Drought and salt stress tolerance of an Arabidopsis glutathione S-transferase U17 knockout mutant are attributed to the combined effect of glutathione and abscisic acid

Although glutathione S-transferases (GSTs) are thought to play major roles in oxidative stress metabolism, little is known about the regulatory functions of GSTs. We have reported that Arabidopsis (Arabidopsis thaliana) GLUTATHIONE S-TRANSFERASE U17 (AtGSTU17; At1g10370) participates in light signaling and might modulate various aspects of development by affecting glutathione (GSH) pools via a coordinated regulation with phytochrome A. Here, we provide further evidence to support a negative role of AtGSTU17 in drought and salt stress tolerance. When AtGSTU17 was mutated, plants were more tolerant to drought and salt stresses compared with wild-type plants. In addition, atgstu17 accumulated higher levels of GSH and abscisic acid (ABA) and exhibited hyposensitivity to ABA during seed germination, smaller stomatal apertures, a lower transpiration rate, better development of primary and lateral root systems, and longer vegetative growth. To explore how atgstu17 accumulated higher ABA content, we grew wild-type plants in the solution containing GSH and found that they accumulated ABA to a higher extent than plants grown in the absence of GSH, and they also exhibited the atgstu17 phenotypes. Wild-type plants treated with GSH also demonstrated more tolerance to drought and salt stresses. Furthermore, the effect of GSH on root patterning and drought tolerance was confirmed by growing the atgstu17 in solution containing l-buthionine-(S,R)-sulfoximine, a specific inhibitor of GSH biosynthesis. In conclusion, the atgstu17 phenotype can be explained by the combined effect of GSH and ABA. We propose a role of AtGSTU17 in adaptive responses to drought and salt stresses by functioning as a negative component of stress-mediated signal transduction pathways.     

Organ-specific expression of glutathione S-transferases and the efficacy of herbicide safeners in Arabidopsis

The functions of plant glutathione S-transferases (GSTs) under normal growth conditions are poorly understood, but their activity as detoxification enzymes has been harnessed in agriculture for selective weed control. Herbicide safeners protect monocot crops from herbicide injury but have little effect on weedy monocot or dicot species. Protection by safeners is associated with expression of herbicide-metabolizing enzymes including GSTs, but the basis for selective action of safeners between monocots and dicots is not known. To address this question we have studied the response of Arabidopsis (Arabidopsis thaliana) to various safeners. Benoxacor, fenclorim, and fluxofenim did not protect Arabidopsis from herbicide injury but did induce RNA expression of the glutathione-conjugate transporters encoded by AtMRP1, AtMRP2, AtMRP3, and AtMRP4. These safeners also induced the organ-specific expression of AtGSTU19 and AtGSTF2, two previously characterized Arabidopsis GSTs from different classes of this enzyme family. RNA hybridization, immunoblot, and reporter gene analyses indicated expression of AtGSTU19 induced by safeners predominated in roots. To test the hypothesis that increased expression of AtGSTU19 would be sufficient to provide tolerance to chloroacetamide herbicides, a chimeric gene was produced containing the open reading frame for this GST driven by a constitutive promoter. Plants containing this transgene had a modest increase in AtGSTU19 protein, predominantly in roots, but this had no effect on tolerance to chloroacetamide herbicides. The localized induction of GSTs by safeners in roots of Arabidopsis may explain why these compounds are unable to provide herbicide tolerance to dicot plant species.     

Induction of glutathione S-transferases in Arabidopsis by herbicide safeners

Herbicide safeners increase herbicide tolerance in cereals but not in dicotyledenous crops. The reason(s) for this difference in safening is unknown. However, safener-induced protection in cereals is associated with increased expression of herbicide detoxifying enzymes, including glutathione S-transferases (GSTs). Treatment of Arabidopsis seedlings growing in liquid medium with various safeners similarly resulted in enhanced GST activities toward a range of xenobiotics with benoxacor, fenclorim, and fluxofenim being the most effective. Safeners also increased the tripeptide glutathione content of Arabidopsis seedlings. However, treatment of Arabidopsis plants with safeners had no effect on the tolerance of seedlings to chloroacetanilide herbicides. Each safener produced a distinct profile of enhanced GST activity toward different substrates suggesting a differential induction of distinct isoenzymes. This was confirmed by analysis of affinity-purified GST subunits by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. AtGSTU19, a tau class GST, was identified as a dominant polypeptide in all samples. When AtGSTU19 was expressed in Escherichia coli, the recombinant enzyme was highly active toward 1-chloro-2,4-dinitrobenzene, as well as chloroacetanilide herbicides. Immunoblot analysis confirmed that AtGSTU19 was induced in response to several safeners. Differential induction of tau GSTs, as well as members of the phi and theta classes by safeners, was demonstrated by RNA-blot analysis. These results indicate that, although Arabidopsis may not be protected from herbicide injury by safeners, at least one component of their detoxification systems is responsive to these compounds.     

Engineering plants for the phytoremediation of RDX in the presence of the co-contaminating explosive TNT

The explosive compounds hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6-trinitrotoluene (TNT) are widespread environmental contaminants commonly found as co-pollutants on military training ranges. TNT is a toxic carcinogen which remains tightly bound to the soil, whereas RDX is highly mobile leaching into groundwater and threatening drinking water supplies. We have engineered Arabidopsis plants that are able to degrade RDX, whilst withstanding the phytotoxicity of TNT. Arabidopsis thaliana (Arabidopsis) was transformed with the bacterial RDX-degrading xplA, and associated reductase xplB, from Rhodococcus rhodochrous strain 11Y, in combination with the TNT-detoxifying nitroreductase (NR), nfsI, from Enterobacter cloacae. Plants expressing XplA, XplB and NR remove RDX from soil leachate and grow on soil contaminated with RDX and TNT at concentrations inhibitory to XplA-only expressing plants. This is the first study to demonstrate the use of transgenic plants to tackle two chemically diverse organic compounds at levels comparable with those found on contaminated training ranges, indicating that this technology is capable of remediating concentrations of RDX found in?situ. In addition, plants expressing XplA and XplB have substantially less RDX available in aerial tissues for herbivory and potential bioaccumulation.     

Leaching of contaminated leaves following uptake and phytoremediation of RDX, HMX, and TNT by poplar

The uptake and fate of 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) by hybrid poplars in hydroponic systems were compared and exposed leaves were leached with water to simulate potential exposure pathways from groundwater in the field. TNT was removed from solution more quickly than nitramine explosives. Most of radioactivity remained in root tissues for 14C-TNT, but in leaves for 14C-RDX and 14C-HMX. Radiolabel recovery for TNT and HMX was over 94%, but that of RDX decreased over time, suggesting a loss of volatile products. A considerable fraction (45.5%) of radioactivity taken up by whole plants exposed to 14C-HMX was released into deionized water, mostly as parent compound after 5 d of leaching. About a quarter (24.0%) and 1.2% were leached for RDX and TNT, respectively, mostly as transformed products. Leached radioactivity from roots was insignificant in all cases (< 2%). This is the first report in which small amounts of transformation products of RDX leach from dried leaves following uptake by poplars. Such behavior for HMX was reported earlier and is reconfirmed here. All three compounds differ substantially in their fate and transport during the leaching process.     

Conjugation of isoprene monoepoxides with glutathione, catalyzed by alpha, mu, pi and theta-class glutathione S-transferases of rat and man

In the present study, the enzymatic conjugation of the isoprene monoepoxides 3,4 epoxy-3-methyl-1-butene (EPOX-I) and 3,4-epoxy-2-methyl-1-butene (EPOX-II) with glutathione was investigated, using purified glutathione S-transferases (GSTs) of the alpha, mu, pi and theta-class of rat and man. HPLC analysis of incubations of EPOX-I and EPOX-II with [35S]glutathione (GSH) showed the formation of two radioactive fractions for each isoprene monoepoxide. The structures of the EPOX-I and EPOX-II GSH conjugates were elucidated with 1H-NMR analysis. As expected, two sites of conjugation were found for both isoprene epoxides. EPOX-II was conjugated more efficiently than EPOX-I. In addition, the mu and theta class glutathione S-transferases were much more efficient than the alpha and pi class glutathione S-transferases, both for rat and man. Because the mu- and theta-class glutathione S-transferases are expressed in about 50 and 40-90% of the human population, respectively, this may have significant consequences for the detoxification of isoprene monoepoxides in individuals who lack these enzymes. Rat glutathione S-transferases were more efficient than human glu tathione S-transferases: rat GST T1-1 showed about 2.1-6.5-fold higher activities than human GST T1-1 for the conjugation of both EPOX-I and EPOX-II, while rat GST M1-1 and GST M2-2 showed about 5.2-14-fold higher activities than human GST M1a-1a. Most of the glutathione S-transferases showed first order kinetics at the concentration range used (50-2000 microM). In addition to differences in activities between GST-classes, differences between sites of conjugation were found. EPOX-I was almost exclusively conjugated with glutathione at the C4-position by all glutathione S-transferases, with exception of rat GST M1-1, which also showed significant conjugation at the C3-position. This selectivity was not observed for the conjugation of EPOX-II. Incubations with EPOX-I and EPOX-II and hepatic S9 fractions of mouse, rat and man, showed similar rates of GSH conjugation for mouse and rat. Compared to mouse and rat, human liver S9 showed a 25-50-fold lower rate of GSH conjugation.     

Biodegradation of nitro-substituted explosives 2,4,6-trinitrotoluene, hexahydro-1,3,5-trinitro-1,3,5-triazine, and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine by a phytosymbiotic Methylobacterium sp. associated with poplar tissues (Populus deltoides x nigra DN34)

A pink-pigmented symbiotic bacterium was isolated from hybrid poplar tissues (Populus deltoides x nigra DN34). The bacterium was identified by 16S and 16S-23S intergenic spacer ribosomal DNA analysis as a Methylobacterium sp. (strain BJ001). The isolated bacterium was able to use methanol as the sole source of carbon and energy, which is a specific attribute of the genus Methylobacterium. The bacterium in pure culture was shown to degrade the toxic explosives 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine (HMX). [U-ring-(14)C]TNT (25 mg liter(-1)) was fully transformed in less than 10 days. Metabolites included the reduction derivatives amino-dinitrotoluenes and diamino-nitrotoluenes. No significant release of (14)CO(2) was recorded from [(14)C]TNT. In addition, the isolated methylotroph was shown to transform [U-(14)C]RDX (20 mg liter(-1)) and [U-(14)C]HMX (2.5 mg liter(-1)) in less than 40 days. After 55 days of incubation, 58.0% of initial [(14)C]RDX and 61.4% of initial [(14)C]HMX were mineralized into (14)CO(2). The radioactivity remaining in solution accounted for 12.8 and 12.7% of initial [(14)C]RDX and [(14)C]HMX, respectively. Metabolites detected from RDX transformation included a mononitroso RDX derivative and a polar compound tentatively identified as methylenedinitramine. Since members of the genus Methylobacterium are distributed in a wide diversity of natural environments and are very often associated with plants, Methylobacterium sp. strain BJ001 may be involved in natural attenuation or in situ biodegradation (including phytoremediation) of explosive-contaminated sites.     

Phytodetoxification of the environmental pollutant and explosive 2,4,6-trinitrotoluene

Our recent study highlights the role of 2 glutathione transferases (GSTs) in the detoxification of the environmental pollutant, 2,4,6-trinitrotoluene (TNT) in Arabidopsis thaliana. TNT is toxic and highly resistant to biodegradation in the environment, raising both health and environmental concerns. Two GSTs, GST-U24 and GST-U25, are upregulated in response to TNT treatment, and expressed predominantly in the root tissues; the site of TNT location following uptake. Plants overexpressing GST-U24 and GST-U25 exhibited significantly enhanced ability to withstand and detoxify TNT, and remove TNT from contaminated soil. Analysis of the catalytic activities of these 2 enzymes revealed that they form 3 TNT-glutathionyl products. Of particular interest is 2-glutathionyl-4,6-dinitrotoluene as this represents a potentially favorable step toward subsequent degradation and mineralization of TNT. We demonstrate how GSTs fit into what is already known about pathways for TNT detoxification, and discuss the short and longer-term fate of TNT conjugates in planta.     

An explosive-degrading cytochrome P450 activity and its targeted application for the phytoremediation of RDX

The widespread presence in the environment of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), one of the most widely used military explosives, has raised concern owing to its toxicity and recalcitrance to degradation. To investigate the potential of plants to remove RDX from contaminated soil and water, we engineered Arabidopsis thaliana to express a bacterial gene xplA encoding an RDX-degrading cytochrome P450 (ref. 1). We demonstrate that the P450 domain of XplA is fused to a flavodoxin redox partner and catalyzes the degradation of RDX in the absence of oxygen. Transgenic A. thaliana expressing xplA removed and detoxified RDX from liquid media. As a model system for RDX phytoremediation, A. thaliana expressing xplA was grown in RDX-contaminated soil and found to be resistant to RDX phytotoxicity, producing shoot and root biomasses greater than those of wild-type plants. Our work suggests that expression of xplA in landscape plants may provide a suitable remediation strategy for sites contaminated by this class of explosives.     

Characterization of two Arabidopsis thaliana glutathione S-transferases

Glutathione S-transferases (GST) are multifunctional proteins encoded by a large gene family, divided on the basis of sequence identity into phi, tau, theta, zeta and lambda classes. The phi and tau classes are present only in plants. GSTs appear to be ubiquitous in plants and are involved in herbicide detoxification and stress response, but little is known about the precise role of GSTs in normal plant physiology and during biotic and abiotic stress response. Two cDNAs representing the two plant classes tau and phi, AtGSTF9 and AtGSTU26, were expressed in vitro and the corresponding proteins were analysed. Both GSTs were able to catalyse a glutathione conjugation to 1-chloro-2,4-dinitrobenzene (CDNB), but they were inactive as transferases towards p-nitrobenzylchloride (pNBC). AtGSTF9 showed activity towards benzyl isothiocyanate (BITC) and an activity as glutathione peroxidase with cumene hydroperoxide (CumHPO). AtGSTU26 was not active as glutathione peroxidase and towards BITC. RT-PCR analysis was used to evaluate the expression of the two genes in response to treatment with herbicides and safeners, chemicals, low and high temperature. Our results reveal that AtGSTU26 is induced by the chloroacetanilide herbicides alachlor and metolachlor and the safener benoxacor, and after exposure to low temperatures. In contrast, AtGSTF9 seems not to be influenced by the treatments employed.     

Structural basis for catalytic differences between alpha class human glutathione transferases hGSTA1-1 and hGSTA2-2 for glutathione conjugation of environmental carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide

The ultimate diol epoxide carcinogens derived from polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (BP), are metabolized primarily by glutathione (GSH) conjugation reaction catalyzed by GSH transferases (GSTs). In human liver and probably lung, the alpha class GSTs are likely to be responsible for the majority of this reaction because of their high abundance. The catalytic efficiency for GSH conjugation of the carcinogenic (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide [(+)-anti-BPDE] is more than 5-fold higher for hGSTA1-1 than for hGSTA2-2. Here, we demonstrate that mutation of isoleucine-11 of hGSTA2-2, a residue located in the hydrophobic substrate-binding site (H-site) of the enzyme, to alanine (which is present in the same position in hGSTA1-1) results in about a 7-fold increase in catalytic efficiency for (+)-anti-BPDE-GSH conjugation. Thus, a single amino acid substitution is sufficient to convert hGSTA2-2 to a protein that matches hGSTA1-1 in its catalytic efficiency. The increased catalytic efficiency of hGSTA2/I11A is accompanied by greater enantioselectivity for the carcinogenic (+)-anti-BPDE over (-)-anti-BPDE. Further remodeling of the H-site of hGSTA2-2 to resemble that of hGSTA1-1 (S9F, I11A, F110V, and S215A mutations, SIFS mutant) results in an enzyme whose catalytic efficiency is approximately 13.5-fold higher than that of the wild-type hGSTA2-2, and about 2.5-fold higher than that of the wild-type hGSTA1-1. The increased activity upon mutations can be rationalized by the interactions of the amino acid side chains with the substrate and the orientation of the substrate in the active site, as visualized by molecular modeling. Interestingly, the catalytic efficiency of hGSTA2-2 toward (-)-anti-BPDE was increased to a level close to that of hGSTA1-1 upon F110V, not I11A, mutation. Similar to (+)-anti-BPDE, however, the SIFS mutant was the most efficient enzyme for GSH conjugation of (-)-anti-BPDE.     

Plant processes important for the transformation and degradation of explosives contaminants

Environmental contamination by explosives is a worldwide problem. Of the 20 energetic compounds, 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) are the most powerful and commonly used. Nitroamines are toxic and considered as possible carcinogens. The toxicity and persistence of nitroamines requires that their fate in the environment be understood and that contaminated soil and groundwater be remediated. This study, written as a minireview, provides further insights for plant processes important for the transformation and degradation of explosives. Plants metabolize TNT and the distribution of the transformation products, conjugates, and bound residues appears to be consistent with the green liver model concept. Metabolism of TNT in plants occurs by reduction as well as by oxidation. Reduction probably plays an important role in the tolerance of plants towards TNT, and, therefore a high nitroreductase capacity may serve as a biochemical criterion for the selection of plant species to remediate TNT. Because the activities and the inducibilities of the oxidative enzymes are far lower than of nitroreductase, reducing processes may predominate. However, oxidation may initiate the route to conjugation and sequestration leading ultimately to detoxification of TNT, and, therefore, particularly the oxidative pathway deserves more study. It is possible that plants metabolize RDX also according to the green liver concept. In the case of plant metabolism of HMX, a conclusion regarding compliance with the green liver concept was not reached due to the limited number of available data.     

Sheep prefer clean forage over forage contaminated with military explosives TNT, RDX and HMX

The common military explosives 2-methyl-1,3,5-trinitrobenzene (TNT), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX) and 1,3,5,7-tetranitro-1,3,5,7-tetrazocane (HMX) are distributed in many military training areas, and are thus encountered by grazing animals. The aim of this study was to examine small ruminant's intake of forage contaminated with explosives. An indoor, experimental setup was used to determine if contamination of forage by these compounds affected intake by sheep. The results clearly demonstrate that contamination by any of the three explosives reduced forage intake in sheep; in order of increasing avoidance: RDX < TNT < HMX. The results are discussed in a risk assessment context.     

Phytotransformation of 2,4-dinitrotoluene in arabidopsis thaliana: toxicity, fate, and gene expression studies in vitro

Basic knowledge of the plant transformation pathways and toxicity of 2,4-dinitrotoluene (2,4-DNT) will assist in the design and assessment of a phytoremediation strategy. This study presents the toxicity and fate of 2,4-DNT and gene expression in response to 2,4-DNT exposure using the model plant Arabidopsis thaliana, an increasingly popular system for genetic and biochemical studies of phytotransformation of explosives. From the results of biomass and root growth assays for toxicity, 2,4-DNT was toxic to the plants at concentrations as low as 1 mg/L. In the uptake study, 95% of the initial 2,4-DNT was removed by 15-day-old seedlings from liquid media regardless of the initial 2,4-DNT concentrations while 30% accounted for the adsorption to the autoclaved plant materials. The mass balance was over 86% using [U-14C]2,4-DNT, and the mineralization by the plants was less than 1% under sterile conditions during 14 days of exposure. The percentage of the bound radioactivity increased from 49% to 72% of the radioactivity in the plants, suggesting transformed products of 2,4-DNT may be incorporated into plant tissues such as lignin and cellulose. Monoaminonitrotoluene isomers and unknown metabolites with short retention times were detected as transformed products of 2,4-DNT by the plants. Most (68%) of the radioactivity taken up by the plants was in the root tissues in nonsterile hydroponic cultures. Glutathione and expression of related genes (GSH1 and GSH2) in plants exposed to 2,4-DNT were 1.7-fold increased compared to untreated plants. Genes of a glutathione S-transferase and a cytochrome P450, which were induced by 2,4,6-trinitrotoluene exposure in previous studies, were upregulated by 10- and 8-fold, respectively. The application of phytoremediation and the development of transgenic plants for 2,4-DNT may be based on TNT phytotransformation pathway characteristics because of the similar fate and gene expression in plants.