Dimerisation of maize glutathione transferases in recombinant bacteria

Two cDNAs encoding novel type III maize (Zea mays) GST subunits, ZmGST VI and ZmGST VII, have been cloned in addition to the previously described ZmGST V. Together with the type I GSTs ZmGST I and ZmGST III, these subunits were expressed in Escherichia coli, both individually and in tandem combinations using a customised pET vector. The GST dimers formed were then characterised. When type I GSTs were co-expressed only the respective homodimers were formed rather than the ZmGST I-III heterodimer. The failure to form this heterodimer, together with the negligible herbicide-detoxifying activity associated with recombinant ZmGST III-III, suggests that the identity of herbicide-detoxifying isoenzymes described in maize as being composed of ZmGST III subunits requires re-evaluation. In contrast, co-expression of the type III GSTs ZmGST V and ZmGST VI resulted in the formation of ZmGST V-V, ZmGST VI-VI and ZmGST V-VI dimers in the ratio 1:1:2 as predicted for random subunit association. ZmGST V-VI had kinetic characteristics intermediate between those of the two homodimers, indicating that the subunits were catalytically independent of one another. Co-expression of ZmGST V and ZmGST VII resulted in the formation of ZmGST V-VII and this isoenzyme was subsequently identified in maize plants. Attempts to dimerise type I GST subunits with type III GST subunits proved unsuccessful. These results demonstrate the utility of co-expressing recombinant GSTs to explore the potential of subunit-subunit associations and to help unravel the complexity of homodimeric and heterodimeric GSTs in plants.     

Binding and protection of porphyrins by glutathione S-transferases of Zea mays L

Glutathione S-transferases (GSTs) are multi-functional enzymes, known to conjugate xenobiotics and degrade peroxides. Herein, we report on the potential of four Zea mays GST isoforms (Zm GST I-I, Zm GST I-II, Zm GST II-II and Zm GST III-III) to act as binding and protection proteins. These isoforms bind protoporphyrin IX (PPIX), mesoporphyrin, coproporphyrin, uroporphyrin and Mg-protoporpyhrin, but do not form a glutathione conjugate. The binding is non-covalent and inhibits GSTs enzymatic activity, dependent on the type of the porphyrin and GST isoform tested. I(50) values are in the range of 1 to 10 microM for PPIX, the inhibition by mesoporphyrin and Mg-protoporphyrin (Mg-PPIX) is two to five times less. The mode of binding is non-competitive for the hydrophobic substrate and competitive for glutathione. Binding affinities (K(D) values) of the GST isoforms are between 0.3 and 0.8 microM for coproporphyrin and about 2 microM for mesoporphyrin.Zm GST III-III prevents the nonenzymatic autoxidation of protoporphyrinogen to the phytotoxic PPIX. Zm GST II-II can reduce the oxidative degradation of hemin. This points to a specific ligand role of distinct GST isoforms to protect tetrapyrroles in the plant cell.     

Forced evolution of a herbicide detoxifying glutathione transferase

Plant Tau class glutathione transferases (GSTUs) detoxify diphenylether herbicides such as fluorodifen, determining their selectivity in crops and weeds. Using reconstructive PCR, a series of mutant GSTUs were generated from in vitro recombination and mutagenesis of the maize sequences ZmGSTU1 and ZmGSTU2 (with the prefix Zm designating Zea mays L.). A screen of 5000 mutant GSTUs identified seven enzymes with enhanced fluorodifen detoxifying activity. The best performing enhanced fluorodifen detoxifying mutant (EFD) had activity 19-fold higher than the parent enzymes, with a single point mutation conferring this enhancement. Further mutagenesis of this residue generated an EFD with a 29-fold higher catalytic efficiency toward fluorodifen as compared with the parents but with unaltered catalysis toward other substrates. When expressed in Arabidopsis thaliana, the optimized EFD, but not the parent enzymes, conferred enhanced tolerance to fluorodifen. Molecular modeling predicts that the serendipitous mutation giving the improvement in detoxification is due to the removal of an unfavorable interaction together with the introduction of a favorable change in conformation of residues 107-119, which contribute to herbicide binding.     

Partial characterization of glutathione S-transferases from wheat (Triticum spp.) and purification of a safener-induced glutathione S-transferase from Triticum tauschii.

Hexaploid wheat (Triticum aestivum L.) has very low constitutive glutathione S-transferase (GST) activity when assayed with the chloroacetamide herbicide dimethenamid as a substrate, which may account for its low tolerance to dimethenamid in the field. Treatment of seeds with the herbicide safener fluxofenim increased the total GST activity extracted from T. aestivum shoots 9-fold when assayed with dimethenamid as a substrate, but had no effect on glutathione levels. Total GST activity in crude protein extracts from T. aestivum, Triticum durum, and Triticum tauschii was separated into several component GST activities by anion-exchange fast-protein liquid chromatography. These activities (isozymes) differed with respect to their activities toward dimethenamid or 1-chloro-2,4-dinitrobenzene as substrates and in their levels of induction by safener treatment. A safener-induced GST isozyme was subsequently purified by anion-exchange and affinity chromatography from etiolated shoots of the diploid wheat species T. tauschii (a progenitor of hexaploid wheat) treated with the herbicide safener cloquintocet-mexyl. The isozyme bound to a dimethenamid-affinity column and had a subunit molecular mass of 26 kD based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme (designated GST TSI-1) was recognized by an antiserum raised against a mixture of maize (Zea mays) GSTs. Amino acid sequences obtained from protease-digested GST TSI-1 had significant homology with the safener-inducible maize GST V and two auxin-regulated tobacco (Nicotiana tabacum) GST isozymes.     

Characterisation of glutathione transferases and glutathione peroxidases in pea (Pisum sativum)

Glutathione peroxidases (GPOXs) and glutathione transferases, also termed glutathione S-transferases (GST, EC 2.5.1.18), with activities toward a range of xenobiotic substrates including herbicides, have been characterized in etiolated pea (Pisum sativum L. cv. Feltham's First) seedlings. Crude extracts showed high activity toward a range of GST substrates including 1-chloro-2,4-dinitrobenzene (GSTC activity) and the herbicide fluorodifen (GSTF) but low activities toward chloroacetanilides and atrazine. Treatment of the pea seedlings with the herbicide safener dichlormid selectively increased the activity of GSTC and the GST which detoxified atrazine. This induction was restricted to the roots and was not observed with any of the other GST or GPOX activities. In contrast, treatment with CuCl₂ increased GPOX activity in the root but had no effect on any GST activity, while treatment of epicotyls with elicitors of the phytoalexin response increased GST activity toward ethacrynic acid, but had no effect on other GST or GPOX activities. The major enzymes with GSTC, GSTF and GPOX activities were purified from pea epicotyls 3609-fold, 1431-fold and 1554-fold, respectively. During purification by hydrophobic interaction chromatography and affinity chromatography using S-hexyl-glutathione as ligand all three activities co-eluted but could be partially resolved by anion exchange chromatography and gel filtration chromatography. Both GSTC and GPOX had a molecular mass of 48 kDa and their activities were associated with a similar 27.5-kDa subunit but distinct 29-kDa subunits. GSTF could be resolved into two isoenzymes with molecular masses of 49.5 and 54 kDa. GSTF activity was associated with a unique 30-kDa subunit in addition to 27.5- and 29-kDa peptides, suggesting that the two isoenzymes were composed of differing subunits. These results demonstrate that peas contain multiple GST isoenzymes some of which have GPOX activity and that the various activities are differentially responsive to biotic and abiotic stress.     

Characterization of Glutathione S-Transferase Isoforms in Three Maize Inbred Lines Exhibiting Differential Sensitivity to Alachlor

Glutathione S-transferases (GSTs) are a family of isozymes that catalyze the conjugation of glutathione to several xenobiotics, including a number of important herbicides. Several GST isoforms have been identified in maize (Zea mays L.). In this study we focused on three isoforms, GST I, II, and IV, derived from homo-or heterodimerization of two subunits GST-29 and GST-27, which have been shown to be responsible for reactivity to alachlor. The expression of these isoforms was examined in three inbred lines of maize that showed tolerance, susceptibility, and intermediate resistance to alachlor (2-Cl-N-[2,6-diethylphenyl]-N-[methoxymethyl]acetamide) treatment. The different isoforms were separated by anion-exchange chromatography and subunits were quantified by western blot analysis. GST assays were performed against both 1-Cl-2,4-dinitrobenzene and alachlor. This analysis showed that the susceptible and intermediate lines exhibit impaired function in the GST-27 and GST-29 subunits, respectively. In addition, this study suggests that GST IV is the principal, detoxifying enzyme for alachlor, although GST I and II are required to achieve tolerance to high rates of the herbicide.     

A genomics approach to the comprehensive analysis of the glutathione S-transferase gene family in soybean and maize

By BLAST searching a large expressed sequence tag database for glutathione S-transferase (GST) sequences we have identified 25 soybean (Glycine max) and 42 maize (Zea mays) clones and obtained accurate full-length GST sequences. These clones probably represent the majority of members of the GST multigene family in these species. Plant GSTs are divided according to sequence similarity into three categories: types I, II, and III. Among these GSTs only the active site serine, as well as another serine and arginine in or near the "G-site" are conserved throughout. Type III GSTs have four conserved sequence patches mapping to distinct structural features. Expression analysis reveals the distribution of GSTs in different tissues and treatments: Maize GSTI is overall the most highly expressed in maize, whereas the previously unknown GmGST 8 is most abundant in soybean. Using DNA microarray analysis we observed increased expression among the type III GSTs after inducer treatment of maize shoots, with different genes responding to different treatments. Protein activity for a subset of GSTs varied widely with seven substrates, and any GST exhibiting greater than marginal activity with chloro-2,4 dinitrobenzene activity also exhibited significant activity with all other substrates, suggesting broad individual enzyme substrate specificity.     

A 2,4-D-inducible glutathione S-transferase from soybean (Glycine max). Purification, characterisation and induction

Glutathione S-transferases (GSTs; EC 2.5.1.18) have recently been proposed to form one large group among the auxin-induced proteins. However. the properties and regulation of such auxin-responsive GSTs in the plant still await detailed investigation. In this study, a 2,4-dichloro-phenoxyacetic acid (2,4-D)-inducible GST isozyme from soybean ( Glycine max [L.] Merr. cv. Williams) was purified to near homogeneity by anion-exchange and affinity chromatography on S-hexylglutathione agarose. The native enzyme had a molecular mass of 49 kDa, as determined by gel filtration, and consisted of 26-kDa subunits. The purified GST conjugated glutathione to 1-chloro-2,4-dinitrobenzene and to the herbicide metolachlor, but not to the other GST substrates atrazine. fluorodifen or trans-cinnamic acid. The N-termmal amino acid sequence shared significant homology with the deduced polypeptide sequences of two 2,4-D-inducible genes from tobacco, par A and CNT107 . The levels of the 26-kDa GST subunit protein in soybean hypocotyls were analysed by immunoblotting. At micromolar concentrations, 2,4-D induced a transient increase in net accumulation of GST, whereas indole-3-acetic acid or I-naphthaleneacetic acid did not increase the GST levels. Known inhibitors of polar auxin transport, including 2.3.5-tri-iodobenzoic acid. N-I-naphthylphthalamic acid and analogues thereof, differed widely in their ability to elicit GST protein accumulation. It is concluded that the induction of soybean GST by 2,4-D and by some of the auxin transport inhibitors is not related to auxin activity or to changes in the endogenous auxin levels.     

转Zm13_Barnase基因玉米的获得及其花粉育性研究

5 mg·L-1 Bialaphos was determined to be the adequate concentration for selection of maize (Zea mays L.) calli on herbicide resistance after evaluating three concentrations of Bialaphos in three genotypes. The chimeric gene Zm13-Barnase was transferred into the embryogenic calli of maize by particle bombardment, and 24 normal plants were regenerated from the 32 herbicide-resistant calli obtained after six cycles of continuous selection under 5 mg·L-1 Bialaphos. PCR assay showed that 18 plants were positive for Barnase gene with a frequency of 75%. I-KI staining and pollen germination revealed that five plants were partially male-sterile. Southern blot analysis indicated integration of Barnase gene into maize genome.     

O-Glucosyltransferase activities toward phenolic natural products and xenobiotics in wheat and herbicide-resistant and herbicide-susceptible black-grass (Alopecurus myosuroides).

Herbicide safeners manipulate herbicide selectivity by enhancing the activities of detoxifying enzymes, such as glutathione transferases (GSTs) and cytochrome P450 mono-oxygenases (CYPs) in cereal crops. As part of a study examining the importance of O-glucosyltransferases (OGTs) in pesticide metabolism in hexaploid bread wheat (Triticum aestivum L.), seedlings were grown in the presence of dichlormid, a safener used in maize and cloquintocet mexyl, a wheat safener. The efficacy of the treatments was confirmed by monitoring changes in the abundance of phi and tau class GSTs. OGT activities in the root and shoot tissue were assayed using phenolics of natural and xenobiotic origin to determine if they were enhanced by safeners. Cloquintocet mexyl selectively increased OGT activities toward xenobiotics (4-nitrophenol and 2,4,5-trichlorophenol) and flavonoids, (quercetin, luteolin, genistein and coumestrol) in both the roots and shoots. However, OGT activity towards simple phenols and phenylpropanoids was not enhanced by cloquintocet mexyl. Dichlormid was a much weaker enhancer of OGT activity, with the same subset of OGT activities increased as determined with cloquintocet mexyl, but with the effect being largely restricted to the roots. OGT activities were also determined in black-grass (Alopecurus myosuroides L.), an agronomically important weed in wheat. Two populations of black-grass differing in their sensitivity to herbicides were analysed. The population Peldon, which is resistant to multiple classes of herbicides due in part to the elevated expression of CYPs and GSTs active in herbicide detoxification, contained higher OGT activities than herbicide sensitive black-grass. Unlike wheat, treatment with cloquintocet mexyl or dichlormid, had no effect on OGT activities in either black-grass population.     

Purification and characterization of a glutathione S-transferase from benoxacor-treated maize (Zea mays).

A glutathione S-transferase (GST) isozyme from maize (Zea mays Pioneer hybrid 3906) treated with the dichloroacetamide herbicide safener benoxacor (CGA-154281) was purified to homogeneity and partially characterized. The enzyme, assayed with metolachlor as a substrate, was purified approximately 200-fold by ammonium sulfate precipitation, anion-exchange chromatography on Mono Q resins, and affinity chromatography on S-hexylglutathione agarose from total GST activity present in etiolated shoots. The purified protein migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) as a single band with a molecular mass of 27 kD. Using nondenaturing PAGE, we determined that the native protein has a molecular mass of about 57 kD and that the protein exists as a dimer. Two-dimensional electrophoresis revealed only a single protein with an isoelectric point of 5.75 and molecular mass of 27 kD. These results further suggest that the protein exists as a homodimer of two identical 27-kD subunits. The enzyme was most active with substrates possessing a chloroacetamide structure. trans-Cinnamic acid and 1-chloro-2,4-dinitrobenzene were not effective substrates. Apparent Km values for the enzyme were 10.8 microM for the chloroacetamide metolachlor and 292 microM for glutathione. The enzyme was active from pH 6 to 9, with a pH optimum between 7.5 and 8. An apparently blocked amino terminus of the intact protein prevented direct amino acid sequencing. The enzyme was digested with trypsin, and the amino acid sequences of several peptide fragments were obtained. The sequence information for the isolated GST we have designated "GST IV" indicates that the enzyme is a unique maize GST but shares some homology with maize GSTs I and III.     

Genome‐wide identification,expression analysis of auxin‐responsive GH3 family genes in maize (Zea mays L.) under abiotic stresses

Auxin is involved in different aspects of plant growth and development by regulating the expression of auxinresponsive family genes. As one of the three major auxinresponsive families, GH3(Gretchen Hagen3) genes participate in auxin homeostasis by catalyzing auxin conjugation and bounding free indole-3-acetic acid(IAA) to amino acids.However, how GH3 genes function in responses to abiotic stresses and various hormones in maize is largely unknown.Here, the latest updated maize(Zea mays L.) reference genome sequence was used to characterize and analyze the Zm GH3 family genes from maize. The results showed that 13 Zm GH3 genes were mapped on fi ve maize chromosomes(total10 chromosomes). Highly diversi fi ed gene structures and tissue-speci fi c expression patterns suggested the possibility of function diversi fi cation for these genes in response to environmental stresses and hormone stimuli. The expression patterns of Zm GH3 genes are responsive to several abiotic stresses(salt, drought and cadmium) and major stress-relatedhormones(abscisic acid, salicylic acid and jasmonic acid)Various environmental factors suppress auxin free IAA contents in maize roots suggesting that these abiotic stresses and hormones might alter GH3-mediated auxin levels. The respon siveness of Zm GH3 genes to a wide range of abiotic stresses and stress-related hormones suggested that Zm GH3 s are involved in maize tolerance to environmental stresses.     

Biochemical characterization of metabolism‐based atrazine resistance in Amaranthus tuberculatus and identification of an expressed GST associated with resistance

Rapid detoxification of atrazine in naturally tolerant crops such as maize (Zea mays) and grain sorghum (Sorghum bicolor) results from glutathione S‐transferase (GST) activity. In previous research, two atrazine‐resistant waterhemp (Amaranthus tuberculatus) populations from Illinois, U.S.A. (designated ACR and MCR), displayed rapid formation of atrazine‐glutathione (GSH) conjugates, implicating elevated rates of metabolism as the resistance mechanism. Our main objective was to utilize protein purification combined with qualitative proteomics to investigate the hypothesis that enhanced atrazine detoxification, catalysed by distinct GSTs, confers resistance in ACR and MCR. Additionally, candidate AtuGST expression was analysed in an F₂ population segregating for atrazine resistance. ACR and MCR showed higher specific activities towards atrazine in partially purified ammonium sulphate and GSH affinity‐purified fractions compared to an atrazine‐sensitive population (WCS). One‐dimensional electrophoresis of these fractions displayed an approximate 26‐kDa band, typical of GST subunits. Several phi‐ and tau‐class GSTs were identified by LC‐MS/MS from each population, based on peptide similarity with GSTs from Arabidopsis. Elevated constitutive expression of one phi‐class GST, named AtuGSTF2, correlated strongly with atrazine resistance in ACR and MCR and segregating F₂ population. These results indicate that AtuGSTF2 may be linked to a metabolic mechanism that confers atrazine resistance in ACR and MCR.     

Identification and characterization of endoplasmic reticulum-associated degradation proteins differentially affected by endoplasmic reticulum stress

The disposal of misfolded proteins from the lumen of the endoplasmic reticulum (ER) is one of the quality control mechanisms present in the protein secretory pathway. Through ER-associated degradation, misfolded substrates are targeted to the cytosol where they are degraded by the proteasome. We have identified four maize (Zea mays) Der1-like genes (Zm Derlins) that encode homologs of Der1p, a yeast (Saccharomyces cerevisiae) protein implicated in ER-associated degradation. Zm Derlins are capable of functionally complementing a yeast Der1 deletion mutant. Such complementation indicates that the Der1p function is conserved among species. Zm Derlin genes are expressed at low levels throughout the plant, but appear prevalent in tissues with high activity of secretory protein accumulation, including developing endosperm cells. Expression of three of the four Zm Derlin genes increases during ER stress, with Zm Derlin1-1 showing the strongest induction. Subcellular fractionation experiments localized Zm Derlin proteins to the membrane fraction of microsomes. In maize endosperm, Zm Derlin proteins were found primarily associated with ER-derived protein bodies regardless of the presence of an ER stress response.     

Induction of glutathione S-transferase activity and glutathione level in plants exposed to glyphosate

Treatment with the herbicide glyphosate led to significantly increased activities of the enzyme gluiathione S-transferase (GST, EC 2.5.1.18) in wheat ( Triticum aestivum L. cv. Kadett and cv. Satu), pea ( Pisum sativum L. ev. Debreceni Világoszöld) and in maize ( Zea mays. L. Pioneer 3839 hybrid) tissues. GST activities in wheat seedlings (cv. Kadett) exposed to 960 μM glyphosate for 4 days were ca 6-fold and 3-fold higher in shoots and roots, respectively, than in the controls. Glyphosate increased the GST activity to a lesser extent in pea and maize than in wheat. In wheat seedlings (cv. Satu) exposed let 120 μM glyphosate gradual increases in the content of non-protein thiols were observed. After 7 days exposure to glyphosate the thiol levels rose to about 360% and 220% of the controls in wheal shoots and roots, respectively. The elevation of thiol content in glyphosate-treated plants was shown to be primarily due increases of glutathione level. These results suggest that the enhanced glutathione metabolism may have a role in the mode of action or degradation of this herbicide.     

Glutathione transferase activity of vacuoles,plastids, and tissue extracts of red beetroot

Glutathione transferase (GST) activity revealed in vacuoles of red beetroot (Beta vulgaris L.) cells was investigated in comparison with the GST activity of plastids and extracts of tissues. The level of GST activity determined by spectrophotometric method proved fairly high in water extracts and membrane fractions of isolated vacuoles and plastids, as well as in water extracts of tissues. In the objects studied, pH dependence of the GST activity slightly differed. Optimal pH for the vacuolar GST activity was in the range 7.0–7.5, for the GST of plastids and tissue extracts it was 7.5. The GSTs differed in specificity to the substrates fluorodifen and ethacrynic acid. The activity of the vacuolar and tissue extract GSTs with fluorodifen was significantly higher than that of the GST from plastids. Ethacrynic acid, often used as a competitive inhibitor of GST, almost completely inhibited the GST activity assayed with 1-chloro-2,4-dinitrobenzene as a main substrate. However, ethacrynic acid was a substrate only for the GSTs of vacuoles and tissue extract, but not for the GST of plastids. Using zymography allowing estimation of the GST activity in a gel after electrophoresis of proteins, several zones of enzymatic activity were revealed in all objects that may correspond to different isozymes. It was found that the composition of the vacuolar GST isoforms and their substrate specificity may differ from the GSTs of other cellular structures. It is assumed that vacuole, having quite high activity of GST, should make a significant contribution to intracellular detoxification processes.     

Purification and characterization of a safener-induced glutathione S-transferase from wheat (Triticum aestivum)

The genome of cultivated wheat is hexaploid, and in consequence a large number of glutathione S-transferase (GSTs, EC 2.5.1.18) isozymes is expected in that organism. Wheat GST subunits were first analyzed by reverse-phase high performance liquid chromatography (RP-HPLC). In root and shoot tissues, subunits 4, 8, and 9 were constitutively expressed whereas subunits 2, 3, and 5 were inducible by the herbicide safener naphthalic anhydride (NA). Significant differences were observed, however, between the distributions of these six major subunits in roots and shoots. A major GST isozyme was purified from the shoots of plants treated by NA. A combination of ammonium sulphate precipitation, hydrophobic interaction chromatography (HIC) and affinity chromatography resulted in purification with an apparent yield of 4.6% and a 48-fold increase in specific activity toward 1-chloro-2,4-dinitrobenzene (CDNB). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single band at 24.5 kDa. Molecular mass estimated by nondenaturing PAGE was 49.5 kDa. These results suggest that the enzyme exists as a dimer. A pI of 5.2 was determined by native isoelectric focusing (IEF). Analysis by 2-D electrophoresis showed a single spot, with a pI of 5.8–5.9. However, further analysis by RP-HPLC revealed that the two subunits were different. They were characterized and identified by electrospray ionization mass spectrometry (ESI-MS) as subunits 2 and 3, molecular masses 24 924±3 and 24 958±5 Da, respectively. Therefore, GST(2–3) is apparently a heterodimer consisting of subunits 2 and 3. Apparent K_M values were 424 μ M for CDNB and 228 μ M for glutathione (GSH). GST(2–3) metabolized the herbicide fluorodifen, and a K _M of 22 μ M was determined for the herbicide.     

Characterization of Cytosolic Glutathione S-Transferase in Spruce Needles

Active glutathione S-transferase (GST) has been purified from needles of Norway spruce (Picea abies L. Karst.). Two isoforms of the enzyme which exhibit different physico-chemical and catalytic properties were separated by (NH₄)₂SO₄ fractionation, affinity chromatography on epoxy-activated 4% cross-linked beaded agarose, using glutathione as the ligand, ion-exchange chromatography, and isoelectric focusing. The isozymes have pI values of 5.5 (GST I) and 4.3 (GST II). Both GST isozymes are homodimeric proteins with subunit sizes of 26 kD (GST I), and 23 kD (GST II). The kinetic properties of the enzymes are described and compared with other plants GSTs. Only GST II is able to conjugate the pesticides fluorodifen and alachlor.     

The expression of a maize glutathione S-transferase gene in transgenic wheat confers herbicide tolerance,both in planta and in vitro

Maize (Zea mays), in common with a number of other important crop species, has several glutathione S-transferase (GST) isoforms that have been implicated in the detoxification of xenobiotics via glutathione conjugation. A cDNA encoding the maize GST subunit GST-27, under the control of a strong constitutive promoter, was introduced into explants of the wheat (Triticum aestivum L.) lines cv. Florida and L88-31 via particle bombardment, using the phosphinothricin acetyltransferase (pat) gene as a selectable marker. All six independent transgenic wheat lines recovered expressed the GST-27 gene. T₁ progeny of these wheat lines were germinated on solid medium containing the chloroacetanilide herbicide alachlor, and tolerance to this herbicide was correlated with GST-27 expression levels. In glasshouse sprays, homozygous T₂ plants were resistant not only to alachlor but also to the chloroacetanilide herbicide dimethenamid and the thiocarbamate herbicide EPTC. These additional GST-27 activities, demonstrated via over-expression in a heterologous host, have not been described previously. T₂ plants showed no enhanced tolerance to the herbicides atrazine (an s-triazine) or oxyfluorfen (a diphenyl ether). In further experiments, T₂ wheat plants were recovered from immature transgenic scutella cultured on medium containing 100 mg/l alachlor, a concentration which killed null segregant and wild-type scutella. These data indicate the potential of the maize GST-27 gene as a selectable marker in wheat transformation.