Diphenyl Ether Herbicides Research Articles

Imazamox and Diphenylether Herbicide Interactions in Soybean (Glycine max)

The effect of three diphenylether herbicides on the control of green foxtail, kochia, velvetleaf, and ivyleaf morningglory with imazamox in soybean was evaluated in a field study conducted near Manhattan, Kansas in 1997 and 1998. Herbicide treatments were: imazamox at 26 and 44 g ai/ha; acifluorfen and fomesafen each at 140 and 280 g ai/ha; lactofen at 70 and 140 g ai/ha; and tank mixtures of imazamox at both rates with both rates of acifluorfen, fomesafen, and lactofen. Tank mixtures of 26 g/ha imazamox with either rate of acifluorfen, or fomesafen were antagonistic on kochia control, as was 44 g/ha imazamox with 280 g/ha acifluorfen 4 weeks after treatment. Tank mixtures of 26 g/ha imazamox and 280 g/ha acifluorfen or fomesafen were antagonistic on velvetleaf control. The only tank mixture that resulted in reduced ivyleaf morningglory control was 26 g/ha imazamox plus 70 g/ha lactofen. The tank mixture of 26 g/ha imazamox plus 280 g/ ha fomesafen was antagonistic for green foxtail. When a diphenylether herbicide was applied with imazamox, 44 g/ha imazamox resulted in fewer antagonistic interactions than mixtures with 26 g/ha.

Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase gene are resistant to diphenyl ether herbicide oxyfluorfen.

Protoporphyrinogen oxidase (Protox), the penultimate step enzyme of the branch point for the biosynthetic pathway of Chl and hemes, is the target site of action of diphenyl ether (DPE) herbicides. However, Bacillus subtilis Protox is known to be resistant to the herbicides. In order to develop the herbicide-resistant plants, the transgenic rice plants were generated via expression of B. subtilis Protox gene under ubiquitin promoter targeted to the cytoplasm or to the plastid using Agrobacterium-mediated gene transformation. The integration and expression of the transgene were investigated at T0 generation by DNA and RNA blots. Most transgenic rice plants revealed one copy transgene insertion into the rice genome, but some with 3 copies. The expression levels of B. subtilis Protox mRNA appeared to correlate with the copy number. Furthermore, the plastidal transgenic lines exhibited much higher expression of the Protox mRNA than the cytoplasmic transgenic lines. The transgenic plants expressing the B. subtilis Protox gene at T0 generation were found to be resistant to oxyfluorfen when judged by cellular damage with respect to cellular leakage, Chl loss, and lipid peroxidation. The transgenic rice plants targeted to the plastid exhibited higher resistance to the herbicide than the transgenic plants targeted to the cytoplasm. In addition, possible resistance mechanisms in the transgenic plants to DPE herbicides are discussed.

Determination of diphenyl-ether herbicides and metabolites in natural waters using high-performance liquid chromatography with diode array tandem mass spectrometric detection

A method for the identification and quantification of neutral diphenyl-ether (DPhE) (aclonifen, bifenox, fluoroglycofen, lactofen, oxyfluorfen) and acid metabolites (acifluorfen, bifenox acid, fomesafen) at a low nanogram per liter concentration level in natural water is presented. These herbicides and their metabolites were determined in drinking water, groundwater, and river water. The analytes were isolated from water samples by solid-phase extraction (SPE) on Carbograph-1 cartridges and analyzed with reversed phase high-performance liquid chromatography (RP-HPLC) using UV detection at a wavelength of 290 nm. The isolation procedure separated the acid DPhE from neutral DPhE during the elution from Carbograph-1 cartridge using dichloromethane:methanol (80:20, v/v) for neutral compounds and dichloromethane:methanol (80:20, v/v) acidified with 25 mmol/l of formic acid for the anionic metabolites. Quantification was performed by generation of an external calibration curve. High recoveries (>85%) of extraction were obtained for all the compounds. The method detection limit ranged from 4.1 to 8.7 ng/l for drinking water, from 5.1 to 9.5 ng/l for groundwater and from 17.5 to 36.2 ng/l for river water. This method involves confirmatory analysis by tandem mass spectrometry (MS-MS) in selected reaction monitoring (SRM) mode. Conditions for MS-MS detection of characteristic daughter ions formed by collision-induced dissociation of the parent ion are described. The final samples were analyzed by HPLC–MS-MS utilizing a heat-assisted electrospray interface (turbo ion spray) (TISP) for acid DPhE and a heated nebulizer (HN) interface for neutral DPhE. Quantification was performed by generation of an internal calibration curve. Excellent method precision was demonstrated with a relative standard deviation of less than 18% for all analytes at all concentration levels. Application of the method for detecting DPhE herbicide residues in a real-world groundwater samples was demonstrated.

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.

Overexpression of plastidic protoporphyrinogen IX oxidase leads to the diphenyl-ether herbicide acifluorfen

Overexpression of plastidic protoporphyrinogen IX oxidase leads to the diphenyl-ether herbicide acifluorfen

Mitotic Index and Cell Proliferation Kinetics as Additional Variables for Assessment of Genotoxic Effect of The Herbicide Modown

·ivikova K. , J . Dianovsk : Mitotic Index and Cell Proliferation Kinetics as Additional Variables for Assessment of Genotoxic Effect of The Herbicide Modown. Acta Vet. Brno 2000, 69: 45–50. The in vitro effect of the herbicide Modown (with active component bifenox) was tested for the ability to influence cell proliferation of PHA-stimulated bovine peripheral lymphocytes. Mitotic (MI) and proliferation (PI) indices were determined as an alternative for the screening of the cytostatic activity. The herbicide Modown exerted a clear effect on the inhibition of MI and PI over a concentration-tested range of 25 μg/ml to 1000 μg/ml. An expressive proliferation delays was found after the treatment with herbicide at a dose of 250 μg/ml (P < 0.001), while the higher doses of 500 and 1000 μg/ml had caused nearly complete mitotic inhibition in each donor (P < 0.05 and P < 0.001, respectively). A correlation between the PI and MI inhibition refers rather to cytostatic than cytotoxic effects of the herbicide. The results support the possibility of immunosuppression by herbicide exposure. Herbicide Modown, bovine peripheral lymphocytes, cell proliferation, cytostatic effect Nitrodiphenylether herbicides are persistent and lipophilic environmental contaminants may contribute to the impairment of animal health and production. The presence of pesticide residues has been demonstrated in raw bovine milk and in different domestic animal tissues (Lioi et al. 1998). All nitrodiphenylether herbicides exert their phytotoxic activity in chlorophyll of plants by the reduction itself to radical species, which initiate destructive reactions in membrane lipids, leading to the cell leakage (Corbet t et al. 1984). Members, as acifluorfen and oxyfluorfen, e.g. are extremely potent inhibitors of protoporphyrinogen oxidase, a membrane–bound enzyme involved in the heme and chlorophyll biosynthesis pathways (Camadro et al. 1995). The later was studied for the induction of haematological diseases in human erythroid progenitor cells but a cytotoxic effect had been seen only at very high concentrations (Rio et al. 1997). Bifenox is a selective herbicide used in control of annual broad-leaved weeds and some grasses, e. g. in cereals, maize, soya beans, rice and other crops. J inno et al. (1999) described the cytotoxic and porphyrogenic effects of bifenox in rat hepatocytes. They found the maximum porphyrin accumulation at 0.25 mM of the herbicide and an inhibion of protoporphyrogen oxidase, resulting in the accumulation of protoporphyrin IX. Francis et al. (1999) evaluated maternal and developmental toxicity of ten diphenylether herbicides (including bifenox) in mice. They found a correlation among the position of chlorine substituents and the potential for inducing prenatal and postnatal syndroms. But no prenatal and postnatal embryotoxicity was shown. ACTA VET. BRNO 2000, 69: 45–50 Address for correspondence: RNDr. Katarina ·ivikova, CSc. Department of Veterinary Genetics, University of Veterinary Medicine Komenskeho 73, 041 81 Ko‰ice, Slovak Republic Phone: +421 95 63 321 11, (ext. 482) Fax: +421 95 63 236 66 E-mail: sivikova@uvm.sk http://www.vfu.cz/acta-vet/actavet.htm

Overexpression of plastidic protoporphyrinogen IX oxidase leads to resistance to the diphenyl-ether herbicide acifluorfen.

The use of herbicides to control undesirable vegetation has become a universal practice. For the broad application of herbicides the risk of damage to crop plants has to be limited. We introduced a gene into the genome of tobacco (Nicotiana tabacum) plants encoding the plastid-located protoporphyrinogen oxidase of Arabidopsis, the last enzyme of the common tetrapyrrole biosynthetic pathway, under the control of the cauliflower mosaic virus 35S promoter. The transformants were screened for low protoporphyrin IX accumulation upon treatment with the diphenyl ether-type herbicide acifluorfen. Leaf disc incubation and foliar spraying with acifluorfen indicated the lower susceptibility of the transformants against the herbicide. The resistance to acifluorfen is conferred by overexpression of the plastidic isoform of protoporphyrinogen oxidase. The in vitro activity of this enzyme extracted from plastids of selected transgenic lines was at least five times higher than the control activity. Herbicide treatment that is normally inhibitory to protoporphyrinogen IX oxidase did not significantly impair the catalytic reaction in transgenic plants and, therefore, did not cause photodynamic damage in leaves. Therefore, overproduction of protoporphyrinogen oxidase neutralizes the herbicidal action, prevents the accumulation of the substrate protoporphyrinogen IX, and consequently abolishes the light-dependent phytotoxicity of acifluorfen.

Differential Susceptibilities of Wheat and Barley to Diphenyl Ether Herbicide Oxyfluorfen

Wheat is known to be relatively tolerant to diphenyl ether herbicides. Growth and physiological responses of wheat to diphenyl ether herbicide oxyfluorfen were examined in comparison with those of oxyfluorfensusceptible barley. Compared to barley, wheat was significantly less susceptible to the herbicide with preemergence and postemergence treatments. The differential susceptibilities of wheat and barley to the herbicide were more apparent with postemergence than with preemergence treatment. The effects of the herbicide on causing cellular leakage, chlorophyll loss, and lipid peroxidation were much lower in wheat than in barley leaves. Protoporphyrin IX accumulated in both wheat and barley leaves treated with oxyfluorfen, in a concentration-dependent manner. However, the magnitude of the protoporphyrin IX accumulation was much lower in wheat than in barley leaves treated with oxyfluorfen at a concentration of higher than 0.33 μM. Protoporphyrinogen oxidase from wheat leaves was found to be less susceptible to oxyfluorfen than that from barley leaves. The I50 concentrations of oxyfluorfen on protoporphyrinogen oxidase activity were approximately 0.2 and 0.06 μM for wheat and barley etioplasts, respectively. Whereas the inhibition of protoporphyrinogen oxidase in barley etioplasts increased with increasing concentration of oxyfluorfen, protoporphyrinogen oxidase in wheat etioplasts was not further inhibited beyond 0.33 μM oxyfluorfen concentrations. In addition, both plant species similarly responded to superoxide anion-generating paraquat and to singlet oxygen-generating rose bengal, implying that the relative tolerance of wheat to oxyfluorfen is not due to protective mechanism against active oxygen species. Our results suggest that the differential susceptibilities of wheat and barley to oxyfluorfen are at least partly due to the differential inhibition of protoporphyrinogen oxidase by the herbicide, and thereby wheat is relatively tolerant to the herbicide.

Basis for Common Chickweed (Stellaria media) Tolerance to Oxyfluorfen

Common chickweed (Stellaria media Vill.) is one of the major weeds in upland and orchard in Japan and has been known to be a species poorly controlled by diphenyl ether herbicides. Laboratory experiments were conducted to determine the physiological basis of the plant's tolerance to the diphenyl ether herbicide oxyfluorfen (2-chloro-4-trifluoromethylphenyl 3-ethoxy-4-nitrophenyl ether). Uptake, translocation, and metabolism of the herbicide, sensitivity of the action site, porphyrin accumulation, and tolerance to singlet oxygen in common chickweed were compared with slender amaranth (Amaranthus viridis) and large crabgrass (Digitaria adscendens), oxyfluorfen-sensitive species. Common chickweed showed a little visible herbicide injury to 10 μM of sprayed oxyfluorfen, while the herbicide significantly reduced the fresh weight of large crabgrass and slender amaranth at 1 μM. Uptake of 14C following [14C]oxyfluorfen application to the adaxial surface of the second leaf for 2 h was similar for common chickweed and large crabgrass, although the initial rate was greater in large crabgrass. No translocation of the herbicide out of the treated leaf was observed in either species. The major part of absorbed 14C was identified as unmetabolized oxyfluorfen in common chickweed and large crabgrass 24 h after application. The herbicide caused protoporphyrin IX accumulation in slender amaranth and large crabgrass. In contrast, no accumulation of the photosensitizing tetrapyrrole was observed in common chickweed. When excised leaf disks of slender amaranth and large crabgrass were treated with the herbicide, the porphyrin accumulation occurred faster than in intact plants. However, no accumulation was detected in leaf disks of common chickweed. Furthermore, vacuum infiltration of oxyfluorfen solution into the leaf disks of common chickweed did not promote the porphyrin accumulation. Protoporphyrinogen oxidase, the target enzyme of the herbicide, in common chickweed was inhibited by oxyfluorfen in vitro. The plant was also more tolerant to the singlet oxygen generating agent, rose bengal, than the susceptible species. However, tolerance of common chickweed to oxyfluorfen is considered to be due mainly to the mechanism which prevents protoporphyrin IX accumulation.

Measurement of Protoporphyrinogen Oxidase Activity

Protoporphyrinogen oxidase catalyzes the oxidation of protoporphyrinogen to protophyrin. It is a membrane-bound mitochondrial enzyme and it is the target of photobleaching herbicides. The basic assay presented in this unit for measuring oxidase activity is based on oxidation of the colorless, nonfluorescent substrate, protoporphyrinogen, to the colored, fluorescent protophyrin. Alternate protocols are provided for the measuring the accumulation of protoporphyrinogen resulting from a decrease in oxidase activity due to treatment with diphenyl ether herbicides or oxidase inhibitor.

Liver preneoplastic changes in mice treated with the herbicide fomesafen.

1. Effect of the diphenyl ether herbicide fomesafen on liver preneoplastic changes and porphyrin biosynthesis was examined in male C57BL/6J mice (0.23% in the diet for 14 months) and ICR mice (0.3% in the diet for 50 weeks). Fomesafen treatment resulted in preneoplastic changes (liver nodules and foci of altered hepatocytes) in both strains, uroporphyria developed only in ICR mice. 2. Iron pretreatment (600 mg/kg as a single dose) accelerated the development of fomesafen-induced preneoplastic changes in both mouse strains. The number of foci containing altered hepatocytes, as well as the number and size of liver nodules, were increased in iron-pretreated animals. 3. A single injection of iron induced marked uroporphyria in C57BL/6J mice after 14 months (liver porphyrin content 102 nmol/g). This uroporphyria was further potentiated by fomesafen administration (208 nmol/g). 4. In ICR mice, liver histology was apparently normal after a 3 month recovery from fomesafen treatment (0.32% for 9 months). Liver porphyrin content (260 nmol/g) started to decrease immediately after fomesafen withdrawal, but was still significantly elevated after 3 months (5 nmol/g), as compared to controls (1 nmol/g). 5. It is concluded that the toxicological evaluation of fomesafen should focus on liver porphyrin biosynthesis.

Responses of Antioxidative Systems to Oxyfluorfen and Their Role in Herbicidal Tolerance of Plnts.

To further investigate the role of antioxidative systems in plant tolerance to diphenylether (DPE) herbicides, the relationship between the antioxidative enzyme activities, antioxidant content and the level of oxidative damage by oxyfluorfen [2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-trifluoromethyl benzene] were investigated in rice, corn, radish and buckwheat. Rice showed the highest tolerance to oxyfluorfen, while buckwheat showed the lowest. However, both plants accumulated a larger amount of protoporphyrin IX (Proto IX) than corn or radish which showed intermediate tolerance to the herbicide. The measurement of antioxidative enzymes, superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APx) and glutathione reductase (GR), and antioxidant contents of carotenoids and reduced glutathione (GSH) in four plants showed that SOD and CAT activities and GSH content were much higher in rice than in the other plants. Furthermore, activities of all the enzymes in rice were increased until Proto IX accumulation reached maximum (4hr after light irradiation). The antioxidative ability of buckwheat was lowest and was not increased by the herbicide treatment at all. The results strongly suggested that antioxidative ability is one of the critical factors determining the tolerance to oxyflourfen in some plant species.

Glutathione transferases involved in herbicide detoxification in the leaves of Setaria faberi (giant foxtail)

Setaria faberi (giant foxtail) is a major grass weed of maize in North America and can prove difficult to control using selective herbicides. In grasses, tolerance to the chloro‐s‐triazine and chloroacetanilide classes of selective herbicides is associated with their rapid detoxification by glutathione conjugation catalysed by glutathione transferases (GSTs, EC 2.5.1.18). We were therefore interested in characterising the GSTs in S. faberi and comparing them with the corresponding enzymes in maize. Four S. faberi GST isoenzymes (SfaGSTs 1 to 4) with activities toward the triazine herbicide atrazine, the chloroacetanilide herbicides metolachlor and alachlor and the diphenyl ether herbicide fluorodifen were purified from the foliage of young plants. These GSTs detoxified herbicides with similar efficiencies to those determined for GST isoenzymes from maize, but their levels of expression in the leaves were 20‐fold lower than those reported in the crop. All SfaGSTs were composed of two subunits and the 28 kDa subunit of the isoenzyme SfaGST1 reacted strongly to an antiserum raised to the maize theta‐type GSTZm GSTI‐II. SfaGST1 also appeared to be very similar in substrate specificity to the major maize GST ZmGSTI‐I. The similarity of SfaGST1 and ZmGSTI subunits was confirmed by RT‐PCR using primers specific to ZmGSTI, with a 370 bp DNA amplification product from S. faberi showing 88% identity at the nucleotide level to the corresponding sequence of ZmGSTI. However, SfaGSTs also differed significantly from ZmGSTs. Unlike maize, only one isoenzyme, SfaGST2, detoxified chloroacetanilides. Also, SfaGST3 and SfaGST4 resembled tau‐type GSTs from maize in showing high activities toward fluorodifen, but these SfaGSTs were not recognised by an antiserum raised to the maize tau‐type GST ZmGSTV‐VI. SfaGST4 also differed from the ZmGSTs described to date in showing high activities toward atrazine. Our results demonstrate that while some GSTs are conserved in grass crops and weeds, others are quite different.

Origin attribution of polychlorinated dibenzo p-dioxins and dibenzofurans in sediment and soil from a Japanese freshwater lake area through congener-specific data analysis

Polychlorinated dibenzo p-dioxins and polychlorinated dibenzofurans (PCDDs/PCDFs) in sediment and soil from a freshwater lake area were congener-specifically determined. The obtained data were examined to estimate the major origins of these compounds, with the aid of principal component analysis (PCA). Four major principal components (PCs) were obtained, and three of them were attributed to PCDDs/PCDFs in the atmosphere, in a diphenyl ether herbicide and in pentachlorophenol, based on congener-specific comparisons with references. One PC remained unattributed.These four were interpreted as the major origins of PCDDs/PCDFs in the area. The relative influence of the origins was also investigated.

Purification of and Kinetic Studies on a Cloned Protoporphyrinogen Oxidase from the Aerobic BacteriumBacillus subtilis

The previously cloned and expressed protoporphyrinogen oxidase fromBacillus subtilishas been purified to homogeneity by Ni2+affinity chromatography using a His6tag and characterized. The enzyme has a molecular weight of approximately 56,000 daltons, a pIof 7.5, a pH optimum (protoporphyrinogen) of 8.7, and a noncovalently bound flavine adenine dinucleotide cofactor. The Michaelis constants (Km) for protoporphyrinogen-IX, coproporphyrinogen-III, and mesoporphyrinogen-IX are 1.0, 5.29, and 4.92 μM, respectively. Polyclonal antibody toB. subtilisprotoporphyrinogen oxidase demonstrated weak cross-reactivity with both human andMyxococcus xanthusprotoporphyrinogen oxidase.B. subtilisprotoporphyrinogen oxidase is not inhibited by the diphenyl ether herbicide acifluorfen at 100 μM and is weakly inhibited by methylacifluorfen at the same concentration. Bilirubin, biliverdin, and hemin are all competitive inhibitors of this enzyme.

Comparative Molecular Field Analysis (CoMFA) of Herbicidal Protoporphyrinogen Oxidase Inhibitors using Standard Steric and Electrostatic Fields and an Alternative LUMO Field

A set of diphenyl ether herbicides [1] was examined with Comparative Molecular Field Analysis (CoMFA) using standard steric and electrostatic fields and alternative frontier orbitals as 3-D fields to explain observed Protoporphyrinogen oxidase (PPO) enzyme inhibition. Significant CoMFA models were obtained utilizing standard CoMFA and the LUMO field both together and separately. These findings support previous QSAR work that identified several electronic properties as important for PPO inhibition. The resulting CoMFA models identify specific 3-D steric and electronic interactions affecting PPO enzyme binding most significantly.

Weed control in soybean (Glycine max) with imazamox and imazethapyr

Field and greenhouse experiments were conducted in 1995 and 1996 to determine soybean injury and weed control differences from imazamox and imazethapyr applied postemergence with a nonionic surfactant or methylated seed oil and with selected tank mixtures. Soybean injury from imazamox at 35 g ai ha−1plus either a methylated seed oil or nonionic surfactant was equal to injury from imazethapyr at 70 g ai ha−1in the greenhouse and field. Imazamox provided greater common lambsquarters control than imazethapyr in the field in 1995 and in the greenhouse. Thifensulfuron tank mixed with imazethapyr increased common lambsquarters control, while soybean response increased when thifensulfuron was tank mixed with imazamox. Common ragweed dry weight was reduced 61 to 64% from 35 g ha−1imazamox and 70 g ha−1imazethapyr in the field; however, imazamox provided greater common ragweed control than imazethapyr in the greenhouse. Tank mixtures of lactofen with imazamox or imazethapyr increased common ragweed control and resulted in greater soybean seed yield in 1996 than when imazamox and imazethapyr were applied alone; however, lactofen antagonized giant foxtail control with imazamox and imazethapyr, and antagonized common lambsquarters control with imazamox. Giant foxtail control in the greenhouse was antagonized more when acifluorfen, fomesafen, and lactofen were tank mixed with 35 g ha−1imazethapyr than with 35 g ha−1imazamox. Giant foxtail control with imazamox or imazethapyr applied alone or with diphenyl ether herbicides increased when 28% urea ammonium nitrate was added with nonionic surfactant compared with nonionic surfactant only. Imazethapyr antagonized giant foxtail control by clethodim in the field and was more antagonistic than imazamox in the greenhouse. A methylated seed oil improved common ragweed control by imazethapyr at 70 g ha−1and imazamox at 18 and 35 g ha−1, while common lambsquarters and velvetleaf control increased when a methylated seed oil was included with 18 g ha−1imazethapyr compared to nonionic surfactant in the greenhouse.

Glutathione transferases and herbicide detoxification in suspension-cultured cells of giant foxtail (Setaria faberi)

Glutathione transferases (GSTs) catalysing the conjugation of 1-chloro-2,4-dinitrobenzene, the chloro-s-triazine herbicide atrazine, the chloroacetanilide herbicides metolachlor and alachlor and the diphenyl ether herbicide fluorodifen have been identified in suspension-cultured cells derived from the grass weed giant foxtail (Setaria faberi Herrm.). In contrast to suspension-cultured cells of maize, where atrazine-conjugating GSTs are lost during de-differentiation, the GSTs active toward this herbicide in S. faberi plants were also expressed in cultures, suggesting that these isoenzymes are subject to different regulation in the crop and weed. As a result, glutathione conjugation was the major route of atrazine metabolism in S. faberi cultures. Activities of these GSTs were maximal three days after sub-culturing when the cells were dividing most actively, when they were determined to be in the order CDNB>alachlor>metolachlor= fluorodifen>atrazine. This indicated that GSTs which are enhanced during cell division can metabolise herbicides. On the basis of activity per mg protein, GST activities in the cultures were between 20 and 60-fold higher than those determined in the foliage of S. faberi seedlings. The GSTs with activity towards CDNB were resolved into three peaks following anion-exchange chromatography at pH 7·8 using Q-Sepharose. Peak 1 GSTs were not retained, while peak 2 and peak 3 were sequentially resolved with an increasing concentration of salt. Peak 1 GSTs showed activity toward metolachlor and atrazine but showed little activity toward fluorodifen. Peak 2 and peak 3 GSTs were active toward atrazine and metolachlor, with peak 3 being particularly associated with activity toward fluorodifen. The GSTs in these peaks were then further purified using S-hexyl-glutathione-agarose affinity chromatography. In each case, the affinity-bound fraction of the GSTs consisted of 28 kDa and 26 kDa polypeptides, suggesting that the GST isoenzymes in S. faberi cultures are composed of related subunits. Our results demonstrate that the GST isoenzymes involved in herbicide metabolism in suspension cultures of a grass weed show a similar level of complexity to that determined in maize cell cultures. © 1998 SCI

Glutathione transferases and herbicide detoxification in suspension‐cultured cells of giant foxtail (Setaria faberi)

Glutathione transferases (GSTs) catalysing the conjugation of 1-chloro-2,4-dinitrobenzene, the chloro-s-triazine herbicide atrazine, the chloroacetanilide herbicides metolachlor and alachlor and the diphenyl ether herbicide fluorodifen have been identified in suspension-cultured cells derived from the grass weed giant foxtail (Setaria faberi Herrm.). In contrast to suspension-cultured cells of maize, where atrazine-conjugating GSTs are lost during de-differentiation, the GSTs active toward this herbicide in S. faberi plants were also expressed in cultures, suggesting that these isoenzymes are subject to different regulation in the crop and weed. As a result, glutathione conjugation was the major route of atrazine metabolism in S. faberi cultures. Activities of these GSTs were maximal three days after sub-culturing when the cells were dividing most actively, when they were determined to be in the order CDNB>alachlor>metolachlor= fluorodifen>atrazine. This indicated that GSTs which are enhanced during cell division can metabolise herbicides. On the basis of activity per mg protein, GST activities in the cultures were between 20 and 60-fold higher than those determined in the foliage of S. faberi seedlings. The GSTs with activity towards CDNB were resolved into three peaks following anion-exchange chromatography at pH 7·8 using Q-Sepharose. Peak 1 GSTs were not retained, while peak 2 and peak 3 were sequentially resolved with an increasing concentration of salt. Peak 1 GSTs showed activity toward metolachlor and atrazine but showed little activity toward fluorodifen. Peak 2 and peak 3 GSTs were active toward atrazine and metolachlor, with peak 3 being particularly associated with activity toward fluorodifen. The GSTs in these peaks were then further purified using S-hexyl-glutathione-agarose affinity chromatography. In each case, the affinity-bound fraction of the GSTs consisted of 28 kDa and 26 kDa polypeptides, suggesting that the GST isoenzymes in S. faberi cultures are composed of related subunits. Our results demonstrate that the GST isoenzymes involved in herbicide metabolism in suspension cultures of a grass weed show a similar level of complexity to that determined in maize cell cultures. © 1998 SCI

Generation of Resistance to the Diphenyl Ether Herbicide, Oxyfluorfen, via Expression of the Bacillus subtilis Protoporphyrinogen Oxidase Gene in Transgenic Tobacco Plants.

In an effort to develop transgenic plants resistant to diphenyl ether herbicides, we introduced the protoporphyrinogen oxidase (EC 1.3.3.4) gene of Bacillus subtilis into tobacco plants. The results from a Northern analysis and leaf disc assay indicate that the expression of the B. subtilis protoporphyrinogen oxidase gene under the cauliflower mosaic virus 35S promoter generated resistance to the diphenyl ether herbicide, oxyfluorfen, in transgenic tobacco plants.