Tau Class Glutathione S-transferase Research Articles

The monoterpenoid (S)-carvone massively up-regulates several classes of glutathione S-transferase genes in tobacco leaf discs

The monoterpenoid (S)-carvone was shown earlier to induce glutathione S-transferase (GST) activity and to attenuate necrotic lesion formation in Tobacco mosaic virus (TMV) inoculated, hypersensitively reacting tobacco Xanthi-nc plants. To explore structure-activity relationships, three monoterpenes and five monoterpenoids were tested for their ability to induce increased GST activity in tobacco leaf discs. (S)-Carvone proved to be the most potent inducer. (S)-Carvone treatments markedly up-regulated the expression of several tobacco GST genes belonging to the phi, tau and theta classes, particularly in the case of tau class GSTs. Furthermore, TMV inoculation slightly induced the transcript abundance of the GSTT2 gene. The expression of three catalase (CAT) genes was also examined in (S)-carvone-treated tobacco leaf discs. The expression of CAT1 was not influenced by (S)-carvone treatments, whereas that of CAT2 was significantly repressed. (S)-Carvone very weakly induced the transcription of the CAT3 gene....

Cloning and expression of a tau class glutathione S-transferase (ZjGSTU1) from Chinese jujube in response to phytoplasma infection

Phytoplasmas are associated with diseases of several hundred plant species. Jujube witches’ broom disease (JWB) is a destructive phytoplasma disease in Chinese jujube (Ziziphus jujuba Mill.). Ziziphus jujuba Mill. ‘J5’, an excellent strain with extremely high resistance to JWB, was selected by us. In our previous study, a GST (EC 2.5.1.18) fragment was sequenced from suppression subtractive hybridization library of ‘J5’ under JWB phytoplasma stress. Based on this result, a GST gene (ZjGSTU1, HM345954) was first isolated from jujube by homology cloning and RACE. ZjGSTU1 contains a complete open reading frame of 702 bp encoding a protein of 233 amino acid residues. Sequence alignment showed that ZjGSTU1 shared a typical conserved structure and high identity with tau GSTs from other plant species. Relative RT-PCR demonstrated that the expression of ZjGSTU1 mRNA in jujube leaves and branches could be triggered by JWB phytoplasma. Moreover, the expression of ZjGSTU1 mRNA in resistant strain ‘J5’ increased sooner and higher than that in sensitive strain ‘J9’ under JWB phytoplasma stress (‘J5’ and ‘J9’ are two strains from the same cultivar). Western blotting analyses showed that the expressions of ZjGSTU1 in ‘J5’ and ‘J9’ were dramatically up-regulated under JWB phytoplasma stress and its expression in ‘J5’ was also higher than that in ‘J9’ at protein level. Collectively, this paper highlights that ZjGSTU1 gene is responsive to phytoplasma infection. The possible roles of this gene were discussed in terms of regulatory process in resistance to phytoplasma infection.

Over-Expression of a Rice Tau Class Glutathione S-Transferase Gene Improves Tolerance to Salinity and Oxidative Stresses in Arabidopsis

Glutathione S-transferases (GSTs) are multifunctional proteins encoded by large gene family in plants, which play important role in cellular detoxification of several endobiotic and xenobiotic compounds. Previously, we suggested the diverse roles of rice GST gene family members in plant development and various stress responses based on their differential expression. In this study, we report the functional characterization of a rice tau class GST gene, OsGSTU4. OsGSTU4 fusion protein was found to be localized in nucleus and cytoplasm. The over-expression of OsGSTU4 in E. coli resulted in better growth and higher GST activity under various stress conditions. Further, we raised over-expression transgenic Arabidopsis plants to reveal its in planta function. These transgenic lines showed reduced sensitivity towards plant hormones, auxin and abscisic acid. Various analyses revealed improved tolerance in transgenic Arabidopsis plants towards salinity and oxidative stresses, which may be attributed to the lower accumulation of reactive oxygen species and enhanced GST activity. In addition, microarray analysis revealed up-regulation of several genes involved in stress responses and cellular detoxification processes in the transgenic plants as compared to wild-type. These results suggest that OsGSTU4 can be used as a good candidate for the generation of stress-tolerant crop plants.

Transformed yeast (Schizosaccharomyces pombe) overexpressing rice Tau class glutathione S-transferase (OsGSTU30 and OsGSTU41) shows enhanced resistance to hexavalent chromium

Extensive use of hexavalent chromium [Cr(VI)] in leather tanning, stainless-steel production, wood preservatives and electroplating industries has resulted in widespread environmental pollution and poses a serious threat to human health. A plant's response to Cr(VI) stress results in growth inhibition and toxicity leading to changes in components of antioxidant systems. In a previous study, we observed that a large number of glutathione S-transferase (GST) genes were up-regulated under Cr(VI) stress in rice. In this study, two rice root-specific Tau class GST genes (OsGSTU30 and OsGSTU41) were introduced into yeast (Schizosaccharomyces pombe). Transformed yeast cells overexpressing OsGSTU30 and OsGSTU41 had normal growth, but had much higher levels of GST activities and showed enhanced resistance to Cr(VI) as compared to control cells (transformed with empty vector). Also, a higher accumulation of chromium was found in the transformed yeast cells as compared to the control cells. Manipulation of glutathione biosynthesis by exogenous application of buthionine sulfoximine abolishes the protective effect of OsGSTs against Cr(VI) stress. These results suggest that Tau class OsGSTs play a significant role in detoxification of Cr(VI), probably by chelating and sequestrating glutathione-Cr(VI) complexes into vacuoles.

Structural and Functional Evolution of Positively Selected Sites in Pine Glutathione S-Transferase Enzyme Family*

Phylogenetic analyses have identified positive selection as an important driver of protein evolution, both structural and functional. However, the lack of appropriate combined functional and structural assays has generally hindered attempts to elucidate patterns of positively selected sites and their effects on enzyme activity and substrate specificity. In this study we investigated the evolutionary divergence of the glutathione S-transferase (GST) family in Pinus tabuliformis, a pine that is widely distributed from northern to central China, including cold temperate and drought-stressed regions. GSTs play important roles in plant stress tolerance and detoxification. We cloned 44 GST genes from P. tabuliformis and found that 26 of the 44 belong to the largest (Tau) class of GSTs and are differentially expressed across tissues and developmental stages. Substitution models identified five positively selected sites in the Tau GSTs. To examine the functional significance of these positively selected sites, we applied protein structural modeling and site-directed mutagenesis. We found that four of the five positively selected sites significantly affect the enzyme activity and specificity; thus their variation broadens the GST family substrate spectrum. In addition, positive selection has mainly acted on secondary substrate binding sites or sites close to (but not directly at) the primary substrate binding site; thus their variation enables the acquisition of new catalytic functions without compromising the protein primary biochemical properties. Our study sheds light on selective aspects of the functional and structural divergence of the GST family in pine and other organisms.

Sulfate supply influences compartment specific glutathione metabolism and confers enhanced resistance to Tobacco mosaic virus during a hypersensitive response

Sufficient sulfate supply has been linked to the development of sulfur induced resistance or sulfur enhanced defense (SIR/SED) in plants. In this study we investigated the effects of sulfate (S) supply on the response of genetically resistant tobacco (Nicotiana tabacum cv. Samsun NN) to Tobacco mosaic virus (TMV). Plants grown with sufficient sulfate (+S plants) developed significantly less necrotic lesions during a hypersensitive response (HR) when compared to plants grown without sulfate (−S plants). In +S plants reduced TMV accumulation was evident on the level of viral RNA. Enhanced virus resistance correlated with elevated levels of cysteine and glutathione and early induction of a Tau class glutathione S-transferase and a salicylic acid-binding catalase gene. These data indicate that the elevated antioxidant capacity of +S plants was able to reduce the effects of HR, leading to enhanced virus resistance. Expression of pathogenesis-related genes was also markedly up-regulated in +S plants after TMV-inoculation. On the subcellular level, comparison of TMV-inoculated +S and −S plants revealed that +S plants contained 55–132 % higher glutathione levels in mitochondria, chloroplasts, nuclei, peroxisomes and the cytosol than −S plants. Interestingly, mitochondria were the only organelles where TMV-inoculation resulted in a decrease of glutathione levels when compared to mock-inoculated plants. This was particularly obvious in −S plants, where the development of necrotic lesions was more pronounced. In summary, the overall higher antioxidative capacity and elevated activation of defense genes in +S plants indicate that sufficient sulfate supply enhances a preexisting plant defense reaction resulting in reduced symptom development and virus accumulation.

A plant-specific tau class glutathione S-transferase from Oryza sativa with very high activity against 1-chloro-2,4-dinitrobenzene and chloroacetanilide herbicides

A plant-specific tau class GST gene homolog was successfully cloned from an Oryza sativa cDNA library by PCR using oligonucleotide primers based on the OsGSTU4 (GenBank Accession No. AF309378) sequence. The cDNA was composed of a 720-bp open reading frame encoding 239 amino acids. The deduced amino acid sequence of this gene shared over 65% sequence identity with the sequences of the tau class TaGST28e45 and ZmGST42. Conversely, the OsGSTU4 sequence showed very low identity to the GST sequences of phi, theta and zeta classes. This gene was expressed in Escherichia coli with the pET vector system, and the gene product was purified to homogeneity using GSH–Sepharose affinity column chromatography. The expressed OsGSTU4 formed a homodimer with subunits of approximately 25.5 kDa. OsGSTU4 displayed very high activity toward 1-chloro-2,4-dinitrobenzene. The activity of the OsGSTU4 was significantly inhibited by S-hexylglutathione and hematin. Plant OsGSTU4 had a unique herbicide specificity and played an important role in the detoxification reaction against fluorodifen and chloroacetanilide herbicides.

Tau class glutathione S-transferases candidates from Brassica rapa: Genetic mapping and sequence analysis

Glutathione S-transferases (GSTs) have medically and agriculturally important roles in plants such as herbicide detoxification, responses to biotic and abiotic stress and catalytic reaction of anticancer compounds. The fully sequenced Arabidopsis thaliana and rice (Oryza sativa) genome revealed identification of 52 and 61 members of GSTs, and 28 and 39 of those belong to the Tau class of GSTs (GSTUs), respectively. Based on the sequences of AtGSTUs, 14 BAC clones derived from Brassica rapa that contain similar sequences of AtGSTUs were identified and 17 unique sequences of open reading frames were detected in the 14 BAC clones using blast search and sequence alignment. Those sequences were designated the Tau class candidate of GST derived from B. rapa (BrGSTUs) and it is confirmed that BrGSTUs are also clustered in tandem as it has been known to be common feature in plant. They were mapped on BrR5, BrR7, BrR8, BrR9, or BrR10, and their nucleotide and amino acid sequences were highly similar to those of AtGSTUs. In addition, in silico analysis of BrGSTUs was performed using Korea Brassica Genome Project 24 K (KBGP-24K) oligochip and microarray database for cold, salt, and drought stresses. The results of this study will facilitate further researches for breeding of Chinese cabbage containing medically and agriculturally important traits.

Conserved Residues in the Subunit Interface of tau Glutathione S‐transferase Affect Catalytic and Structural Functions

The tau class glutathione S-transferases (GSTs) have important roles in stress tolerance and the detoxification of herbicides in crops and weeds. Structural investigations of a wheat tau GST (TaGSTU4) show two subunit interactions: a hydrogen bond between the Tyr93 and Pro65 from another subunit of the dimer, and two salt bridges between residues Glu78 and side chains of Arg95 and Arg99 in the opposite subunit. By investigating enzyme activities, kinetic parameters and structural characterizations, this study showed the following results: (i) the hydrogen bond interaction between the Tyr93 and Pro65 was not essential for dimerization, but contributed to the enzyme's catalytic activity, thermal stability and affinity towards substrates glutathione and 1-chloro-2, 4-dinitrobenzene; and (ii) two salt bridges mainly contributed to the protein structure stability and catalysis. The results of this study form a structural and functional basis for rational design of more selective and environmentally friendly herbicides.

Expression of SbGSTU (tau class glutathione S-transferase) gene isolated from Salicornia brachiata in tobacco for salt tolerance

Tau class glutathione transferases (GSTU) genes are plant specific, induced by different abiotic stress, and important for protecting plants against oxidative damage. GST gene was isolated using 5' RACE from an extreme halophyte Salicornia brachiata, cloned, sequenced and its protein structure was predicted. Transcript profiling of SbGST gene expression was studied under different abiotic stress conditions and plant growth regulator treatments, viz. salt, cold, drought, ABA and salicylic acid, with time period point and concentration point. The expression of SbGST gene was up-regulated in all stress conditions, except SA treatment. Seed germination percentage, GST enzyme assay, fresh weight and other growth parameters (root length, shoot length and leaf area) were studied and results indicate that over-expression of SbGST gene in transgenic tobacco leads to enhanced seed germination and growth under salt stress. Transgenic lines were evaluated for their performance under salt stress and tobacco plants over-expressing SbGST showed higher seed germination and survival compared to wild type. These results confirm that expression of SbGST gene is up-regulated by different stresses and over-expression of tau class SbGST gene in transgenic tobacco plays a vital role in abiotic stress tolerance. SbGST gene expressed conspicuously under salt stress leading to enhance seed germination and better growth. Furthermore, GST is a potential candidate gene to be used in genetic engineering for enhancing abiotic stress tolerance.

Elevated antioxidant response and induction of tau-class glutathione S-transferase after glyphosate treatment in Vigna radiata (L.) Wilczek

Glyphosate (N-(phosphonomethyl) glycine) is a broad-spectrum herbicide, acting on the shikimic acid pathway inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), thus obstructing the synthesis of tryptophan, phenylalanine, tyrosine and other secondary products. It has also been reported to generate oxidative stress which influences the antioxidant response of target plants. The effect of glyphosate application on total protein, CAT, POD and GST activities was investigated and elevated expression of the oxidative stress enzymes was obtained after glyphosate treatment. Tau-class GSTs are plant-specific, and are chiefly involved in xenobiotics and oxidative stress metabolisms. Many herbicides and safeners have been known to selectively induce tau-class GSTs in different plant species. Here we also report the induction of tau-class GSTs after glyphosate treatment in the seedling roots of two Vigna radiata varieties (PDM11 and PDM54). GSH-agarose affinity chromatography and mass spectrometry revealed that the tau-class GSTs induced in the two varieties were different; the tau-class GSTs present in the untreated controls were also different in the two varieties. The present study highlights the elevated antioxidant response, the induction of tau-class GST and the genotypic variation in the type of tau-GST in control and glyphosate treated varieties of V. radiata.

Gene isolation and expression analysis of two distinct sweet orange [ Citrus sinensis L. (Osbeck)] tau-type glutathione transferases

Glutathione S-transferases (GSTs) represent a multifunctional family of enzymes grouped into four main classes (tau, phi, theta, and zeta) conjugating endobiotic and xenobiotic compounds to glutathione. In plants, this is considered to be a crucial step in the detoxification process as the S-glutathionylated metabolites are tagged for vacuolar sequestration. In this work, we have isolated two glutathione S-transferases belonging to the tau class GSTs from sweet orange leaves. The cDNA clones contained a complete open reading frame of 651 bp encoding two 216 amino acid proteins. Homology search and sequence alignment showed that the deduced amino acid sequences shared high identity with GSTs from other plant sources, including several strictly conservative motifs and distinctive amino acid residues specific of the tau class GSTs. The genomic clones of both isoforms were also isolated and the analysis of the gene organization confirmed the membership of both enzymes to the tau class GSTs. The encoded proteins differ only for three amino acids: the triplet R89, E117 and I172 found in the isoform named GSTU1 is replaced by the triplet P89, K117 and V172 in the GSTU2 isoform. The successful in vitro expression of the proteins led to the functional active form of both enzymes which showed different specific activity against CDNB as substrate, the GSTU1 showing values three fold lower than that observed for the GSTU2 enzyme. The analysis of the gene expressions suggested that the GST isoforms show either different distribution between leaf and flesh, the isoforms being decidedly expressed in the leaf, or cultivar related specificity, the U2 being highly expressed in the leaves of red orange whereas the U1 in the blond orange leaves. Furthermore, we also showed that the expression of U1 gene was remarkably induced in response to cadmium sulphate, CDNB and cyhalothrin treatments as well as to cold stress. On the contrary, the U2 isoform was constitutively expressed probably playing some sort of “ default scavenging” activity in vivo. Taken together these results suggested that GSTU1 is a stress responsive gene and can be considered as potential target that is genetically modified so as create novel germoplasm with enhanced stress tolerance.

Lipoxygenase and glutathione peroxidase activity in tobacco leaves inoculated with tobacco mosaic virus

Fatty acid hydroperoxide-producing lipoxygenase (LOX) and hydroperoxide-degrading glutathione peroxidase (GPOX) enzyme activities were studied in leaves of virus resistant Xanthi-nc and susceptible Samsun-nn tobacco cultivars after inoculation with Tobacco mosaic virus (TMV). Total LOX activity showed a maximum at pH 5.5 in cell-free extracts of uninfected leaves. LOX activity markedly increased at this pH after TMV inoculation, but a substantial induction was detected also in the basic pH range with an emerging peak around pH = 8.5. TMV-elicited LOX induction was weaker and appeared later in Samsun-nn than in Xanthi-nc leaves. GPOX activity was also substantially induced by TMV infection. However, this induction appeared only 4 days post-inoculation in resistant Xanthi-nc plants in tissues surrounding the localized necrotic lesions. In contrast, GPOX activity did not change in TMV-inoculated, susceptible Samsun-nn leaves. Several glutathione S-transferase (GST) isoenzymes also display GPOX activity. The expression of a tau class GST gene was markedly induced by TMV inoculation in Xanthi-nc leaves. This tobacco GST gene was partially cloned and sequenced.

Molecular Cloning and Characterization of a Senescence-induced Tau-class Glutathione S-transferase from Barley Leaves

Glutathione S-transferases (GSTs) (EC 2.5.1.18) are multifunctional proteins involved in such diverse intracellular events as primary and secondary metabolism, signaling and stress metabolism. In this study, we found a senescence-induced tau-class GST (SIGST) in senescent leaves of barley (Hordeum vulgare L.). The SIGST was purified 19-fold to homogeneity from initial crude extracts by three steps of chromatography with a yield of 5%. The purified SIGST had a GSH-conjugating activity and peroxidase (POD) activity at the same level of 1.7 micromol min(-1) mg protein(-1), although restricted substrate selectivity could be seen in POD activity. Barley SIGST is a slightly acidic protein with a molecular weight of 49 k and is composed of two subunits. The enzyme exhibited a single pH optimum at pH 8.3. The K(m) values were 0.285 mM for GSH and 0.293 mM for 1-chloro-2,4-dinitrobenzene. In most respects, the barley enzyme resembles those that have been reported from other higher plants. The SIGST gene was cloned from cDNA of senescent barley leaves. DNA sequence analysis shows that the cloned SIGST had only one base different from the barley embryo GST, ECGST. The obtained sequence indicates that SIGST is classified into the plant-specific tau class. mRNA expression analysis showed that in addition to senescence, SIGST was strongly induced by treatment with a herbicide and low temperature. The responses to these stresses suggest that SIGST may be involved at least partly in the secondary metabolism as an antioxidant and enhancement of enzymatic activity during senescence.

Differential roles of tau class glutathione S-transferases in oxidative stress.

The plant glutathione S-transferase BI-GST has been identified as a potent inhibitor of Bax lethality in yeast, a phenotype associated with oxidative stress and disruption of mitochondrial functions. Screening of a tomato two-hybrid library for BI-GST interacting proteins identified five homologous Tau class GSTs, which readily form heterodimers between them and BI-GST. All six LeGSTUs were found to be able to protect yeast cells from prooxidant-induced cell death. The efficiency of each LeGSTU was prooxidant-specific, indicating a different role for each LeGSTU in the oxidative stress-response mechanism. The prooxidant protective effect of all six proteins was suppressed in the absence of YAP1, a transcription factor that regulates hydroperoxide homeostasis in Saccharomyces cerevisiae, suggesting a role for the LeGSTUs in the context of the YAP1-dependent stress-responsive machinery. The different LeGSTUs exhibited varied substrate specificity and showed activity against oxidative stress by-products, indicating that their prooxidant protective function is likely related to the minimization of oxidative damage. Taken together, these results indicate that Tau class GSTs participate in a broad network of catalytic and regulatory functions involved in the oxidative stress response.

Abiotic stress alters transcript profiles and activity of glutathione S-transferase, glutathione peroxidase, and glutathione reductase in Euphorbia esula.

Glutathione S-transferase (GST), glutathione peroxidase (GPX), and glutathione reductase (GR) are enzymes that utilize glutathione to play an important role in plant defense mechanisms. In leafy spurge (Euphorbia esula L.), transcript and activity profiles for these enzymes are differentially influenced in tissue exposed to xenobiotic (diclofop-methyl) and environmental stress (cold and drought). Five different EeGST cDNA (including phi, tau, theta, and zeta class GSTs), one EeGPX cDNA, and one EeGR cDNA showed differential expression patterns in leafy spurge plants exposed to diclofop-methyl-, cold- and drought-stress. Tissue treated with diclofop-methyl also had increased GST, GPX and GR activities that were preceded or paralleled by increased gene expression. Transcript profiles resulting from drought-stressed plants were similar to transcript profiles from diclofop-methyl-treated plants but not cold-stressed plants. GPX activity in leafy spurge protein extracts was not bound to either S-hexylglutathione- or glutathione-agarose columns but instead co-migrated with fractions of GST activity that also were not bound by affinity chromatography. Fractions of GST proteins that were bound to S-hexylglutathione revealed that increased GST activity in diclofop-methyl-treated tissue could be identified as phi- and tau-type GSTs.

Osgstu3 and osgtu4, encoding tau class glutathione S-transferases, are heavy metal- and hypoxic stress-induced and differentially salt stress-responsive in rice roots

Glutathione S-transferases (GSTs) have poorly understood roles in plant responses to environmental stresses. A polyethylene glycol (PEG)-induced tau class GST was identified in rice roots by protein microsequencing. PEG and the heavy metals Cd (20 μM), Zn (30 μM), Co and Ni rapidly and markedly induced osgstu4 and osgstu3 in rice seedling roots. Osgstu4 and osgstu3 were also induced in roots by hypoxic stress but not by cold nor heat shock. Salt stress and abscisic acid (ABA) rapidly induced osgstu3 in rice roots, whereas osgstu4 exhibited a late salt stress and no ABA response. Salicylic acid, jasmonic acid and the auxin α-naphthalene acetic acid triggered osgstu4 and osgstu3 expression. Osgstu4 and osgstu3 were rapidly and markedly induced by the antioxidant dithiothreitol and the strong oxidant hydrogen peroxide, which suggested that redox perturbations and reactive oxygen species are involved in their stress response regulations.

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.

Tandemly duplicated Safener-induced glutathione S-transferase genes from Triticum tauschii contribute to genome- and organ-specific expression in hexaploid wheat.

Glutathione S-transferase (GST) gene expression was examined in several Triticum species, differing in genome constitution and ploidy level, to determine genome contribution to GST expression in cultivated, hexaploid bread wheat (Triticum aestivum). Two tandemly duplicated tau class GST genes (TtGSTU1 and TtGSTU2) were isolated from a single bacterial artificial chromosome clone in a library constructed from the diploid wheat and D genome progenitor to cultivated wheat, Triticum tauschii. The genes are very similar in genomic structure and their encoded proteins are 95% identical. Gene-specific reverse transcriptase-polymerase chain reaction analysis revealed differential transcript accumulation of TtGSTU1 and TtGSTU2 in roots and shoots. Expression of both genes was induced by herbicide safeners, 2,4-dichlorophenoxyacetic acid and abscisic acid, in the shoots of T. tauschii; however, expression of TtGSTU1 was always higher than TtGSTU2. In untreated seedlings, TtGSTU1 was expressed in both shoots and roots, whereas TtGSTU2 expression was only detected in roots. RNA gel-blot analysis of ditelosomic, aneuploid lines that are deficient for 6AS, 6BS, or 6DS chromosome arms of cultivated, hexaploid bread wheat showed differential genome contribution to safener-induced GST expression in shoots compared with roots. The GST genes from the D genome of hexaploid wheat contribute most to safener-induced expression in the shoots, whereas GSTs from the B and D genomes contribute to safener-induced expression in the roots.

Structure of a tau class glutathione S-transferase from wheat active in herbicide detoxification.

Glutathione S-transferases (GSTs) from the phi (GSTF) and tau (GSTU) classes are unique to plants and play important roles in stress tolerance and secondary metabolism as well as catalyzing the detoxification of herbicides in crops and weeds. We have cloned and functionally characterized a group of GSTUs from wheat treated with fenchlorazole-ethyl, a herbicide safener. One of these enzymes, TaGSTU4-4, was highly active in conjugating the chemically distinct wheat herbicides fenoxaprop and dimethenamid. The structure of TaGSTU4-4 has been determined at 2.2 A resolution in complex with S-hexylglutathione. This enzyme is the first tau class GST structure to be determined and most closely resembles the omega class GSTs, but without the unique N-terminal extension or active site cysteine. The X-ray structure identifies key amino acid residues in the hydrophobic binding site and provides insights into the substrate specificity of these enzymes.