-high-resolution Fourrier-Transform Ion-Cyclotron-Resonance Mass Spectrometry ((-)FT-ICR-MS), ultra-high-performance liquid chromatography coupled to a Quadrupole-Time of Flight mass spectrometer (UHPLC-Q-ToF-MS) and HPLC/Diode Detector Array (DAD)-Fluorescence spectroscopy was applied to three inactivated dry yeasts soluble fractions, with increasing intracellular glutathione concentration, in order to explore the chemical diversity released in different synthetic media. Using the mean of size exclusion chromatography/DAD and fluorescence detection we report than most of the signals detected were below the 5-75 kDa-calibrated region of the chromatogram, indicating that most of the soluble protein fraction is composed of low molecular weight soluble peptides. In light of these results, high-resolution mass spectrometry was used to scan and annotate the low molecular weight compounds from 50 to 1500 Da and showed that GSH level of enrichment in IDYs was correlated to a discriminant chemical diversity of the corresponding soluble fractions. Our results clearly show an impact of the GSH accumulation process not only visible on the glutathione itself, but also on the global diversity of compounds. Within the 1674 ions detected by (-)FT-ICR-MS, the ratio of annotated elemental formulas containing carbon, hydrogen, oxygen, nitrogen and sulfur (CHONS) to annotated elemental formulas containing carbon, hydrogen, oxygen (CHO) increased from 0.2 to 2.1 with the increasing levels of IDYs GSH content and 36 unique CHONS annotated formulas were unique to the IDY with the highest concentration of GSH. Amongst the 1674 detected ions 193 were annotated as potential peptides (from 2 to 5 residues), 61 ions were annotated as unique amino acid combinations and 46% of which being significantly more intense in GSH-rich IDY. Thus, the process leading to the accumulation of glutathione also involves other metabolic pathways which contribute to an increase in CHONS containing compounds potentially released in wine, notably peptides.

14-3-3s function in plant cadmium response by changes of glutathione and glutathione synthesis in Arabidopsis

It is well-known that plants respond to the toxin cadmium (Cd), a widespread soil contaminant, by accumulating glutathione (GSH) and phytochelatin (PC). However, the factor(s) in the regulation of GSH and PC synthesis remain to be further identified. Here, we report that 14-3-3 κ and λ negatively regulate plant Cd tolerance because the double mutant 14-3-3κλ with higher Cd accumulation exhibited enhanced Cd tolerance, while overexpressing 14-3-3κ or 14-3-3λ resulted in increased Cd sensitivity in terms of changes of primary root elongation and fresh weight under Cd stress. The expression induction of the genes for GSH and PC synthesis and thus GSH and PC accumulation were further promoted in the 14-3-3κλ mutant but reduced in 14-3-3κ or 14-3-3λ overexpression lines compared with those in the stressed wild-type plants, suggesting the involvement of GSH and PC accumulation in 14-3-3s-mediated plant Cd response. This note was further supported by our observation that higher Cd tolerance was compromised in 14-3-3κλ seedlings treated simultaneously with Cd and buthionine sulfoximine, an inhibitor of GSH synthesis, compared with that in 14-3-3κλ seedlings treated with Cd alone. In addition, Cd-induced ROS accumulation was suppressed in 14-3-3κλ plants but further enhanced in 35S::14-3-3κ and 35S::14-3-3λ transgenic lines compared with that in wild-type plants, in consistent with GSH accumulations in these plants. Together, 14-3-3 s function in plant Cd response by changes of glutathione and glutathione synthesis in Arabidopsis.

Eugenol Confers Cadmium Tolerance via Intensifying Endogenous Hydrogen Sulfide Signaling in Brassica rapa

Eugenol, a plant-derived small compound, shows great medicinal potential. However, whether and how eugenol regulates crop physiology remains elusive. Here we reported that eugenol induced Cd (cadmium) tolerance in the root of Brassica rapa. Roots were treated with eugenol and CdCl2 simultaneously (eugenol + Cd) or pretreated with eugenol followed by CdCl2 treatment (eugenol → Cd). Eugenol significantly attenuated Cd-induced growth inhibition, ROS accumulation, oxidative injury, and cell death, which were confirmed by in vivo histochemical analysis. Eugenol remarkably decreased free Cd2+ accumulation in root. Eugenol intensified GSH (glutathione) accumulation in roots upon CdCl2 exposure, which explained the decrease in free Cd2+ and attenuation of oxidative injury. Eugenol stimulated endogenous H2S (hydrogen sulfide) generation by upregulating the expression of BrLCD ( l-cysteine desulfhydrase) and BrDCD ( d-cysteine desulfhydrase) as well as their enzymatic activities in CdCl2-treated root. Application of H2S biosynthesis inhibitor or H2S scavenger led to the decrease in endogenous H2S level in Cd-treated root, which further compromised all the above effects of eugenol. These findings suggested that eugenol triggered H2S → GSH signaling cassette in plants to combat Cd stress, which shed new light on eugenol-modulated plant physiology and the interaction between eugenol and H2S.

G6PDH regulated NADPH production and reactive oxygen species metabolism to enhance disease resistance against blue mold in apple fruit by acibenzolar-S-methyl

Apple fruit were treated with 100 mg L−1 acibenzolar-S-methyl (ASM) and 100 μM dehydroepiandrosterone (DHEA) to investigate its effect on the lesion development inoculated with Penicillium expansum. Effects of ASM on the glucose-6-phosphate dehydrogenase (G6PDH) activity, nicotinamide ademine dinucleotidephosphate (NADPH) production, and reactive oxygen species (ROS) metabolism in apple fruit were also studied. The results indicated that ASM treatment decreased the lesion development of blue mold in apple fruit. ASM treatment enhanced the content of H2O2, the activities of NADPH oxidase (NOX), superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and peroxidase (POD) in apple fruit. ASM treatment increased the gene expression of SOD, APX, DHAR as well as ascorbic acid (AsA) and reduced glutathione (GSH) content whereas it inhibited MDHAR expressions. The result also indicated that ASM treatment significantly inhibited CAT activity in apple fruit. In addition, the higher activity of G6PDH and content of NADPH was observed in ASM-treated apple fruit. In contrast, lesion diameter in ASM + DHEA-treated apple fruit developed more quickly than the control fruit. Also, the accumulation of H2O2, NADPH, AsA and GSH were reduced by DHEA treatment while inhibiting the activities of NOX, SOD, POD, APX, GR, MDHAR and G6PDH. Gene expressions of SOD, APX and DHAR were also inhibited by DHEA treatment. These results suggest that G6PDH played a crucial role in ROS metabolism and NADPH production in apple fruit to enhance resistance against P. expansum.

Protective Effect of Increased Zinc Supply against Oxidative Damage of Sublingual Gland in Chronic Exposure to Cadmium: Experimental Study on Rats.

Cadmium is one of the main chemical pollutants found in the daily environment of developed countries. Cigarettes are a significant source of that metal, which makes it important in terms of oral cavity health. The aim of this study was to determine if increased supply of zinc in chronic exposure to cadmium might protect the sublingual gland structure against oxidative damage. The experiment took 12 months and was conducted on 72 adult male rats. They were randomized into 9 groups. Eight groups received cadmium in drinking water (as CdCl2) at 5 or 50 mg Cd/dm3 and/or zinc (as ZnCl2) at 30 or 60 mg Zn/dm3. The control group received regular water. In the sublingual gland of all animal groups, levels of oxidative parameters were measured. The oxidative stress index was calculated as a TOS/TAS ratio. Cadmium exposure at 5 mg and 50 mg Cd/dm3 induced oxidative stress in the sublingual glands of the rats. Cadmium reduced the TAS and GSH levels and increased LPO, H2O2, TOS, and OSI. In cadmium exposure conditions, increasing the supply of zinc by 79% or 151%, as compared to the standard dietary intake of this microelement, completely prevented the reduction of TAS and GSH levels and accumulation of LPO, H2O2, and TOS in the examined gland at both exposure levels to that metal. The outcome data confirm the protective effect of increased zinc intake on the sublingual gland tissue in chronic cadmium exposure.

MicroRNA-144 modulates oxidative stress tolerance in SH-SY5Y cells by regulating nuclear factor erythroid 2-related factor 2-glutathione axis.

Genome-wide analysis of miRNA expression has revealed increased levels of miR-144 in the brains of Alzheimer's disease (AD) patients. Nuclear factor erythroid 2-related factor 2 (NRF2), a potential target of miR-144, is a central regulator of antioxidant response, and plays an important role in glutathione (GSH) biosynthesis and recycling. In this study, miR-144 mimic was used to over express miR-144. Aβ (1-42) was used to induce oxidative stress in SH-SY5Y cells. Cell viability and intracellular reactive oxygen species (ROS) were assessed to identify the effects of miR-144 on oxidative stress status. GSH and glutathion peroxidase (GPX) activities were detected to reveal the effect of miR-144 on GSH accumulation. To understand the effects of miR-144 on GSH biosynthesis and recycling, intracellular GPX1, glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM), glutathione reductase (GR), and NRF2 expression were detected by western blot and Real-time PCR. In oxidative stress conditions, miR-144 increased the intracellular accumulation of ROS, reduced cell viability, reduced the activities of GSH and antioxidant enzymes, GPX1, and decreased the expression of GCLC, GCLM, GR and NRF2. In conclusion, miR-144 modulates oxidative stress tolerance by regulating NRF2 expression and GSH generation, which may contribute to the pathogenesis of AD.

GR1-like gene expression in Lycium chinense was regulated by cadmium-induced endogenous jasmonic acids accumulation.

The G1-like gene from the Lycium chinensewas cloned and transferred into N. tabacum. Evidence showed that endogenous JA accumulation was crucial to LcGRgene expression in cadmium-stressed L. chinense. Glutathione reductase (GR) plays a vital role in glutathione-ascorbate metabolism and is a key enzyme in maintaining the redox state in plants. Jasmonic acids (JA) are important hormones regulating protective responses against bacteria and mechanic damage in plants. At present, the relationship between the endogenous JA accumulation, the glutathione (GSH) content and GR gene expression in plants under cadmium (Cd) stress has not been elucidated. This study primarily aims to explore their interconnected relations. First, we isolated theGR1-like gene fromLycium chinense (LcGR). Real-time PCR showed that gene LcGR and allene oxide cyclase (LcAOC) (a JA synthesis gene) expression in L. chinense plants was significantly enhanced by CdCl2 and reduced by CdCl2 cotreatment with 12,13-epoxy-octadecenoic acid (EOA), a JA synthesis inhibitor. Meanwhile, the JA content in plants strongly increased under Cd stress and decreased under Cd+EOA treatment, which was in accordance with expression pattern of LcAOC. The function of gene LcGR was confirmed in vitro with E. coli expression system. The subcellular localization in chloroplasts of LcGR gene was proved in Nicotiana tabacum leaves with transient transfection system of Agrobacterium tumefaciens. Furthermore, the overexpression of gene LcGR in the transgenic tabacum led to great Cd-tolerance and higher GSH accumulation. Overall, the results showed that the endogenous JA accumulation in Cd-stressed plants affects the GR expression which is crucial to the GSH accumulation and GSH-dependent tolerance to cadmium in LcGR transformants.

Beneficial effects of melatonin in overcoming drought stress in wheat seedlings

Melatonin plays an important role in abiotic stress in plant, but its role in wheat drought tolerance is less known. To verify its role, wheat seedlings (Triticum aestivum L. ‘Yan 995’) at 60% and 40% of field capacity were treated with 500 μM melatonin in this study. Melatonin treatment significantly enhanced the drought tolerance of wheat seedlings, as demonstrated by decreased membrane damage, more intact grana lamella of chloroplast, higher photosynthetic rate, and maximum efficiency of photosystem II, as well as higher cell turgor and water holding capacity in melatonin-treated seedlings. Besides, melatonin markedly decreased the content of hydrogen peroxide and superoxide anion in melatonin-treated seedlings, which is attributed to the increased total antioxidant capacity, GSH and AsA contents, as well as enzyme activity including ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione peroxidase (GPX), and glutathione transferase (GST). The GSH-AsA related genes including APX, MDHAR, and DHAR were commonly upregulated by melatonin and correlated to the antioxidant enzyme activity as well as the content of GSH and AsA, indicating that the increase of GSH and AsA was attributed to the expression of these genes. Our result confirmed the mitigation potential of melatonin in drought stress and certain mechanisms of melatonin-induced GSH and AsA accumulation, which could deepen our understanding of melatonin-induced drought tolerance in wheat.

Regional differences in glutathione accumulation pathways in the rat cornea: Mapping of amino acid transporters involved in glutathione synthesis

In this study we have sought to complete the identification and localisation of uptake pathways involved in accumulating precursor amino acids involved in GSH synthesis in the rat cornea. To do this, we performed reverse transcription PCR (RT-PCR) to identify the Excitatory Amino Acid Transporters (EAAT 1–5) responsible for glutamate uptake, and glycine transporters (GLYT 1–2) at the transcript level. Western blotting was used to verify protein expression, while immunolabelling of sagittal sections was used to localise transporters to the different layers of the cornea. Immunolabelling of en face sections was used to examine the subcellular distribution of proteins in the corneal endothelium. Our findings revealed EAAT 1–5 and GLYT 1–2 to be expressed at the transcript and protein level in the rat cornea. Immunohistochemistry revealed all amino acid transporters to be localised to the epithelium. In the majority of cases, labelling was restricted to the epithelium, and labelling absent from the stroma or endothelium. However, EAAT 4 and GLYT 2 labelling was detected in the stroma with EAAT 4 labelling also present in the endothelium. Overall, the identification of amino acid transporters strongly supports the existence of an intracellular GSH synthesis pathway in the rat corneal epithelium. This suggests that regional differences in GSH accumulation pathways exist, with direct uptake of GSH and intracellular synthesis of GSH restricted to the endothelial and epithelial cell layers, respectively. This information is important in the design of targeted strategies to enhance GSH levels in specific layers of the cornea to prevent against oxidative damage, corneal swelling and loss of corneal transparency.

Thioredoxin (Trxo1) interacts with proliferating cell nuclear antigen (PCNA) and its overexpression affects the growth of tobacco cell culture

Thioredoxins (Trxs), key components of cellular redox regulation, act by controlling the redox status of many target proteins, and have been shown to play an essential role in cell survival and growth. The presence of a Trx system in the nucleus has received little attention in plants, and the nuclear targets of plant Trxs have not been conclusively identified. Thus, very little is known about the function of Trxs in this cellular compartment. Previously, we studied the intracellular localization of PsTrxo1 and confirmed its presence in mitochondria and, interestingly, in the nucleus under standard growth conditions. In investigating the nuclear function of PsTrxo1 we identified proliferating cellular nuclear antigen (PCNA) as a PsTrxo1 target by means of affinity chromatography techniques using purified nuclei from pea leaves. Such protein–protein interaction was corroborated by dot-blot and bimolecular fluorescence complementation (BiFC) assays, which showed that both proteins interact in the nucleus. Moreover, PsTrxo1 showed disulfide reductase activity on previously oxidized recombinant PCNA protein. In parallel, we studied the effects of PsTrxo1 overexpression on Tobacco Bright Yellow-2 (TBY-2) cell cultures. Microscopy and flow-cytometry analysis showed that PsTrxo1 overexpression increases the rate of cell proliferation in the transformed lines, with a higher percentage of the S phase of the cell cycle at the beginning of the cell culture (days 1 and 3) and at the G2/M phase after longer times of culture (day 9), coinciding with an upregulation of PCNA protein. Furthermore, in PsTrxo1 overexpressed cells there is a decrease in the total cellular glutathione content but maintained nuclear GSH accumulation, especially at the end of the culture, which is accompanied by a higher mitotic index, unlike non-overexpressing cells. These results suggest that Trxo1 is involved in the cell cycle progression of TBY-2 cultures, possibly through its link with cellular PCNA and glutathione.