Proteomic characterization of herbicide safener-induced proteins in the coleoptile of Triticum tauschii seedlings

Proteomic methods such as two-dimensional gel electrophoresis and liquid chromatography tandem mass spectrometry, as well as immunoblotting, were used to identify herbicide safener-induced proteins in the coleoptile of Triticum tauschii, a diploid wheat containing the D genome also found in the cultivated, hexaploid wheat Triticum aestivum. The herbicide safener fluxofenim dramatically increased protein abundance in the molecular weight (M(r)) range of 24 to 30 kDa, as well as a few higher M(r) proteins, in the coleoptile of T. tauschii seedlings. In total, twenty proteins were identified in this study. Eleven proteins were highly safener induced and only weakly expressed in the control; seven proteins were new safener induced proteins that were not detected in the control. Two other proteins were constitutively expressed in both the control and safener-treated coleoptiles. Among the eighteen inducible proteins, fifteen were glutathione S-transferase (GST) subunits that fall into three subclasses: eight proteins were from the tau subclass, six proteins were from the phi subclass, and one protein was from the lambda class. Another three safener inducible proteins showed homology to the aldo/keto reductase family and with proteins that have roles in glycolysis and the Krebs cycle. Two constitutively expressed proteins were identified, one having highest homology to the dehydroascorbate reductase subclass of GSTs and one with an ascorbate peroxidase. Immunoblot analyses, using two different antisera raised against the same GST protein but differing in their specificity, were used to further characterize the GST proteins expressed in response to safener treatment. Results from immunoblotting, combined with mass spectral analysis, showed that post-translational modification of GST proteins in control and safener-treated coleoptiles may occur.     

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.     

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.     

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.     

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,regulation and cloning of a glutathione transferase (GST) from maize resembling the auxin-inducible type-III GSTs

The glutathione transferases (GSTs) from maize (Zea mays L.) with activities toward the chloroacetanilide herbicide metolachlor and the diphenyl ether herbicide fluorodifen were fractionated into two pools based on binding to affinity columns. Pool 1 GSTs were retained on Orange A agarose and were identified as isoenzymes Zea mays (Zm) GST I-I, Zm GST I-II and Zm GST I-III, which have been described previously. Pool 2 GSTs selectively bound to S-hexyl-glutathione-Sepharose and were distinct from the pool 1 GSTs, being composed of a homodimer of 28.5 kDa subunits, termed Zm GST V-V, and a heterodimer of the 28.5 kDa polypeptide and a 27.5 kDa subunit, termed Zm GST V-VI. Using an antibody raised to Zm GST V-VI, a cDNA expression library was screened and a Zm GST V clone identified showing sequence similarity to the type-III auxin-inducible GSTs previously identified in tobacco and other dicotyledenous species. Recombinant Zm GST V-V showed high GST activity towards the diphenyl ether herbicide fluorodifen, detoxified toxic alkenal derivatives and reduced organic hydroperoxides. Antibodies raised to Zm GST I-II and Zm GST V-VI were used to monitor the expression of GST subunits in maize seedlings. Over a 24 h period the Zm GST I subunit was unresponsive to chemical treatment, while expression of Zm GST II was enhanced by auxins, herbicides, the herbicide safener dichlormid and glutathione. The Zm GST V subunit was more selective in its induction, only accumulating significantly in response to dichlormid treatment. During development Zm GST I and Zm GST V were expressed more in roots than in shoots, with Zm GST II expression limited to the roots.     

Characterization of the safener-induced glutathione S-transferase isoform II from maize

The safener-induced maize (Zea mays L.) glutathione S-transferase, GST II (EC 2.5.1.18) and another predominant isoform, GST I, were purified from extracts of maize roots treated with the safeners R-25788 (N,N-diallyl-2-dichloroacetamide) or R-29148 (3-dichloroace-tyl-2,2,5-trimethyl-1,3-oxazolidone). The isoforms GST I and GST II are respectively a homodimer of 29-kDa (GST-29) subunits and a heterodimer of 29 and 27-kDa (GST-27) subunits, while GST I is twice as active with 1-chloro-2,4-dinitrobenzene as GST II, GST II is about seven times more active against the herbicide, alachlor. Western blotting using antisera raised against GST-29 and GST-27 showed that GST-29 is present throughout the maize plant prior to safener treatment. In contrast, GST-27 is only present in roots of untreated plants but is induced in all the major aerial organs of maize after root-drenching with safener. The amino-acid sequences of proteolytic fragments of GST-27 show that it is related to GST-29 and identical to the 27-kDa subunit of GST IV.Abbreviations CDNB 1-chloro-2,4-dinitrobenzene - DEAE di-ethylaminoethyl - FPLC fast protein liquid chromatography - GSH reduced glutathione - GST glutathione S-transferase - GST-26 26-kDa subunit of maize GST - GST-27 27-kDa subunit of maize GST - GST-29 29-kDa subunit of maize GST - R-25788 safener N,N-diallyl-2-dichloroacetamide - R-29148 safener 3-dichloroacetyl-2,2,5-trimethyl-1,3-oxazolidone - RPLC reverse phase liquid chromatography We are grateful to M-M. Lay, ZENECA AG Products (formerly ICI Americas), Richmond, Calif., USA for providing [¹⁴C] R-25788. ZENECA Seeds in the UK is part of ZENECA Limited.     

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.     

Cloning and characterization of a glutathione S-transferase induced by a herbicide safener in barley (Hordeum vulgare)

The effect of the herbicide safener mefenpyr-diethyl on glutathione S -transferase (GST, EC 2.5.1.18) activities of dark-grown barley ( Hordeum vulgare cv. Alexis) was examined. Mefenpyr-diethyl treatment increased the GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) and the herbicide fenoxaprop as substrates. Glutathione (GSH) peroxidase activity was markedly increased. GST subunits were analysed by high performance liquid chromatography (HPLC). The quantities of two major subunits were increased by the safener treatment, while the quantities of two other major subunits remained constant. A cDNA encoding the most abundant inducible GST (HvGST6) was cloned and expressed in E. coli . The purified enzyme exhibited a low activity with herbicides as substrates. By contrast, it exhibited a strong GSH peroxidase activity.     

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.     

Partial Characterization of Glutathione S-Transferase Isozymes Induced by the Herbicide Safener Benoxacor in Maize

The effects of the dichloroacetamide safener benoxacor on maize (Zea mays L. var Pioneer 3906) growth and glutathione S-transferase (GST) activity were evaluated, and GST isozymes induced by benoxacor were partially separated, characterized, and identified. Protection from metolachlor injury was closely correlated with GST activity, which was assayed with metolachlor as a substrate, as benoxacor concentration increased from 0.01 to 1 [mu]M. GST activity continued to increase at higher benoxacor concentrations (10 and 100 [mu]M), but no further protection was observed. Total GST activity with metolachlor as a substrate increased 2.6- to 3.8-fold in response to 1 [mu]M benoxacor treatment. Total GST activity from maize treated with or without 1 [mu]M benoxacor was resolved by fast protein liquid chromatography anion-exchange chromatography into four major activities, designated activity peaks A, B, C, and D in their order of elution. These GST activity peaks were enhanced to varying degrees by benoxacor. Activity peak B showed the least induction, whereas activity peak A was absent constitutively and thus highly induced by benoxacor. In contrast to earlier reports, there appear to be not one, but at least two, major constitutive isozymes (activity peaks A and D) having activity with metolachlor as substrate; there were at least three such isozymes in benoxacor-treated maize (activity peaks A, C, and D). The elution volumes of activity peaks A, B, C, and D were compared with those of partially purified maize GST I and GST II; also, the reactivity of polypeptides in these activity peaks with antisera to GST I or GST I/III (mixture) was evaluated. Evidence from these experiments indicated that activity peak B contained GST I, and activity peak C contained GST II and GST III. Activity peaks A and D contained unique GSTs that may play a major role in metolachlor metabolism and in the safening activity of benoxacor in maize. Isozymes present in activity peaks A and D were not detected in earlier reports because of the very low activity with the artificial substrate 1-chloro-2,4-dinitrobenzene. Immunoblotting experiments also indicated the presence of numerous unidentified GST subunits, including multiple subunits in chromatography fractions containing single peaks of GST activity; this is indicative of the likely complexity and diversity of the maize GST enzyme family.     

Cloning,characterization and regulation of a family of phi class glutathione transferases from wheat

Six phi (F) class glutathione transferases (GSTs) were cloned from bread wheat (Triticum aestivum L.) treated with the herbicide safener fenchlorazole ethyl and named TaGSTF1–6. Recombinant TaGSTFs were assayed for glutathione conjugating activity towards xenobiotics including herbicides and for glutathione peroxidase (GPOX) activity. TaGSTF1, which resembled ZmGSTF1, the dominant GST in maize (Zea mays), was highly active in conjugating 1-chloro-2,4-dinitrobenezene (CDNB) but had low activities towards chloroacetanilide, diphenyl ether and aryloxphenoxypropionate herbicides. TaGSTF2, TaGSTF3 and TaGSTF4 all resembled the safener-inducible ZmGSTF2, with TaGSTF2 and TaGSTF3 being highly active GPOXs and rapidly detoxifying chloroacetanilides. TaGSTF5 resembled ZmGSTF3, having limited conjugating and GPOX activity. TaGSTF6 contained both ZmGSTF1- and ZmGSTF2-like sequences but was most similar to ZmGSTF1 in detoxifying activity. The expression of TaGSTFs in wheat seedlings was enhanced upon exposure to fenchlorazole ethyl, herbicides or other chemical inducing treatments. TaGSTFs were also enhanced by treatment with the natural products caffeic acid, 7,4-dihydroxyflavone and naringenin. The CDNB-conjugating activity of TaGSTF1, and to a lesser extent TaGSTF6, was highly sensitive to inhibition by flavonoids, particularly the chalcone isoliquiritigenin. The other TaGSTFs were much less sensitive to such inhibition. It was subsequently determined that isoliquiritigenin underwent glutathione conjugation, though this reversible reaction did not require the intervention of any TaGSTF. The potential importance of GSTFs and glutathione conjugation in flavonoid metabolism is discussed.     

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.     

Selective induction of glutathione S-transferase subunits in wheat plants exposed to the herbicide acifluorfen

Exposure to the herbicide acifluorfen resulted in marked increase of glutathione S-transferase (GST) enzyme activity in wheat seedlings, primarily in shoot tissues. From the six major, constitutively expressed GST subunits found in untreated wheat shoots subunits 2 and 3 were selectively induced by acifluorfen. No new subunit could be detected. The induced subunits belong to those GST isoenzymes, which metabolize diphenyl ether herbicides.     

Specificity of the glutathione S-transferases in the conversion of leukotriene A4 to leukotriene C4

We have synthesized the 5,6-LTA4, 8,9-LTA4, and 14,15-LTA4 as methyl esters by an improved biomimetic method with yields as high as 70-80%. We have investigated the catalytic efficiency of the purified cytosolic glutathione S-transferase (GST) isozymes from rat liver in the conversion of these leukotriene epoxides to their corresponding LTC4 methyl esters. Among various rat liver GST isozymes, the anionic isozyme, a homodimer of Yb subunit, exhibited the highest specific activity. In general, the isozymes containing the Yb subunit showed better activity than the isozymes containing the Ya and/or Yc subunits. Interestingly, all three different LTA4 methyl esters gave comparable specific activities with a given GST isozyme indicating that regiospecificity of GSTs was not the factor in determining their ability to catalyze this reaction. Surprisingly, purified GSTs from sheep lung and seminal vesicles showed little activity toward these leukotriene epoxides, indicating a lack of the counterpart of rat liver anionic GST isozyme in these tissues.