Induction of PR-1 accumulation accompanied by runaway cell death in the lsd1 mutant of Arabidopsis is dependent on glutathione levels but independent of the redox state of glutathione

The lesions simulating disease (lsd) mutants of Arabidopsis spontaneously develop hypersensitive-response-like lesions in the absence of pathogens. To address the function of the redox regulator glutathione in disease resistance, we examined the relationship between endogenous glutathione and PR-1 accumulation using one of these mutants, lsd1, as a disease resistance model. Lesion formation on lsd1 was suppressed by weak light and initiated by the subsequent transition to normal light. The application of buthionine sulfoximine, a specific inhibitor of glutathione biosynthesis, suppressed conditionally induced runaway cell death and expression of the PR-1 gene, suggesting that glutathione regulates the conditional cell death and PR-1 gene expression. The application of reduced (GSH) or oxidized (GSSG) glutathione to lsd1 upregulated the level of total glutathione ([GSH]+[GSSG]) accompanied by hastened accumulation of PR-1, and the basal level of total glutathione in lsd1 was higher than that in wild-type plants. The glutathione redox state defined as [GSH]/([GSH]+[GSSG]) decreased following the conditional transition, but the suppression of this decrease by the application of GSH did not inhibit the accumulation of PR-1. Taken together, conditional PR-1 accumulation in lsd1 is regulated not by the redox state but by the endogenous level of glutathione.     

Wheat Chloroplastic Glutathione Reductase Activity is Regulated by the Combined Effect of pH, NADPH and GSSG

To study the mechanism of regulation of chloroplastic glutathionereductase (GR) under photooxidative conditions, GR activity,and the levels of NADPH, GSH and GSSG were measured in wheatchloroplasts under photooxidative light. GR was extremely labile,and the concentrations of GSH and GSSG progressively diminishedin chloroplasts prepared without ascorbate. The NADPH leveldid not significantly change during photooxidative treatment.The addition of 10 mM ascorbate to the incubation medium preventedthe decrease of GSH and GSSG and strongly protected GR activity.However, ascorbate had no effect on NADPH-dependent inhibitionof the chloroplastic GR purified from wheat leaves. We studiedthe effect of NADPH, temperature, pH and GSSG on the purifiedenzyme. The inhibition by NADPH was greatly dependent on temperatureand pH. NADPH inhibited GR by around 93% up to pH 7.5, but withina range of 8.0 to 9.5 the inhibition was only marginal. ThepH dependence of the NADPH inhibitory effect could be due, atleast in part, to different rates in the generation of NADPH-X,a derivative of NADPH which inactivates several pyridin nucleotidedehydrogenases. Furthermore, the NADPH-dependent inhibitionwas almost completely prevented by GSSG, but not by GSH. (Received July 9, 1998; Accepted April 30, 1999)     

Disruption of a glutathione reductase encoding gene in Acremonium chrysogenum leads to reduction of its growth, cephalosporin production and antioxidative ability which is recovered by exogenous methionine

Glutathione is a ubiquitous thiol in eukaryotic cells, and its high intracellular ratio of reduced form (GSH) to oxidized form (GSSG) is largely maintained by glutathione reductase (GR) using NADPH as electron donor. glrA, a glutathione reductase encoding gene, was found and cloned from Acremonium chrysogenum by searching its genomic sequence based on similarity. Its deduced protein exhibits high similarity to GRs of other eukaryotic organisms. Disruption of glrA resulted in lack of GR activity and accumulation of a high level of GSSG in A. chrysogenum. Overexpression of glrA dramatically enhanced GR activity and the ratio of GSH/GSSG in this fungus. The spore germination and hyphal growth of glrA disruption mutant was strongly reduced in chemical defined medium. Meanwhile, the mutant was more sensitive to hydrogen peroxide than the wild-type strain. We found that the glrA mutant recovered normal germination and growth by adding exogenous methionine (Met). Exogenous Met also enhanced the antioxidative ability of both the mutant and wild-type strain. GSH determination indicated that the total GSH and ratio of GSH/GSSG in the mutant or wild-type strain were significantly increased when addition of Met into the medium. The glrA mutant grew poorly and could not produce detectable cephalosporin in the fermentation medium without Met. However, its growth and cephalosporin production was restored with addition of exogenous Met. These results indicate that glrA is required for the normal growth and protection against oxidative damage in A. chrysogenum, and its absence can be complemented by exogenous Met.     

Mechanism of RPE cell death in α-crystallin deficient mice: a novel and critical role for MRP1-mediated GSH efflux

Absence of α-crystallins (αA and αB) in retinal pigment epithelial (RPE) cells renders them susceptible to oxidant-induced cell death. We tested the hypothesis that the protective effect of α-crystallin is mediated by changes in cellular glutathione (GSH) and elucidated the mechanism of GSH efflux. In α-crystallin overexpressing cells resistant to cell death, cellular GSH was >2 fold higher than vector control cells and this increase was seen particularly in mitochondria. The high GSH levels associated with α-crystallin overexpression were due to increased GSH biosynthesis. On the other hand, cellular GSH was decreased by 50% in murine retina lacking αA or αB crystallin. Multiple multidrug resistance protein (MRP) family isoforms were expressed in RPE, among which MRP1 was the most abundant. MRP1 was localized to the plasma membrane and inhibition of MRP1 markedly decreased GSH efflux. MRP1-suppressed cells were resistant to cell death and contained elevated intracellular GSH and GSSG. Increased GSH in MRP1-supressed cells resulted from a higher conversion of GSSG to GSH by glutathione reductase. In contrast, GSH efflux was significantly higher in MRP1 overexpressing RPE cells which also contained lower levels of cellular GSH and GSSG. Oxidative stress further increased GSH efflux with a decrease in cellular GSH and rendered cells apoptosis-prone. In conclusion, our data reveal for the first time that 1) MRP1 mediates GSH and GSSG efflux in RPE cells; 2) MRP1 inhibition renders RPE cells resistant to oxidative stress-induced cell death while MRP1 overexpression makes them susceptible and 3) the antiapoptotic function of α-crystallin in oxidatively stressed cells is mediated in part by GSH and MRP1. Our findings suggest that MRP1 and α crystallin are potential therapeutic targets in pathological retinal degenerative disorders linked to oxidative stress.     

Effects of glutathione reductase inhibition on cellular thiol redox state and related systems

Although inhibition of glutathione reductase (GR) has been demonstrated to cause a decrease in reduced glutathione (GSH) and increase in glutathione disulfide (GSSG), a systematic study of the effects of GR inhibition on thiol redox state and related systems has not been noted. By employing a monkey kidney cell line as the cell model and 2-acetylamino-3-[4-(2-acetylamino-2-carboxy-ethylsulfanylthio carbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as a GR inhibitor, an investigation of the effects of GR inhibition on cellular thiol redox state and related systems was conducted. Our study demonstrated that, in addition to a decrease in GSH and increase in GSSG, 2-AAPA increased the ratios of NADH/NAD⁺ and NADPH/NADP⁺. Significant protein glutathionylation was observed. However, the inhibition did not affect the formation of reactive oxygen species or expression of antioxidant defense enzyme systems [GR, glutathione peroxidase, catalase, and superoxide dismutase] and enzymes involved in GSH biosynthesis [γ-glutamylcysteine synthetase and glutathione synthetase].     

Glutathione reductase plays an anti-apoptotic role against oxidative stress in human hepatoma cells

The cellular roles of glutathione reductase (GR) in the reactive oxygen species (ROS)-induced apoptosis were studied using the HepG2 cells transfected with GR. The overexpression of GR caused a marked enhancement in reduced and oxidized glutathione (GSH/GSSG) ratio, and significantly decreased ROS levels in the stable transfectants. Hydrogen peroxide (H₂O₂), under the optimal condition for apoptosis, significantly decreased cellular viability and total GSH content, and rather increased ROS level, apoptotic percentage and caspase-3 activity in the mock-transfected cells. However, hydrogen peroxide could not largely generate these apoptotic changes in cellular viability, ROS level, apoptotic percentage, caspase-3 activity and total GSH content in the cells overexpressing GR. Taken together, GR may play a protective role against oxidative stress.     

Increased recovery of brain acetylcholinesterase activity in dichlorvos-intoxicated European eels Anguilla anguilla by bath treatment with N-acetylcysteine

Organophosphate (OP) pesticides are widely used as antiparasitic chemicals in finfish aquaculture. However, current antidotes cannot be applied to treat intoxicated fish. We showed in previous studies the importance of glutathione (GSH) metabolism in pesticide resistance of the European eel Anguilla anguilla L. The present work studied the effects of the antioxidant and glutathione pro-drug N-acetyl-L-cysteine (NAC) on the recovery of European eels exposed for 96 h to a sublethal concentration (0.17 mg l(-1); 20% of its 96 h LC50) of the OP pesticide dichlorvos (2,2-dichlorovinyl dimethyl phosphate; DDVP). This insecticide and acaricide decreased muscular GSH content and increased oxidised glutathione (GSSG), lowering the GSH:GSSG ratio, which is indicative of a condition of oxidative stress. Acetylcholinesterase (AChE) and glutathione reductase (GR) activities in the brain, which were biomarkers of neurotoxicity and oxidative stress, respectively, were also highly inhibited. Recovery in a 0.5 mM (81.6 mg l(-1)) NAC concentration ameliorated muscular GSH depletion, GSH:GSSG ratio, and the inhibition of brain AChE and GR activities. Hence, this is the first evidence of improved recovery of organophosphate-poisoned fish by bath treatments.     

Inhibition of glutathione synthesis reduces chilling tolerance in maize

 The role of glutathione (GSH) in protecting plants from chilling injury was analyzed in seedlings of a chilling-tolerant maize (Zea mays L.) genotype using buthionine sulfoximine (BSO), a specific inhibitor of γ-glutamylcysteine (γEC) synthetase, the first enzyme of GSH synthesis. At 25 °C, 1 mM BSO significantly increased cysteine and reduced GSH content and GSH reductase (GR: EC 1.6.4.2) activity, but interestingly affected neither fresh weight nor dry weight nor relative injury. Application of BSO up to 1 mM during chilling at 5 °C reduced the fresh and dry weights of shoots and roots and increased relative injury from 10 to almost 40%. Buthionine sulfoximine also induced a decrease in GR activity of 90 and 40% in roots and shoots, respectively. Addition of GSH or γEC together with BSO to the nutrient solution protected the seedlings from the BSO effect by increasing the levels of GSH and GR activity in roots and shoots. During chilling, the level of abscisic acid increased both in controls and BSO-treated seedlings and decreased after chilling in roots and shoots of the controls and in the roots of BSO-treated seedlings, but increased in their shoots. Taken together, our results show that BSO did not reduce chilling tolerance of the maize genotype analyzed by inhibiting abscisic acid accumulation but by establishing a low level of GSH, which also induced a decrease in GR activity. Received: 9 November 1999 / Accepted: 17 February 2000     

Glutathione metabolism in soybean callus-cultures as affected by salinity

Induction and growth of soybean callus cultures were influenced by NaCl, especially at the highest concentration tested (150 mM). Protein content was raised as NaCl was increased in the Murashige and Skoog medium. Total sulfhydryl group (-SH) and glutathione (GSH) concentrations were also increased in NaCl treated cultures. The affinity (Km) of glutathione reductase (GR) for oxidized glutathione (GSSG) was gradually increased as NaCl level was raised in the medium. The GSH/GSSG ratio was raised significantly as the result of GR activity. The increase in GR activity may constitute an adaptive response of soybean callus to NaCl. This revised version was published online in July 2006 with corrections to the Cover Date.     

High level of reduced glutathione contributes to detoxification of lipid peroxide‐derived reactive carbonyl species in transgenic Arabidopsis overexpressing glutathione reductase under aluminum stress

Lipid peroxide‐derived reactive carbonyl species (RCS), generated downstream of reactive oxygen species (ROS), are critical damage‐inducing species in plant aluminum (Al) toxicity. In mammals, RCS are scavenged primarily by glutathione (reduced form of glutathione, GSH), but in plant Al stress, contribution of GSH to RCS detoxification has not been evaluated. In this study, Arabidopsis plants overexpressing the gene AtGR1 (accession code At3g24170), encoding glutathione reductase (GR), were generated, and their performance under Al stress was examined. These transgenic plants (GR‐OE plants) showed higher GSH levels and GSH/GSSG (oxidized form of GSH) ratio, and an improved Al tolerance as they suffered less inhibition of root growth than wild‐type under Al stress. Exogenous application of 4‐hydroxy‐2‐nonenal, an RCS responsible for Al toxicity in roots, markedly inhibited root growth in wild‐type plants. GR‐OE plants suffered significantly smaller inhibition, indicating that the enhanced GSH level increased the capacity of RCS detoxification. The generation of H₂O₂ due to Al stress in GR‐OE plants was lower by 26% than in wild‐type. Levels of various RCS, such as malondialdehyde, butyraldehyde, phenylacetaldehyde, (E)‐2‐heptenal and n‐octanal, were suppressed by more than 50%. These results indicate that high levels of GSH and GSH/GSSG ratio by GR overexpression contributed to the suppression of not only ROS, but also RCS. Thus, the maintenance of GSH level by overexpressing GR reinforces dual detoxification functions in plants and is an efficient approach to enhance Al tolerance.     

Preliminary studies on the involvement of glutathione metabolism and redox status against zinc toxicity in radish seedlings by 28-Homobrassinolide

The effect of exogenous application of 28-Homobrassinolide (HBR) on radish (Raphanus sativus L.) seedlings under zinc (Zn²⁺) stress on glutathione (GSH) production, consumption and changes in redox status was investigated. Zinc toxicity resulted in oxidative burst as evidenced by increased accumulation of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) content. These stress indices were significantly decreased by HBR supplementation. Under Zn²⁺ stress, GSH pool was decreased, while the contribution of oxidized glutathione (GSSG) to total GSH increased (GSSH/GSH ratio), this translated into significant reduction of GSH redox homeostasis. In addition, an increase of phytochelatins (PCs) was observed. In radish seedlings under Zn²⁺ stress, the activities of gamma-glutamylcysteine synthetase (γ-ECS), glutathione synthetase (GS), glutathione peroxidase (GPX), glutathione-S-transferase (GST) and cysteine (Cys) levels increased but the activity of glutathione reductase (GR) decreased. However, application of HBR increased the GSH pool and maintained their redox ratio by increasing the enzyme activities of GSH biosynthesis (γ-ECS and GS) and GSH metabolism (GR, GPX and GST). The results of present study are novel in being the first to demonstrate that exogenous application of HBR modulates the GSH synthesis, metabolism and redox homeostasis to confer resistance against Zn²⁺ induced oxidative stress.     

Genetic study of glutathione accumulation during cold hardening in wheat

The effect of cold hardening on the accumulation of glutathione (GSH) and its precursors was studied in the shoots and roots of wheat (Triticum aestivum L.) cv. Cheyenne (Ch, frost-tolerant) and cv. Chinese Spring (CS, moderately frost-sensitive), in a T. spelta L. accession (Tsp, frost-sensitive) and in chro- mosome substitution lines CS (Ch 5A) and CS (Tsp 5A). The fast induction of total glutathione accumulation was detected during the first 3 d of hardening in the shoots, especially in the frost-tolerant Ch and CS (Ch 5A). This observation was corroborated by the study of de novo GSH synthesis using [³⁵S]sulfate. In Ch and CS (Ch 5A) the total cysteine, γ-glutamylcysteine (precursors of GSH), hydroxymethylglutathione and GSH contents were greater during the 51-d treatment than in the sensitive genotypes. After 35 d hardening, when the maximum frost tolerance was observed, greater ratios of reduced to oxidised hydroxymethylglutathione and glutathione were detected in Ch and CS (Ch 5A) compared to the sensitive genotypes. A correspondingly greater glutathione reductase (EC 1.6.4.2) activity was also found in Ch and CS (Ch 5A). It can be assumed that chromosome 5A of wheat has an influence on GSH accumulation and on the ratio of reduced to oxidised glutathione as part of a complex regulatory function during hardening. Consequently, GSH may contribute to the enhancement of frost tolerance in wheat. Received: 24 March 1999 / Accepted: 19 July 1999     

Parameters related to oxygen free radicals in erythrocytes,plasma and epidermis of the hairless rat

The following parameters related to oxygen free radicals (OFR) were determined in erythrocytes and the epidermis of hairless rats: catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), reduced (GSH) and oxidized (GSSG) glutathione, glutathione S-transferase (GST), superoxide dismutase (SOD) and thiobarbituric acid reactive substances (TBARS). GSH, GSSG and TBARS were also analyzed in plasma. In erythrocytes, the Pearson correlation coefficients (r) were significant (p < 0.001) between glutathione and other parameters as follows: GSH correlated negatively with GSSG (r = -0.665) and TBARS (r = -0.669); GSSG correlated positively with SOD (r = 0.709) and TBARS (r = 0.752). Plasma GSSG correlated negatively with erythrocytic thermostable GST activity (r = -0.608; p=0.001) and with erythrocytic total GST activity (r = -0.677; p < 0.001). In epidermis (p < 0.001 in all cases), GSH content correlated with GSSG (r = 0.682) and with GPx (r = 0.663); GSSG correlated with GPx (r = 0.731) and with GR (r = 0.794). By multiple linear regression analysis some predictor variables (R(2)) were found: in erythrocytes, thermostable GST was predicted by total GST activity and GSSG, GSSG content was predicted by GSH and by the GSH/GSSG ratio and GPx activity was predicted by GST, CAT and SOD activities; in epidermis, GSSG was predicted by GR and SOD activities and GR was predicted by GSSG, TBARS and GPx. It is concluded that the hairless rat is a good model for studying OFR-related parameters simultaneously in blood and skin, and that it may provide valuable information about other animals under oxidative stress.     

Manipulation of Glutathione and Amino Acid Biosynthesis in the Chloroplast

Poplars (Populus tremula × Populus alba) were transformed to overexpress Escherichia coli γ-glutamylcysteine synthetase (γ-ECS) or glutathione synthetase in the chloroplast. Five independent lines of each transformant strongly expressed the introduced gene and possessed markedly enhanced activity of the gene product. Glutathione (GSH) contents were unaffected by high chloroplastic glutathione synthetase activity. Enhanced chloroplastic γ-ECS activity markedly increased γ-glutamylcysteine and GSH levels. These effects are similar to those previously observed in poplars overexpressing these enzymes in the cytosol. Similar to cytosolic γ-ECS overexpression, chloroplastic overexpression did not deplete foliar cysteine or methionine pools and did not lead to morphological changes. Light was required for maximal accumulation of GSH in poplars overexpressing γ-ECS in the chloroplast. High chloroplastic, but not cytosolic, γ-ECS activities were accompanied by increases in amino acids synthesized in the chloroplast. We conclude that (a) GSH synthesis can occur in the chloroplast and the cytosol and may be up-regulated in both compartments by increased γ-ECS activity, (b) interactions between GSH synthesis and the pathways supplying the necessary substrates are similar in both compartments, and (c) chloroplastic up-regulation of GSH synthesis is associated with an activating effect on the synthesis of specific amino acids formed in the chloroplast.