The Effects of Methylglyoxal on Glutathione S-Transferase from Nicotiana tabacum

Methylglyoxal (MG) is one of the aldehydes that accumulate in plants under environmental stress. Glutathione S-transferases (GSTs) play important roles, including detoxification, in the stress tolerance systems of plants. To determine the effects of MG, we characterized recombinant GST. MG decreased GST activity and thiol contents with increasing K _m. GST can serve as a target of MG modification, which is suppressed by application of reduced glutathione.     

Nitric oxide modulates antioxidant defense and the methylglyoxal detoxification system and reduces salinity-induced damage of wheat seedlings

The present study investigates the possible regulatory role of exogenous nitric oxide (NO) in antioxidant defense and methylglyoxal (MG) detoxification systems of wheat seedlings exposed to salt stress (150 and 300 mM NaCl, 4 days). Seedlings were pre-treated for 24 h with 1 mM sodium nitroprusside, a NO donor, and then subjected to salt stress. The ascorbate (AsA) content decreased significantly with increased salt stress. The amount of reduced glutathione (GSH) and glutathione disulfide (GSSG) and the GSH/GSSG ratio increased with an increase in the level of salt stress. The glutathione S-transferase (GST) activity increased significantly with severe salt stress (300 mM). The ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT) and glutathione peroxidase (GPX) activities did not show significant changes in response to salt stress. The glutathione reductase (GR), glyoxalase I (Gly I), and glyoxalase II (Gly II) activities decreased upon the imposition of salt stress, especially at 300 mM NaCl, with a concomitant increase in the H₂O₂ and lipid peroxidation levels. Exogenous NO pre-treatment of the seedlings had little influence on the non-enzymatic and enzymatic components compared to the seedlings of the untreated control. Further investigation revealed that NO pre-treatment had a synergistic effect; that is, the pre-treatment increased the AsA and GSH content and the GSH/GSSG ratio, as well as the activities of MDHAR, DHAR, GR, GST, GPX, Gly I, and Gly II in most of the seedlings subjected to salt stress. These results suggest that the exogenous application of NO rendered the plants more tolerant to salinity-induced oxidative damage by enhancing their antioxidant defense and MG detoxification systems.     

Methylglyoxal levels in plants under salinity stress are dependent on glyoxalase I and glutathione

Methylglyoxal (MG), a cytotoxic by-product produced mainly from triose phosphates, is used as a substrate by glyoxalase I. In this paper, we report on the estimation of MG level in plants which has not been reported earlier. We show that MG concentration varies in the range of 30-75 microM in various plant species and it increases 2- to 6-fold in response to salinity, drought, and cold stress conditions. Transgenic tobacco underexpressing glyoxalase I showed enhanced accumulation of MG which resulted in the inhibition of seed germination. In the glyoxalase I overexpressing transgenic tobacco, MG levels did not increase in response to stress compared to the untransformed plants, however, with the addition of exogenous GSH there was a decrease in MG levels in both untransformed and transgenic plants. The exogenous application of GSH reduced MG levels in WT to 50% whereas in the transgenic plants a 5-fold decrease was observed. These studies demonstrate an important role of glyoxalase I along with GSH concentration in maintaining MG levels in plants under normal and abiotic stress conditions.     

Transgenic tobacco plants overexpressing glyoxalase enzymes resist an increase in methylglyoxal and maintain higher reduced glutathione levels under salinity stress

The mechanism behind enhanced salt tolerance conferred by the overexpression of glyoxalase pathway enzymes was studied in transgenic vis-à-vis wild-type (WT) plants. We have recently documented that salinity stress induces higher level accumulation of methylglyoxal (MG), a potent cytotoxin and primary substrate for glyoxalase pathway, in various plant species [Yadav, S.K., Singla-Pareek, S.L., Ray, M., Reddy, M.K. and Sopory, S.K. (2005) MG levels in plants under salinity stress are dependent on glyoxalase I and glutathione. Biochem. Biophys. Res. Commun. 337, 61-67]. The transgenic tobacco plants overexpressing glyoxalase pathway enzymes, resist an increase in the level of MG that increased to over 70% in WT plants under salinity stress. These plants showed enhanced basal activity of various glutathione related antioxidative enzymes that increased further upon salinity stress. These plants suffered minimal salinity stress induced oxidative damage measured in terms of the lipid peroxidation. The reduced glutathione (GSH) content was high in these transgenic plants and also maintained a higher reduced to oxidized glutathione (GSH:GSSG) ratio under salinity. Manipulation of glutathione ratio by exogenous application of GSSG retarded the growth of non-transgenic plants whereas transgenic plants sustained their growth. These results suggest that resisting an increase in MG together with maintaining higher reduced glutathione levels can be efficiently achieved by the overexpression of glyoxalase pathway enzymes towards developing salinity stress tolerant plants.     

Engineering stress tolerance in transgenic plants

Research in our laboratory has focused on the analysis of the functions of a variety of enzymes that are involved in the scavenging of reactive oxygen intermediates (ROI) such as superoxide radicals (·O ₂ ⁻ ) and hydrogen peroxide (H₂O₂). Recent work has been on transgenic plants that over-express glutathione S-transferases (GST) that also have glutathione peroxidase activity. Transgenic tobacco plants that contain gene constructs that encode two different tobacco GST’s had elevated levels of both GST and GPX activity. Analysis of mature vegetative transgenic tobacco plants that over-express GST/GPX failed to show any increase in paraquat tolerance or protection from photooxidative stress. However, seeds of these GST/GPX-expressing tobacco lines are capable of more rapid germination and seedling growth at low temperatures and at elevated salt concentrations. Reduced levels of lipid peroxidation were noted in GST/GPX-expressing seedling compared to control seedlings under both stressful and non-stressful conditions. In addition, GST/GPX-expressing seedlings significantly accumulated more oxidized glutathione (GSSG) than control seedlings during stress. These characteristics clearly indicate that over-expression of GST/GPX in transgenic seedlings can have substantial effects on their stress tolerance. Furthermore, it appears that this effect is due primarily to the elevated levels of GPX activity.     

Overexpression of a GST gene (ThGSTZ1) from Tamarix hispida improves drought and salinity tolerance by enhancing the ability to scavenge reactive oxygen species

Although plant glutathione transferase (GST) genes are reported to be involved in responses to abiotic stress, few GST genes have been functionally characterized in woody halophytes. In the present study, a GST gene from Tamarix hispida, designated ThGSTZ1, was cloned and functionally characterized. Expression of ThGSTZ1 was downregulated by drought and salinity stress, and abscisic acid. Transgenic Arabidopsis thaliana plants with constitutive expression of ThGSTZ1 showed increased survival rates under drought and salinity stress. These transgenic Arabidopsis plants exhibited increased levels of GST, glutathione peroxidase, superoxide dismutase and peroxidase activity, along with decreased malondialdehyde content, electrolyte leakage rates and reactive oxygen species (ROS) levels under salt and drought stress conditions. Transgenic T. hispida that transiently overexpressed ThGSTZ1 showed increased GST and GPX activities under NaCl and mannitol treatments, as well as improved ROS scavenging ability. These results suggest that ThGSTZ1 can improve drought and salinity tolerance in plants by enhancing their ROS scavenging ability. Therefore, ThGSTZ1 represents a candidate gene with potential applications for molecular breeding to increase stress tolerance in plants.     

Methylglyoxal detoxification by a DJ-1 family protein provides dual abiotic and biotic stress tolerance in transgenic plants

Methylglyoxal (MG) is a key signaling molecule resulting from glycolysis and other metabolic pathways. During abiotic stress, MG levels accumulate to toxic levels in affected cells. However, MG is routinely detoxified through the action of DJ1/PARK7/Hsp31 proteins that are highly conserved across kingdoms and mutations in such genes are associated with neurodegenerative diseases. Here, we report for the first time that, similar to abiotic stresses, MG levels increase during biotic stresses in plants, likely contributing to enhanced susceptibility to a wide range of stresses. We show that overexpression of yeast Heat shock protein 31 (Hsp31), a DJ-1 homolog with robust MG detoxifying capabilities, confers dual biotic and abiotic stress tolerance in model plant Nicotiana tabacum. Strikingly, overexpression of Hsp31 in tobacco imparts robust stress tolerance against diverse biotic stress inducers such as viruses, bacteria and fungi, in addition to tolerance against a range of abiotic stress inducers. During stress, Hsp31 was targeted to mitochondria and induced expression of key stress-related genes. These results indicate that Hsp31 is a novel attractive tool to engineer plants against both biotic and abiotic stresses.     

外源甲基乙二醛对干旱胁迫下板栗幼苗的影响

甲基乙二醛(MG)是一种在植物中具有多种功能的新型信号分子.为探究MG对板栗幼苗干旱胁迫的影响,以两年生'黄棚'板栗幼苗为试材,通过聚乙二醇(PEG)模拟干旱胁迫并进行MG及其清除剂N-乙酰半胱氨酸(NAC)处理,分析板栗幼苗叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(...     

紫外线强烈诱导的谷胱甘肽转移酶基因的功能鉴定

植物谷胱甘肽转移酶（glutathione S-transferases,GSTs)基因家族在逆境反应和植物生长发育过程中都起着非常重要的作用。为了阐明GST在紫外辐射下是否对植物有保护作用,以紫外强烈诱导表达的GST、cDNA为探针,筛选拟南芥cDNA文库,获得了这种GST的全长cDNA;利用此cDNA构建植物表达载体,并通过农杆菌介导法转化拟南芬,使其在拟南芥中得到大量表达;通过对转基因植株的紫外辐射耐性分析,证实了该GST的过量表达可明显增强拟南芥对紫外辐射损伤作用的耐受性。     

Selenium-Induced Up-Regulation of the Antioxidant Defense and Methylglyoxal Detoxification System Reduces Salinity-Induced Damage in Rapeseed Seedlings

The present study investigates the regulatory role of exogenous selenium (Se) in the antioxidant defense and methylglyoxal (MG) detoxification systems in rapeseed seedlings exposed to salt stress. Twelve-day-old seedlings, grown in Petri dishes, were supplemented with selenium (25 μM Na₂SeO₄) and salt (100 and 200 mM NaCl) separately and in combination, and further grown for 48 h. The ascorbate (AsA) content of the seedlings decreased significantly with increased salt stress. The amount of reduced glutathione (GSH) and glutathione disulfide (GSSG) increased with an increase in the level of salt stress, while the GSH/GSSG ratio decreased. In addition, the ascorbate peroxidase (APX) and glutathione S-transferase (GST) activity increased significantly with increased salt concentration (both at 100 and 200 mM NaCl), while glutathione peroxidase (GPX) activity increased only at moderate salt stress (100 mM NaCl). Glutathione reductase (GR) activity remained unchanged at 100 mM NaCl, while it was decreased under severe (200 mM NaCl) salt stress. Monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT), glyoxalase I (Gly I), and glyoxalase II (Gly II) activities decreased upon the imposition of salt stress, whereas a sharp decrease of these activities was observed under severe salt stress (200 mM NaCl). Concomitant increases in the levels of H₂O₂ and lipid peroxidation (MDA) were also measured. Exogenous Se treatment alone had little effect on the non-enzymatic and enzymatic components. However, further investigation revealed that Se treatment had a synergistic effect: in salt-stressed seedlings, it increased the AsA and GSH contents; GSH/GSSG ratio; and the activities of APX, MDHAR, DHAR, GR, GST, GPX, CAT, Gly I, and Gly II. As a result, addition of Se in salt-stressed seedlings led to a reduction in the levels of H₂O₂ and MDA as compared to salt stress alone. These results suggest that the exogenous application of Se rendered the plants more tolerant to salt stress-induced oxidative damage by enhancing their antioxidant defense and MG detoxification systems.     

转入盐地碱蓬谷胱甘肽转移酶和过氧化氢酶基因增强水稻幼苗对低温胁迫的抗性

以水稻（Oryza sativa L.）品种中花11号成熟种子为材料,利用农杆菌介导法将盐地碱蓬的GST（谷胱甘肽转移酶）单基因和GST＋CAT1（catalase 1）双基因转入低温敏感水稻品种中花11号,并对T4代转基因水稻幼苗的抗低温特性进行了分析。结果显示,低温处理后,转基因植株的GST和CAT活性都比未转入这两种基因的对照高;且PSⅡ最大光化学效率也高于非转基因对照;而H2O2和MDA（malondialdehyde）含量及细胞膜透性则低于对照。说明转基因水稻幼苗GST和GST＋CAT1的表达提高了对低温胁迫的抗性。     

The metabolite methylglyoxal-mediated gene expression is associated with histone methylglyoxalation

Methylglyoxal (MG) is a byproduct of glycolysis that functions in diverse mammalian developmental processes and diseases and in plant responses to various stresses, including salt stress. However, it is unknown whether MG-regulated gene expression is associated with an epigenetic modification. Here we report that MG methylglyoxalates H3 including H3K4 and increases chromatin accessibility, consistent with the result that H3 methylglyoxalation positively correlates with gene expression. Salt stress also increases H3 methylglyoxalation at salt stress responsive genes correlated to their higher expression. Following exposure to salt stress, salt stress responsive genes were expressed at higher levels in the Arabidopsis glyI2 mutant than in wild-type plants, but at lower levels in 35S::GLYI2 35S::GLYII4 plants, consistent with the higher and lower MG accumulation and H3 methylglyoxalation of target genes in glyI2 and 35S::GLYI2 35S::GLYII4, respectively. Further, ABI3 and MYC2, regulators of salt stress responsive genes, affect the distribution of H3 methylglyoxalation at salt stress responsive genes. Thus, MG functions as a histone-modifying group associated with gene expression that links glucose metabolism and epigenetic regulation.     

Tobacco chloroplast transformants expressing genes encoding dehydroascorbate reductase, glutathione reductase, and glutathione-S-transferase, exhibit altered anti-oxidant metabolism and improved abiotic stress tolerance

One approach to understanding the Reactive Oxygen Species (ROS)-scavenging systems in plant stress tolerance is to manipulate the levels of antioxidant enzyme activities. In this study, we expressed in the chloroplast three such enzymes: dehydroascorbate reductase (DHAR), glutathione-S-transferase (GST) and glutathione reductase (GR). Homoplasmic chloroplast transformants containing either DHAR or GST, or a combination of DHAR:GR and GST:GR were generated and confirmed by molecular analysis. They exhibited the predicted changes in enzyme activities, and levels or redox state of ascorbate and glutathione. Progeny of these plants were then subjected to environmental stresses including methyl viologen (MV)-induced oxidative stress, salt, cold and heavy metal stresses. Overexpression of these different enzymes enhanced salt and cold tolerance. The simultaneous expression of DHAR:GR and GST:GR conferred MV tolerance while expression of either transgene on its own didn't. This study provides evidence that increasing part of the antioxidant pathway within the chloroplast enhances the plant's ability to tolerate abiotic stress.     

Arsenic tolerance in mesquite (Prosopis sp.): Low molecular weight thiols synthesis and glutathione activity in response to arsenic

The effects of arsenic stress on the production of low molecular weight thiols (LMWT), glutathione S-transferase activity (GST) and sulfur metabolism of mesquite plant (Prosopis sp.) were examined in hydroponic culture at different arsenic [As(III) and (V)] concentrations. The production of LMWT was dependent on As speciation and concentration in the growth medium. The roots of As(III) treated plants produced significantly higher LMWT levels than As(V) treated roots at the same concentration of As applied. In leaves, the thiols content increased with increasing As(III) and (V) concentrations in the medium. Hypersensitivity of the plant to high As concentrations was observed by a significant decrease of LMWT produced in the roots at 50 mg/L treatment in both As(III) and (V) treatments. Sulfur was translocated from roots and accumulated mainly in the shoots. In response to As-induced phytotoxicity, the plants slightly increased the sulfur content in the roots at the highest As treatment. Compared with As(V)-treated plants, As(III)-treated roots and leaves showed significantly higher GST activity. The roots of both As(III) and (V) treated plants showed an initial increase in GST at low As concentration (5 mg/L), followed by significant inhibition up to 50 mg/L. The leaves had the highest GST activity, an indication of the ability of the plant to detoxify As in the leaves than in the roots. The correlation between LMWT content, S content and GST activity may be an indication these parameters may be used as biomarkers of As stress in mesquite.     

Transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> Plants Expressing Grape Glutathione S-Transferase Gene (<Emphasis Type="Italic">VvGSTF13</Emphasis>) Show Enhanced Tolerance to Abiotic Stress

Although glutathione S-transferase (GST, EC 2.5.1.18) is thought to play important roles in abiotic stress, limited information is available regarding the function of its gene in grapes. In this study, a GST gene from grape, VvGSTF13, was cloned and functionally characterized. Transgenic Arabidopsis plants containing this gene were normal in terms of growth and maturity compared with control plants but had enhanced resistance to salt, drought, and methyl viologen stress. The increased tolerance of the transgenic plants correlated with changes in activities of antioxidative enzymes. Our results indicate that the gene from grape plays a positive role in improving tolerance to salinity, drought, and methyl viologen stresses in Arabidopsis.     

Over-expressing GsGST14 from Glycine soja enhances alkaline tolerance of transgenic Medicago sativa

Glutathione-S-transferases (GSTs) are ubiquitous enzymes that play a key role in stress tolerance and cellular detoxification. The GST gene GsGST14 selected from the gene expression profiles of Glycine soja under alkaline stress was transformed into alfalfa (Medicago sativa L.). Transgenic alfalfa plants showed 1.73–1.99 times higher GST activity than wild-type plants. Transgenic alfalfa grew well in the presence of 100 mM NaHCO₃, while wild-type plants exhibited chlorosis and stunted growth, even death. There were marked changes in malondialdehyde content and relative membrane permeability caused by alkaline stress in non-transgenic lines compared to transgenic lines. The results indicate that the gene GsGST14 could enhance alkaline resistance in transgenic alfalfa.