Physiological responses of maize and sorghum to four different safeners for metazachlor

The ability of the herbicide safeners, BAS-145138 (1-dichloroacetyl-hexahydro-3,3,8a-trimethyl-pyrrolo(1,2a)pyrimidin-6(2H)-one), dichlormid (N,N-diallyl-2,2-dichloroacetamide), flurazole (phenylmethyl ester), and MG-191 (2-dichloromelhyl-2-methyl-1,3-dioxolane) for preventing metazachlor injury to maize (Zea mays L.) and sorghum (Sorghum bicolor L.) seedlings were compared with their effects on ¹⁴C-metazachlor metabolism to a glutathione (GSH) conjugate, effects on non-protein thiol contents (mainly GSH) and effects on Glutathione S-transferase (GST) activity in these two species. Sorghum shoot growth was reduced by 41% and maize shoot growth was reduced by 54%, by metazachlor concentrations in vermiculite nutrient culture of 0·6 μM and 7·5μM, respectively. In this system, all four compounds had significant activity as safeners for metazachlor in both sorghum and maize seedlings. BAS-145138 and flurazole were the most effective safeners in maize and sorghum, respectively. In the absence of safeners, the rate of non-enzymatic conjugation of metazachlor and GSH was much greater than the enzymatic rate. However, the rate of enzymatic conjugation of metazachlor with GSH was increased by safener treatment in both maize and sorghum. Safener effectiveness was highly correlated with increases in ¹⁴C-metazachlor uptake and metabolism in both species. Safener effectiveness was more highly correlated with safener effects on GST activity in maize or sorghum when ¹⁴C-metazachlor was used as the substrate than when the non-specific CDNB (1-chloro-2,4-dinitrobenzene) was used as the substrate. Safener effectiveness was also strongly correlated with safener effects on GSH levels in sorghum, but not in maize, possibly because of the greater importance of non-enzymatic conjugation of metazachlor with GSH in sorghum as compared to maize.     

Potential safeners for protecting sorghum (Sorghum bicolor (L.) Moench) against chlorsulfuron,fluazifop-butyl and sethoxydim*

In greenhouse studies, the efficacy of the herbicide safeners NA(1,8-naphthalic anhydride), R-25788 (N,N-diallyl-2,2-dichloroacetamide), cyometrinil and CGA-92194 [N-(1,3-dioxolan-2-yl-methoxy)imino-benzeneaceto-nitrile] in protecting grain sorghum (Sorghum bicolor (L.) Moench, cv. ‘Funk G623’) against injury from pre-emergence or early post-emergence applications of the herbicides chlorsulfuron, fluazifop-butyl and sethoxydim was examined. NA as a seed dressing at 0·5 or 1·0% (w/w) was the most effective of the four safeners and offered partial to good protection to sorghum against injury from the lower rates of pre-emergence applications of all three herbicides. R-25788 was totally ineffective as a sorghum protectant against fluazifop-butyl injury but it did antagonize partially the injurious effects of the lower rates of sethoxydim and chlorsulfuron on sorghum. Cyometrinil and CGA-92194 offered partial protection to sorghum against injury from the lowest rate of all herbicides but their efficacy against higher rates of the three herbicides was very limited. None of the four safeners was effective in protecting grain sorghum against injury from post-emergence applications of the three herbicides tested.     

Dichloroacetamide antidotes enhance thiocarbamate sulfoxide detoxification by elevating corn root glutathione content and glutathione S-transferase activity

Glutathione (GSH) content and GSH S-transferase activity are consistently increased in corn roots on 24-hr exposure of corn seedlings to part per million levels of N,N-diallyl-2,2-dichloroacetamide (R-25788) and related antidotes for thiocarbamate herbicide injury in susceptible corn varieties. This combined enhancement of enzyme activity and cofactor level leads to rapid detoxification of thiocarbamate sulfoxides, which are proposed to be the active herbicidal compounds formed on metabolic sulfoxidation. S-(N,N-Dipropylcarbamyl)-GSH is formed by this enzyme-catalyzed detoxification of EPTC sulfoxide. This hypothesis on antidote mode of action is supported by studies on 32 dichloroacetamides and related compounds and on the concentration- and time-dependent relationships of R-25788 action. The liver GSH content is normal in mice injected with high doses of R-25788, but the content is reduced when EPTC or EPTC sulfoxide is administered. EPTC sulfoxide also carbamoylates the thiol group of coenzyme A in neutral aqueous medium.     

Measuring cysteine biosynthesis activity from serine in extracts from sorghum, corn and grass weeds, and their metolachlor susceptibility

In vitro assay procedures for measuring the activity of cysteine biosynthesis from serine (CBS), which is a coupled reaction catalyzed by serine acetyltransferase and cysteine synthase, were developed using crude extracts from sorghum shoots. Cysteine biosynthesis from serine activity was dependent on acetyl‐CoA concentrations (up to 1.5 mmol L^?1), serine (at least up to 20 mmol L^?1) and sulfide (up to 0.25 mmol L^?1), respectively, and was proportional to the protein concentration in the reaction mixture below 0.4 mg mL^?1. The reaction rate was 6.6 nmol min^?1 per mg of protein during the first 5 min, but increased to 45.6 nmol min^?1 per mg of protein between 30 and 45 min after reaction initiation. Sorghum had the highest CBS total activity (222.4 nmol min^?1 per g of fresh weight), and large crabgrass had the lowest CBS total activity (4.7 nmol min^?1 per g of fresh weight) when CBS activity in shoots was extracted from sorghum, corn, johnsongrass, barnyardgrass, goosegrass, green foxtail and large crabgrass. Similar results were obtained for CBS specific activity (nmol min^?1 per mg of protein). There was no correlation between total CBS activity and susceptibility to metolachlor; however, when corn was excluded, a correlation of R² = 0.690 was found. Flurazole seed treatment (1.25 g per kg of seed) conferred metolachlor resistance by sorghum, and enhanced total CBS activity and non‐protein thiol content by 27 and 61%, respectively. The increase in thiol content presumably contributed to metolachlor tolerance in sorghum. From these results, the difference in CBS activity partially contributes to the selectivity to metolachlor among certain grass species, and to the safening action of flurazole by increasing thiol content.     

Purification and characterisation of a family of glutathione transferases with roles in herbicide detoxification in soybean (Glycine max L.); selective enhancement by herbicides and herbicide safeners

Glutathione transferases in soybean (GmGSTs) involved in herbicide detoxification in cell suspension cultures were purified by S-hexylglutathione affinity chromatography and resolved by a combination of HPLC and SDS-PAGE into 11 polypeptides. Analysis by Western blotting using antisera raised to three previously characterised tau (GmGSTU) class subunits demonstrated that five polypeptides were related to GmGSTU1, three to GmGSTU2, and one to Gm GSTU3. Plants contained a simpler profile of polypeptides, with a single GmGSTU2-like polypeptide predominating. With respect to herbicide detoxification, two GmGSTU2-related polypeptides dominated the activity toward the chloroacetanilide acetochlor, while an unclassified subunit was uniquely associated with the detoxification of diphenyl ethers (acifluorfen, fomesafen). The inducibility of the different GST subunits was determined in soybean plants exposed to photobleaching diphenyl ethers and the safeners naphthalic anhydride and dichlormid. GmGSTU3, a GmGSTU1-like polypeptide, and thiol (homoglutathione) content were induced by all chemical treatments, while two uncharacterised subunits were only induced in plants showing photobleaching.     

Herbicide antidotes—A review

The history of herbicide antidotes is reviewed, beginning with the exploration of compounds to protect wheat (Triticum aestivum L.) against barban in the early 1960s, and the later introduction of naphthalic anhydride (NA, naphthalene-1, 8-dicarboxy-licanhydride) as a seed dressing for protecting maize (Zea mays L.) against EPTC. This compound was largely replaced by Stauffer's R-25788 (N, N-diallyl-2, 2-dichloroacetamide) which has continued to be widely used in conjunction with EPTC and butylate in maize. This compound is highly specific to maize and can thus be applied in admixture with the herbicide, but has not proved of practical value on other crop species. NA on the other hand is less specific and is of potential value on sorghum [Sorghum bicolor (L.) Moench] and rice (Oryza sativa L.); experimental work continues on these crops. The only other antidote to be marketed so far is cyometrinil as a seed dressing for protecting sorghum against metolachlor and related herbicides. Other compounds are under development. Mode of action and structure-activity relations are discussed, as well as the current and future potential for antidotes in respect of the control of weed species in closely related crops, the increased options for herbicide use in minor crops and the possibility of reduced costs for broad spectrum weed control in major crops.     

Safener effects on acetochlor toxicity, uptake, metabolism and glutathione S-transferase activity in maize

The effect of five herbicide safeners on preventing maize (Zea mays L.) injury by acetochlor [N-(2-ethyl-6-methyl-phenyl)-N-(ethoxymethyl)-chloroacetamide], their influence on herbicide uptake and metabolism to a glutathione (GSH) conjugate as well as on GSH content and glutathione S-transferase activity (GST) in untreated and herbicide with/without safener-pre-treated 4-day-old seedlings were determined. The safeners studied were: AD-67 (N-di-chloroacetyl-1-oxa-4-azaspiro-4-5-decane), BAS-145138 [1-dichloroacetyl-hexahydro-3,3,8a-trimethyl-pyrrolo(l,2-a)pyrimidin-6(2H)-one], dichlormid (N, A,-diallyl-2,2-dichlo-roacetamide), DKA-24 (N, N²-diallyl-N²dichloroacetylglycineamide) and MG-191 (2-dichloromethyl-2-methyl-1,3-dioxolane). All safeners significantly increased [¹⁴C]acetochlor uptake and metabolism rate, maize GSH content and GST activity. Seedlings receiving BAS-145138 pre-treatment metabolized almost 70% of the absorbed [¹⁴C]acetochlor within 10 min. Safener-enhanced GST activity was always found to be higher when [¹⁴C]acetochlor was used as the substrate compared with CDNB (1-chloro-2,4-dinitrobenzene). Although DKA-24 had a significantly lower influence on both herbicide metabolism and GST activity, it was nearly as effective a safener as BAS-145138, while the others provided no or poor protection to maize from acetochlor injury when they were not incorporated in the soil. Effets d'antidotes sur la toxicité, la pénétration et le métabolisme de I'acétochlore chez le mats etsur l'activité glutathion S-transférase Cinq antidotes d'herbicides diminuaient la toxicité de 1'acétochlore [N-(2-éthyl-6-méthyl-phényl)-N-(éthoxyméthyl)-chloroacétamide] à I'égard du maïs (Zea mays L.). Lew effet sur l'absorption de l'herbicide et sur son métabolisme en un conjugué avec le glutathion (GSH), de même que leur effet sur la teneur en GSH et 1'activité glutathion 5-transférase (GST) ont étéétudiés sur des maïs âgés de 4 jours. Les antidotes étaient: l'AD-67 (N-dichloroacétyl-1-oxa-4-azaspiro-4,5-décane), le BAS-145138 [1 -dichloroacétyl-hexahydro-3,3,8a-triméthyl-pyrrolo(l,2-a) pyrimidine-6(2H)-one], le di-chlormide (N, N-diallyl-2,2-dichloracétamide), le DKA-24 (N, N²-diallyl-N²-dichloracétylgly-cineamide] et le MG-191 (2-dichlorométhyl-2-méthyl-l,3-dioxolane). Tous les antidotes augmentaient de manière significative 1'absorption et le métabolisme de I'acétochlore ¹⁴C, ainsi que la teneur du maïs en GSH et son activité GST. Les jeunes plantes prétraitées avec le BAS-145138 métabolisaient en 10 min près de 70% de I'acétochlore ¹⁴C absorbé. L'activité GST stimulée par 1'antidote était toujours plus élevée avec I'acétochlore ¹⁴C comme substrat qu'avec le CDNB (1-chloro-2,4-dinitrobenzène). Le DKA-24 avait un effet significativement plus faible que le BAS-145138 sur le métabolisme de l'herbicide et sur l'activité GSH, mais son action antidote était presque aussi importante. Les autres produits n'apportaient au maïs qu'une protection faible ou nulle contre l'acétochlore quand ils n'étaient pas incorporés au sol. Wirkung von Safenern auf die Phytotoxizität, Aufnahme und Metabolismus von Acetochlor und die Glutathion-S-Transferase-Aktivität in Mais Die Wirkung von 5 Safenern (AD-67, BAS-145138, Dichlormid, DKA-24 und MG-191; chemische Bezeichnungen s.o.) auf die Phytotoxizität von Acetochlor für Mais (Zea mays L.) und ihr Einfluß auf die Aufnahme des Herbizids und Metabolismus zu einem Glutathion-(GSH-) Konjugat und auf die Glutathion-S-Transferase-(GST-)Aktivität wurde an 4 Tage alten Keimpflanzen untersucht. Durch die Vorbehandlung mit den Safenern wurden die [¹⁴C]-Acetochlor-Aufnahme- und -Metabolierungsrate, der GSH-Gehalt und die GST-Ak-tivität signifikant erhöht. Mit BAS-145138 vorbehandelte Keimpflanzen metabolisierten fast 70% des absorbierten [¹⁴C]-Acetochlor innerhalb von 10 min. Die durch die Safener erhöhte GST-Aktivität bei [¹⁴C]-Acetochlor als Substrat war im Vergleich zu Chlordinitroben-zen immer höher. Obwohl DKA-24 einen signifikant geringeren Einfluß sowohl auf den Herbizidmetabolismus als auch auf die GST-Aktivität hatte, war es fast so wirkungsvoll wie BAS-145138, während die anderen ohne Einarbeitung in den Boden für den Mais keinen oder geringen Schutz gegen Schäden durch Acetochlor boten.     

Phytotoxicity of solanapyrones A and B produced by the chickpea pathogen Ascochyta rabiei(Pass.) Labr. and the apparent metabolism of solanapyrone A by chickpea tissues

When chickpea shoots were placed in solanapyrone A, the compound could not be recovered from the plant and symptoms developed. These consisted of loss of turgor, shrivelling and breakage of stems and flame-shaped, chlorotic zones in leaflets. In similar experiments with solanapyrone B, only 9.4% (22 μ g) of the compound taken up was recovered and stems remained turgid but their leaflets became twisted and chlorotic and some abscized.Cells isolated from leaflets of 12 chickpea cultivars differed by up to five-fold in their sensitivity to solanapyrone A and this compound was 2.6–12.6 times more toxic than solanapyrone B, depending on cultivar.Glutathione reacted with solanapyrone A in vitro reducing its toxicity in a cell assay and forming a conjugate. Measurement of reduced glutathione concentration and glutathione-S-transferase (GST) activity among cultivars showed that the differences of their means were highly significant and both were negatively and significantly correlated with their sensitivity to solanapyrone A. Treatment of shoots with solanapyrone A enhanced total, reduced and oxidized glutathione content as well as GST activity 1.26-, 1.23-, 1.50- and 1.94-fold, respectively. Similarly, treatment of shoots with the safener, dichlormid, also raised total, oxidized and reduced glutathione levels and GST activity 1.42-, 1.07-, 1.43-, 1.42-fold, respectively. Cells isolated from shoots treated with dichlormid at 150 and 300 μ g per shoot were 2.45 and 2.66 times less sensitive to solanapyrone A, with LD₅₀values of 71.5 and 77.8 μ g ml⁻¹, respectively, as compared to 29.2 μ g ml⁻¹for controls.     

Absorption and distribution of flurazole and acetochlor in grain sorghum

The site of uptake, absorption, and distribution of a safener, flurazole [2-chloro-4-(trifluoromethyl)-5-thiazolecarboxylic acid, (phenylmethyl ester)], and a herbicide, acetochlor [2-chloro-N-(ethoxymethyl)-6′-ethyl-O-acetoluidide], in grain sorghum [Sorghum bicolor (L.) Moench “G-522 DR”] were investigated in laboratory and growth chamber studies. Acetochlor was absorbed through shoots while flurazole was taken up primarily by roots. Uptake of [¹⁴C]acetochlor into the plant was rapid, linear, and the ¹⁴C was concentrated in primary roots by 7 days. Absorption of [¹⁴C]flurazole by sorghum was immediate, leveled off at 4 days, and the ¹⁴C was concentrated in primary roots by 7 days. Absorption and distribution of either chemical were not affected by the presence of the other. Flurazole had a slight effect on acetochlor metabolism at 3 days, but by 6 days no differences were noted.     

Glutathione transferase activities toward herbicides used selectively in soybean

Using extracts from suspension-cultured cells of soybean (Glycine max cv. Mandarin) as a source of active enzymes, the activities of glutathione transferases (GSTs) catalysing the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and selective herbicides were determined to be in the order CDNB≫ fomesafen>metolachlor=acifluorfen>chlorimuron-ethyl. GST activities showed a thiol dependence in a substrate-specific manner. Thus, GST activities toward acifluorfen and fomesafen were greater when homoglutathione (hGSH), the endogenously occurring thiol in soybean, was used as the co-substrate rather than glutathione (GSH). Compared with GSH, hGSH addition either reduced or had no effect on GST activities toward other substrates. In the absence of enzyme, the rates of hGSH conjugation with acifluorfen, chlorimuron-ethyl and fomesafen were negligible, suggesting that rapid hGSH conjugation in soybean must be catalysed by GSTs. GST activities were subsequently determined in 14-day-old plants of soybean and a number of annual grass and broadleaf weeds. GST activities of the plants were then related to observed sensitivities to post-emergence applications of the four herbicides. When enzyme activity was expressed on a mg^-1 protein basis, all grass weeds and Abutilon theophrasticontained considerably higher GST activity toward CDNB than soybean. With fomesafen as the substrate, GST activities were determined to be in the order soybean≫Echinochloa crus-galli>Digitaria sanguinalis>Sorghum halepense=Setaria faberi with none of the broadleaf weeds showing any activity. This order related well to the observed selectivity of fomesafen, with the exception of A. theophrasti, which was partially tolerant to the herbicide. Using metolachlor as the substrate the order of the GST activities was soybean>A. theophrasti≫S. halepense>Amaranthus retroflexus>Ipomoea hederacea, with the remaining species showing no activity. GST activities toward metolachlor correlated well with the selectivity of the herbicide toward the broadleaf weeds but not toward the grass weeds. Acifluorfen and chlorimuron-ethyl were selectively active on these species, but GST activities toward these herbicides could not be detected in crude extracts from whole plants. © 1997 SCI     

Molecular cloning, expression, and characterization of a phi-type glutathione S-transferase from Oryza sativa

The structural gene for glutathione S-transferase in Oryza sativa was successfully cloned from a cDNA library by the polymerase chain reaction method. The deduced amino acid sequence of this gene showed 44-66% similarity to the sequences of the class phi GSTs from Arabidopsis thaliana and Zea mays. This gene was expressed in Escherichia coli with the pET vector system and the gene product was purified to homogeneity by GSH-Sepharose affinity column chromatography. The expressed OsGSTF3-3 was a homo-dimer composed of 24 kDa subunit and its pI value was approximately 7.3. The OsGSTF3-3 was retained on GSH affinity column and its K_m value for GSH was 0.28 mM. The OsGSTF3-3 displayed high activity toward 1-chloro-2,4-dinitrobenzene, a general GST substrate and also had high activities towards acetanilide herbicides, alachlor, and metolachlor. The OsGSTF3-3 was highly sensitive to inhibition by benastatin A and S-hexyl-GSH. From these results, the expressed OsGSTF3-3 is a phi class GST and seems to play an important role in the conjugation of the chloroacetanilide herbicides.     

Characterisation of Multiple Glutathione Transferases Containing the GST I Subunit with Activities toward Herbicide Substrates in Maize (Zea mays)

The glutathione transferases (GSTs) of maize with activities toward chloroacetanilide herbicides are relatively well characterised, but their range of substrate specificities has not been determined in detail. GST activities toward an extensive range of chemically diverse xenobiotic substrates, including the herbicides atrazine, alachlor, metolachlor and fluorodifen, have been determined in crude and purified preparations from the roots and shoots of dark-grown maize seedlings treated with and without the herbicide-safener dichlormid. With the exception of the activity toward atrazine, specific activities were higher in the roots than in the shoots in all cases. In untreated shoots activities were in the order atrazine=alachlor=metolachlor>fluorodifen with safener–treatment selectively increasing the activity toward the chloroacetanilides and fluorodifen. In the roots the highest GST activities toward herbicides were toward the chloroacetanilides. Dichlormid treatment resulted in an increase in activities toward all four herbicides in the roots of one maize cultivar (Pioneer 3394) but only enhanced the activities toward the chloroacetanilides and fluorodifen in cultivar Artus. Using the non-herbicide 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, anion-exchange chromatography showed that the roots and shoots contained a similar range of GST isoenzymes. All of these isoenzymes were enhanced in response to safeners, though the extent of this induction was organ-dependent. GST isoenzymes containing the GST I subunit were purified from safener-treated roots by a combination of hydrophobic interaction chromatography and affinity chromatography using Orange A agarose. Three isoenzymes could then be purified following resolution by anion-exchange chromatography. The three GSTs were termed GST I/I, GST I/II and GST I/III with GST I, II and III referring to the presence of 29 kDa, 27 kDa and 26 kDa subunits respectively. This revised nomenclature for the maize GSTs was considered necessary in view of the continued discovery of new isoenzymes, such as GST I/III, composed of subunits which have been previously described. GST I/I had measurable activity toward atrazine, low activities toward the other herbicides and appreciable activities toward a range of other xenobiotic substrates. GST I/II and the novel GST I/III isoenzymes both showed high activities toward the chloroacetanilides and fluorodifen but lower activities toward the other substrates and negligible activities toward atrazine. The GST II subunit of GST I/II also had activity as a glutathione peroxidase. Our results show that the GST I subunit can form dimers with the GST III subunit in addition to the GST I and GST II subunits and that the degree of specificity toward herbicide substrates of the respective isoenzymes is greater than previously reported. Our results also suggest that the safener-inducible GST II subunit has additional activities as a glutathione peroxidase. © 1997 SCI.     

Dichloroacetamide herbicide antidotes enhance sulfate metabolism in corn roots

The herbicide antidotes N,N-diallyl-2,2-dichloroacetamide (R25788) and 3-dichloroacetyl-2,2,5-trimethyloxazolidine (R29148) at ppm levels slightly enhance the uptake of [³⁵S]sulfate in corn roots and greatly increase its metabolism to “bound sulfide”, cysteine, and glutathione (GSH). The decrease in free sulfate content of the roots with R25788 is closely associated with an increase in GSH level. The sulfate content is decreased with an 8-hr exposure to R25788 and R29148 at 3 ppm and its decline continues through 48 hr to about 5% of the control level. Effects on sulfate content are evident at 24 hr even with 0.3–1 ppm of these antidotes. Several other mono- or dichloroacetamide antidotes at 30 ppm also decrease the free sulfate content of corn roots to about 34–60% of control levels within 24 hr. R25788 at 30 ppm has little or no effect at 24 hr on sulfate levels in corn leaves whether the plants are grown in the light or in the dark. R25788 and R29148 decrease sulfate levels in the leaves of milo and in whole pigweed plants, but not in barley, lambsquarters, water grass, wheat, or wild mustard. In increasing GSH biosynthesis, the antidote acts in corn prior to the reduction step to form bound sulfide; in fact, R25788 increases the specific activity of ATP sulfurylase, the first enzyme involved in sulfate assimilation. Thus, dichloroacetamides such as R25788 and R29148 provide a means to experimentally, and perhaps even practically, manipulate sulfate utilization in corn and some other plants.     

Synergism of diazinon toxicity and inhibition of diazinon metabolism in the house fly by tridiphane: Inhibition of glutathione S-transferase activity

Diazinon toxicity to a susceptible strain of house fly (Musca domestica L.) was synergized by tridiphane [2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethyl)oxirane], a herbicide synergist. Both diazinon and tridiphane were partially metabolized in the house fly by glutathione (GSH) conjugation. Synergism appeared to be due to inhibition of diazinon metabolism/detoxification. Crude glutathione S-transferase (GST) preparations from the house fly catalyzed GSH conjugation of diazinon, tridiphane, 3,4-dichloronitrobenzene (DCNB), and chloro-2,4-dinitrobenzene (CDNB). Tridiphane and the GSH conjugate of tridiphane appeared to inhibit diazinon GSH conjugation, but diazinon did not inhibit tridiphane GSH conjugation. The enzymatic rate of tridiphane GSH conjugation was 22 times the rate of diazinon GSH conjugation; therefore, attempts to assay tridiphane as an inhibitor of diazinon GSH conjugation were inconclusive because of the high concentration of tridiphane GSH conjugate produced during the assay. CDNB underwent enzymatic GSH conjugation at a rate 240 times faster than that of tridiphane and 5000 times faster than that of diazinon. GSH conjugation of CDNB was not inhibited by tridiphane, but was inhibited by the GSH conjugate of tridiphane. In vivo, the GSH conjugate of tridiphane was produced in sufficient concentration to cause the observed inhibition of diazinon metabolism and synergism of diazinon toxicity. However, the possibility that parent tridiphane caused or contributed to the inhibition of diazinon metabolism and synergism of diazinon toxicity could not be excluded. Inhibition of diazinon metabolism did not appear to be due to depletion of either GSH or GST.     

Tridiphane [2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethyl)oxirane] an atrazine synergist: Enzymatic conversion to a potent glutathione S-transferase inhibitor

Glutathione S-transferases (GST) from corn, giant foxtail, onion, pea, house fly, and equine liver catalyzed conjugation of tridiphane with glutathione (GSH). The conjugate was characterized by soft ionization mass spectral methods. Tridiphane and the GSH conjugate of tridiphane both inhibited GSH conjugation of atrazine in vitro (corn and giant foxtail). Tridiphane did not inhibit GSH conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) in corn or giant foxtail; however, the GSH conjugate of tridiphane was a competitive inhibitor with respect to GSH and was four times more effective with extracts from giant foxtail (K_i = 2 μM) than from corn (K_i = 8 μM). The GSH conjugate of tridiphane inhibited a variety of GST enzymes with several different substrates. When compared to other inhibitors of GST, only triphenyl tin chloride was more effective than the GSH conjugate of tridiphane in inhibition of GST from giant foxtail. Both GST and GSH decreased in corn and increased in giant foxtail as tissues matured. The catabolism of the GSH conjugate of tridiphane was compared in crude enzyme systems from corn, giant foxtail, and onion. The rate of catabolism was much greater in extracts from corn leaves than from giant foxtail leaves. Inhibition of GSH conjugation of CDNB was reversed as the GSH conjugate of tridiphane was catabolized. The possibility that synergism of atrazine toxicity by tridiphane is mediated by conversion of tridiphane to a GSH conjugate is discussed in relationship to the relative rates of GSH conjugation of tridiphane and atrazine, concentrations of GSH, K_i values, tissue age, and stability of the conjugate in different tissues.     

Physiological interactions between the herbicide EPTC and selected analogues of the antidote naphthalic anhydride on two hybrids of maize

The efficacies of nine structural analogues of the herbicide antidote naphthalene-1,8-dicarboxylic acid anhydride (naphthalic anhydride, NA) for the protection of maize (Zea mays L. cv. DeKalb XL72AA and DeKalb XL67) against injury by the herbicide S-ethyl dipropyl(thiocarbamate) (EPTC) were elevated under greenhouse conditions. The chemical analogues of NA tested were: acenaphthenequinone (ACQ); 4-aminonaphthalene-1,8-dicarboxylic acid anhydride (NH₂NA); 1,8:4,5-naphthalenetetracarboxylic acid dianhydride (NDiA); naphthalene- 1,8-carboximide (NHNA); 4-chloronaphthalene-1,8-dicarboxylic acid anhydride (C1NA); biphenyl-2,2′-dicarboxylic acid anhydride (diphenic anhydride; DA); 2-phenylglutaric anhydride (PGA); phthalic anhydride (PHA); phenalen-1-one (PA). Pre-plant incorporated applications of EPTC at 2.2, 4.5, 6.7, and 9.0 kg ha^?1 were highly toxic to XL67 maize. Appreciable injury to XL72AA maize by EPTC was observed only with the high rates of EPTC (6.7 and 9.0 kg ha^?1). Of the analogues tested PGA and PA were very toxic and inhibited germination of both maize hybrids. NA, ACQ, NH₂NA, NDiA, NHNA, C1NA, DA, and PHA applied as seed dressings at 5.0 and 10 g per kg of seed offered satisfactory protection to XL72AA maize against EPTC rates higher than 6.7 kg ha^?1. The same antidotes significantly antagonised the EPTC activity against XL67 maize but the overall protection obtained was partial and not agronomically important. The presence of the dicarboxylic anhydride group and of at least one aromatic ring attached directly to the anhydride appeared to be essential for the exhibition of protective activity by the structural analogues of NA. NA was slightly toxic to both hybrids of maize and chlorination of NA increased the phytotoxicity of this molecule. A genetic component that is present in the thiocarbamate-tolerant XL72AA hybrid but absent from the thiocarbamate-susceptible XL67 hybrid of maize appeared to be important for the phytotoxic activity of EPTC and may be involved in the protective activity of NA and its structural analogues.     

Glutathione transferases in herbicide-resistant and herbicide-susceptible black-grass (Alopecurus myosuroides)

Glutathione transferase (GST) activities toward the selective herbicide fenoxaprop-ethyl, together with thiol contents, have been compared in seedlings of wheat (Triticum aestivum) and two populations of black-grass (Alopecurus myosuroides) which are resistant to a range of herbicides (Peldon and Lincs E1), and a black-grass population which is susceptible to herbicides (Rothamsted). GST activities toward the non-cereal herbicides metolachlor and fluorodifen were also determined. On the basis of enzyme specific activity, GST activities toward fenoxaprop-ethyl in the leaves were in the order wheat>Peldon=Lincs E1>Rothamsted, while with fluorodifen and metolachlor the order was Peldon=Lincs E1>Rothamsted>wheat. Using an antibody raised to the major GST from wheat, which is composed of 25-kDa subunits, it was shown that the enhanced GST activities in both Peldon and Lincs E1 correlated with an increased expression of a 25-kDa polypeptide and the appearance of novel 27-kDa and 28-kDa polypeptides. Leaves of both wheat and black-grass contained glutathione and hydroxymethylglutathione, with the concentrations of glutathione being in the order Peldon>Lincs E1=Rothamsted=wheat. However, in glasshouse dose-response assays, the Lincs E1 population showed much greater resistance to fenoxaprop-ethyl than Peldon. We conclude that high GST activities and the availability of glutathione may contribute partially to the relative tolerance of black-grass to herbicides detoxified by glutathione conjugation. Although herbicide-resistant populations show enhanced GST expression, in the case of fenoxaprop-ethyl the associated increased detoxifying activities alone cannot explain the differences between populations in the degree of resistance seen at the whole plant level. ©1997 SCI     

The effect of temperature on glutathione S-transferase activity and glutathione content in Alopecurus myosuroides (black grass) biotypes susceptible and resistant to herbicides

Herbicide‐resistant populations of Alopecurus myosuroides (black grass) have become widespread throughout the UK since the early 1980s. Previous observations in this laboratory have demonstrated that natural climatic fluctuations caused increases in endogenous glutathione S‐transferase (GST) enzyme activity in A. myosuroides plants as they mature, which is thought to be linked to herbicide resistance in this species. The present study has investigated the effects of plant growth at 10°C and 25°C, and reports GST specific activity and glutathione (GSH) pool size in resistant and susceptible A. myosuroides biotypes. Findings demonstrate differences in GST activity between resistant and susceptible populations, which are transient at lower growth temperatures. The GSH pool size was elevated at lower growth temperature in both biotypes. We speculate that these endogenous responses are part of a natural mechanism of acclimation to environmental change in this species and suggest that resistant plants are more able to adapt to environmental stress, as indicated in this instance by temperature change. These observations imply that the control of resistant A. myosuroides by graminicides may be more effective when applied at lower temperatures and at earlier growth stages.     

Metolachlor influence on growth and terpenoid synthesis

Height and fresh weight of sorghum (Sorghum bicolor L. var GA 522 DR) grown in sand were reduced by metolachlor (2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl) acetamide) (0, 0.125, 0.25, 0.5, or 1 ppmw) applied preemergence. Significantly different responses were obtained from plants grown at two light intensities (270 and 27 μein/m²/sec). When grown in nutrient solution containing 0, 0.0156, 0.0625, 0.25, 1, 16, or 64 ppmw metolachlor, shoot and root lengths were inhibited by metolachlor; fresh and dry weights of shoot, root, and total plant decreased as metolachlor concentration increased. Carotene content (micrograms per gram fresh weight) in sorghum leaves was decreased by metolachlor. Specific activity of carotene synthesized from [2-¹⁴C] mevalonic acid by carrot (Daucus carota L.) disks was reduced 50% by 10^?5 and 10^?4M metolachlor. Thus, terpenoid biosynthesis is influenced by metolachlor. Gibberellins are terminal products of plant terpenoid biosynthesis, and GA₃ reverses metolachlor inhibition of growth at specific ratios of GA₃ and metolachlor but not at other concentrations. Thus, one effect of metolachlor on plants may be an inhibition of GA synthesis that results in shoot and root growth reductions as metolachlor concentration increases. Other growth responses of plants to metolachlor are unexplained.