Potential involvement of alkoxyl and hydroxyl radicals in the peroxidative action of oxyfluorfen

The potential involvement of hydroxyl and alkoxyl radicals in the peroxidative action of the p-nitro diphenyl ether herbicides acifluorfen (5-[2-chloro-4-(trifluoromethyl)phenoxyl]-2-nitrobenzoic acid), acifluorfen-methyl (methyl ester of acifluorfen), nitrofen [2,4-dichloro-1-(4-nitrophenoxy)benzene], nitrofluorfen [2-chloro-1-(4-nitrophenoxy)-4-(trifluoromethyl)benzene], and oxyfluorfen [2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl)benzene] was evaluated under laboratory conditions. Methional was added to illuminated thylakoids from peas (Pisum sativum L., cv Little Marvel) and its oxidation to ethylene was used as an indicator of hydroxyl and alkoxyl radical production. Oxyfluorfen stimulated the rate of methional oxidation by 138% at 10 μM and 175% at 1 mM. This oxyfluorfen-induced stimulation of the rate of methional oxidation was dependent on light, photosynthetic electron transport, and hydrogen peroxide since it was not observed under dark conditions or in the presence of DCMU and catalase. Addition of Fe-EDTA, a catalyst of the Fenton reaction, stimulated the oxyfluorfen-induced enhancement of methional oxidation sixfold, suggesting that hydroxyl radicals are synthesized through a Fenton reaction. Acifluorfen, nitrofen, and nitrofluorfen inhibited the rate of methional oxidation whereas acifluorfen-methyl had no effect on the rate of methional oxidation, even at high concentrations (1 mM). Nitrofluorfen at 1 mM was the only p-nitro diphenyl ether herbicide tested to inhibit photosynthetic electron transport of pea thylakoids. In experiments with pea leaf disks, acifluorfen at low concentrations stimulated the rate of methional oxidation, whereas acifluorfen-methyl, nitrofen, and nitrofluorfen had no effect. These data indicate that hydroxyl and alkoxyl radicals could be involved in the mechanism of cellular damage caused by oxyfluorfen but they are not important for the activity of the diphenyl ether herbicides acifluorfen, acifluorfen-methyl, nitrofen, and nitrofluorfen.     

Characterization of herbicidal injury by acifluorfen-methyl in excised cucumber (Cucumis sativus L.) cotyledons

Initial signs of herbicidal injury by several diphenyl ether herbicides were monitored by following the efflux of ⁸⁶Rb⁺ from treated cucumber (Cucumis sativis L.) cotyledons after exposure to light (600 μE m^?2 sec^?1; measured as PAR, i.e., photosynthetically active radiation between 400 and 700 nm). This very sensitive, rapid, and quantitative bioassay proved quite useful in (a) a structure-activity correlations study of the diphenyl ether compounds investigated and (b) an examination of herbicidal characteristics. The following diphenyl ether herbicides were analyzed: acifluorfen, sodium 5-[2-chloro-4-(trifluormethyl)phenoxy]-2-nitrobenzoate; acifluorfen-methyl (MC-10108), methyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate; bifenox, methyl 5-(2,4-dichlorophenoxy)-2-nitrobenzoate; nitrofen, 2,4-dichlorophenyl p-nitrophenyl ether; nitrofluorfen, 2-chloro-1-(4-nitrophenoxy)-4-(trifluoromethyl)benzene; oxyfluorfen, 2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl)benzene; MC-7783, potassium 5-(2,4-dichlorophenoxy)-2-nitrobenzoate; and MC-10982, ethyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate. Of the compounds investigated, acifluorfen-methyl (AFM) had the greatest degree of herbicidal activity. Cucumber cotyledons placed in high light (600 μE m^?2 sec^?1; PAR) with 10 nM AFM showed a significant increase in the efflux of ⁸⁶Rb⁺ within 2 to 4 hr. Light was required for herbicidal activity by AFM, and when treated cotyledons were returned to darkness, no further damage to the tissue occurred. By decreasing the quantity of light, the effect of the compound was delayed, although the magnitudes of the responses at the different intensities (600, 300, 150, and 75 μE m^?2 sec^?1; PAR) were nearly equal. By increasing the length of time of dark pretreatment with 1 μM AFM, ⁸⁶Rb⁺ efflux could be detected as early as 10 to 15 min after exposure to light (600 μE m^?2 sec^?1; PAR). Following light activation of AFM there was a simultaneous efflux of ⁸⁶Rb⁺, ³⁶Cl^?, ⁴⁵Ca²⁺, 3-O-methyl-[¹⁴C]glucose, and [¹⁴C]methylamine⁺. These data suggest the initial response to the herbicidal activity of AFM is expressed as a general increase in membrane permeability.     

Effects of LS 82556 on thylakoid activities and photosynthesis: A comparison with paraquat and acifluorfen

LS 82556 is a new phytotoxic compound inducing photodependent herbicidal effect. In isolated cucumber cotyledons maintained under our experimental conditions, on water without sucrose and under continuous light, LS 82556 induces a photodependent degradation of biological membranes, the symptoms of which are near those of paraquat under the same conditons. The inhibition of the photosynthetic electron transfer by diuron, atrazine, or phenmedipham prevents this phytotoxic action. Using class C and class A chloroplasts isolated from spinach leaves and from cucumber cotyledons, it was shown that LS 82556 is neither an uncoupler nor an inhibitor of the electron transfer, nor is it an electron acceptor. Moreover, it is without effect on the CO₂-dependent O₂ evolution of intact chloroplasts (class A) under light. When isolated thylakoids were subjected to a 400 μE m⁻² sec⁻¹ photosynthetically active radiation light for 10 to 25 min, LS 82556, up to 100 μM, did not change the thylakoids activities. All these results show that the photodependent toxic effect of LS 82556 is quite different from that of paraquat, but it is near that of the diphenyl ethers such as acifluorfen. They demonstrate that the direct involvement of the thylakoid electron transfer in the photodependent mode of action of LS 82556 is unlikely. In isolated cucumber cotyledon fragments, under our conditions, this phytotoxic effect of LS 82556 seems to depend on the presence of sufficient amounts of carbohydrates, which are normally provided through photosynthesis.     

Studies into the action of the diphenyl ether herbicides acifluorfen and oxyfluorfen. Part II: The interaction with photosynthetic electron transport reactions

The effects of acifluorfen and oxyfluorfen on photosynthetic electron transport reactions of pea chloroplasts were compared with those induced by paraquat and monuron. Monuron inhibited electron flow between photosystems I and II, and paraquat acted as an electron acceptor for photosystem I, promoting superoxide formation by illuminated chloroplasts. Neither acifluorfen nor oxyfluorfen at concentrations up to 50 μM affected non-cyclic electron flow or promoted superoxide formation. Both herbicides were shown to repress ferredoxin-dependent NADP⁺ reduction by illuminated chloroplasts. Further experiments showed that, in the presence of ferredoxin-NADP⁺ reductase and chloroplast membranes maintained in the dark, p-nitro diphenyl ether (DPE) herbicides promoted the rate of ferredoxin-dependent oxidation of NADPH, implying that these herbicides can accept electrons from reduced ferredoxin. The interaction between acifluorfen, ferredoxin and chloroplast membranes was examined further by following the effect of this herbicide on the peroxidation of illuminated thylakoids. Lipid peroxidation was promoted by acifluorfen, although this effect was abolished if thylakoids were washed prior to use. The effect of washing could be reversed by adding exogenous ferredoxin. These data demonstrate that interaction of DPE herbicides with photosynthetic electron transport in the vicinity of ferredoxin is necessary for light-dependent herbicide activation.     

Photoreceptors and respiratory electron flow involvement in the activity of acifluorfen-methyl and LS 82-556 on nonchlorophyllous soybean cells

The diphenyl ether acifluorfen-methyl [AFM; methyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate] and the pyridine derivative LS 82-556 [(S)-3-N-(methylbenzyl)carbamoyl-5-propionyl-2,6-lutidine] induce light-dependent polyunsaturated fatty acid peroxidation, leading to general membrane disruption. Although devoid of functional chloroplasts, cultured soybean cells are sensitive to AFM and LS 82-556 only in the light. The possible involvement of carotenoids and respiratory electron flow was examined by monitoring ethane evolution, fluorescein release, and dry weight/fresh weight ratio alteration. Herbicide effects on cells exposed to white light or blue light (380–540 nm) remain unchanged when the cells are deprived of carotenoids by treatment with norflurazon [4-chloro-5-(methylamino)-2-(α,α,α-trifluoro-m-tolyl)-3(2H)-pyridazinone]. Treatment with antimycin A reduced sensitivity to AFM and LS 82-556. In nonchlorophyllous cells, chromophores, other than carotenoids, absorbing between 380 and 540 nm are involved as photoreceptors. Furthermore, respiratory electron flow also participates in the toxic process.     

Nitro free radical formation of diphenyl ether herbicides is not necessary for their toxic action

The diphenyl ether herbicides MC 15608 {5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-chloromethylbenzoate} and MC 10878 {5-[2-chloro-4-(trifluoromethyl)phenoxy]methyl benzoate} are structurally similar to acifluorfen-methyl (methyl ester of 5-[2-chloro-4-(trifluoromethyl)phenoxy]-nitrobenzoic acid), except that the NO₂ is replaced by a Cl and H, respectively. These diphenyl ether herbicides required light for herbicide toxicity to the green unicellular alga Chlamydomonas eugametos and three major weeds (Xanthium pennsylvanicum, Abutilon theophrasti, and Ipomoea sp.). Acifluorfen-methyl and MC 15608 toxicity in Chlamydomonas decreased in an atmosphere of nitrogen, and in the presence of the free radical scavengers α-tocopherol and ethanol. Therefore, the mechanism of toxic action of these three different diphenyl ether herbicides is similar and appears to involve some type of free radical reaction. As confirmed by cyclic voltammetry studies, MC 15608 and MC 10878, unlike AFM, cannot readily accept electrons to become free radicals. Therefore, initiation of free radical reactions in polyunsaturated fatty acids of membranes does not necessarily involve direct reduction and reoxidation of the diphenyl ether molecule.     

Quantitative structure-activity relationships of the inhibition of photosynthetic electron flow by substituted diphenyl ethers

The effect of 41 substituted diphenyl ethers (derivatives of nitrofen and chloroxuron) on photosynthesis in isolated spinach chloroplasts has been studied. All the chemicals were found to be inhibitors of non-cyclic electron transport; the pI₅₀ values varied from 3.17 to 7.16 (I₅₀ is the molar concentration causing 50% inhibition; pI₅₀= -log I₅₀). Based on their structures, the compounds were divided into four groups; for most groups, a correlation between the inhibition of photosynthesis and physicochemical parameters was found. Lipophilicity proved to be the most important parameter; electronic effects did not play a role. Introduction of substituents into the nitrophenyl ring of nitrofen lowered activity considerably. Nitrofen and and chloroxuron analogues seemed to inhibit at different sites in the electron transport chain. A relationship between inhibition of photosynthesis and herbicidal activity was not clear.     

Mitochondrial involvement in the mode of action of acifluorfen

The herbicidal action of acifluorfen {5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid} was studied with greened and expanded discs from cotyledons of cucumber (Cucumis sativus L.). Discs were floated on various treatment solutions for 20 hr in darkness before exposure to 400 μE m^?2 sec^?1 of white light. Herbicide damage, as measured by electrolyte leakage, began in the light after a 1- to 2-hr lag period. Cytochemical methods at the ultrastructural level indicated that acifluorfen caused marked increases in production of superoxide radical and hydrogen peroxide in the mitochondrion, but not in the plastid. The mitochondrial inhibitors antimycin A, rotenone, CCCP, and DNP antagonized the action of acifluorfen, lengthening the lag period and reducing the rate of electrolyte leakage. Ethanol, α-tocopherol, N-[2-(2-oxo-1-imidazolidinyl)ethyl]-N′-phenylurea, and copper-penicillin also lengthened the lag phase and slowed the rate of damage, indicating that acifluorfen damage involves toxic oxygen species. PS II-inhibiting levels of DCMU, atrazine, or bentazon did not affect acifluorfen-induced ion leakage. Yellow tissue produced by treatment with tentoxin was supersensitive to acifluorfen, but white tissue produced by treatment with norflurazon was relatively insensitive. These data indicate that, after an initial carotenoid-acifluorfen interaction, the mitochondrion is involved in production of toxic oxygen species and that this process is closely tied to the mechanism of action of this herbicide.     

Effects of bifenox and oxadiazon on isolated chloroplasts,plant mitochondria and leaf pieces

The effects of bifenox (methyl 5-(2,4-dichlorophenoxy)-2-nitrobenzoate) and oxadiazon (5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2(3H)-one on photosynthetic activity were investigated in isolated chloroplasts, and on respiratory activity in isolated mitochondria. The global effects of these chemicals were also investigated on cucumber cotyledon pieces. It was found that, in vitro, bifenox and oxadiazon acted on cotyledon pieces as typical diphenyl ether herbicides, causing complete pigment bleaching, even at low concentrations. In addition, bifenox and oxadiazon were shown to inhibit the photosynthesis process at the chloroplast level. At concentrations of up to 40-50 μM, oxadiazon and bifenox were observed to inhibit fully the light-dependent oxygen evolution of spinach class A chloroplasts, oxadiazon acting preferentially on electron transfer at the PS II level whereas bifenox acted on the photophosphorylation process. Comparison of the amounts of herbicide needed to inhibit photosynthesis and to cause bleaching of cucumber pieces leads to the conclusion that photosynthesis inhibition by bifenox and oxadiazon is only a secondary effect.     

三氟羧草醚类似物的合成及除草活性

以2,5-二羟基苯甲酸甲酯和三氟羧草醚为起始原料,设计合成了3个系列20个新的三氟羧草醚类似物,通过核磁共振氢谱、碳谱对其结构进行了表征。分别采用小杯法和室内盆栽法测定了目标化合物的除草活性。结果表明,化合物 III-02 [5-(2-氯-4-三氟甲基苯氧基)-2-硝基苯甲酸-(6-甲基苯并噻唑-2-基)酯]对单子叶杂草的除草活性明显高于对照药剂三氟羧草醚,其对稗草Echinochloa crusgalli和马唐Digitaria sanguinalis根茎生长的EC₅₀值分别为2.03、0.93 μg/mL和1.49、0.52 μg/mL;在有效成分100 g/hm2的施药剂量下,化合物 III-02 对单子叶杂草稗草、马唐及狗尾草Setaria viridis的防治效果均在85%以上,明显高于三氟羧草醚,对阔叶杂草马齿苋Portulaca oleracea、反枝苋Amaranthus retroflexus及苘麻Abutilon theophrasti的防治效果可达100%。初步构效关系表明,2-硝基苯甲酰衍生物的除草活性明显优于其2-甲氧基衍生物,三氟羧草醚苯甲酸酯衍生物对单子叶杂草的除草活性明显高于其苯甲酰胺衍生物。     

Acifluorfen metabolism in soybean: Diphenylether bond cleavage and the formation of homoglutathione, cysteine, and glucose conjugates

Metabolism of the substituted diphenylether herbicide, acifluorfen [sodium 5-(2-chloro-4-trifluoromethylphenoxy)-2-nitrobenzoate], was studied in excised leaf tissues of soybean [Glycine max (L.) Merr. ‘Evans’]. Studies with [chlorophenyl-¹⁴C]- and [nitrophenyl-¹⁴C]acifluorfen showed that the diphenylether bond was rapidly cleaved. From 85 to 95% of the absorbed [¹⁴C]acifluorfen was metabolized in less than 24 hr. Major polar metabolites were isolated and purified by solvent partitioning, adsorption, thin layer, and high-performance liquid chromatography. The major [chlorophenyl-¹⁴C]-labeled metabolite was identified as a malonyl-β- -glucoside (I) of 2-chloro-4-trifluoromethylphenol. Major [nitrophenyl-¹⁴C]-labeled metabolites were identified as a homoglutathione conjugate [S-(3-carboxy-4-nitrophenyl) γ-glutamyl-cysteinyl-β-alanine] (II), and a cysteine conjugate [S-(3-carboxy-4-nitrophenyl)cysteine] (III).     

9种除草剂对花生白绢病菌的影响

在实验室条件下测定了氟乐灵、乙草胺、异丙甲草胺、异恶草酮、乳氟禾草灵、乙氧氟草醚、三氟羧草醚、恶草酮、二甲戊乐灵等9种除草剂对花生白绢病菌Sclerotium rolfsi Sacc.的影响.结果表明:9种除草剂对花生白绢病病菌的毒力有较大差异,三氟羧草醚和乙氧氟草醚的毒力较高,IC50分别为7.88mg·L-1和18.91mg·L-1;9种除草剂对菌丝干重均有抑制作用,且随剂量的升高而升高,乙氧氟草醚和三氟羧草醚抑制作用最明显,在100mg·L-1和50mg·L-1时抑制率均达90%以上;除乙草胺和异丙甲草胺部分剂量外,其他除草剂对菌核数量均有不同程度的抑制作用,三氟羧草醚作用最为明显,在供试剂量下抑制率均达96%以上;除乙草胺、氟乐灵在供试剂量下对菌核单重有抑制作用外,其他除草剂在多数剂量下对菌核单重均有刺激作用,三氟羧草醚在50mg·L-1时,是对照菌核单重的8.34倍;而各种除草剂在供试剂量下,对菌核萌发均没有影响.     

Studies into the action of the diphenyl ether herbicides acifluorfen and oxyfluorfen. Part I: Activation by light and oxygen in leaf tissue

The effect of acifluorfen and oxyfluorfen on chlorophyll bleaching, lipid peroxidation and photosynthesis in pea leaf discs was studied. Both her- bicides induced light-dependent bleaching and lipid peroxidation, the level of damage being greater at higher light intensities. Photosynthetic carbon dioxide fixation was only partially inhibited in treated leaf discs incubated in darkness, thus indicating that these herbicides did not inhibit photo- synthesis as a primary mode of action. Leaf discs maintained in darkness showed no visible signs of injury, and light-dependent herbicide-induced damage was reduced by incubating discs under nitrogen, orpre-incubating them with the electron-transport inhibitor monuron. It is suggested that acifluorfen and oxyfluorfen are activated by a light-dependent process, which requires photosynthetic electron transport.     

Pyrazole phenyl ether herbicides inhibit protoporphyrinogen oxidase

Two isomeric pairs of pyrazole phenyl ether herbicides [AH 2.429, 4-chloro-1-methyl-5-(4-nitrophenoxy)-3-(trifluoromethyl)-1H-pyrazole; AH 2.430, 4-chloro-1-methyl-3-(4-nitrophenoxy)-5-(trifluoromethyl)-1H-pyrazole; AH 2.431, 5-((4-chloro-1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)oxy)-2-nitrobenzoic acid; and AH 2.432, 5-((4-chloro-1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)oxy)-2-nitrobenzoic acid were evaluated for herbicidal activity in both intact plants and in tissue sections. Their capacity to induce accumulation of porphyrins in tissue sections and to inhibit protoporphyrinogen oxidase (Protox) in vitro were determined. In whole plant tests, the order of herbicidal activity was AH 2.430 AH 2.431 > AH 2.429 > AH 2.432. AH 2.430 consistently caused light-dependent membrane leakage in both green and far-red light grown cucumber cotyledon and barley primary leaf tissue sections after incubation for 20 hr in darkness in 0.1 mM solutions. The same treatment caused marked increases in protoporphyrin IX (PPIX) content during the 20-hr dark incubation. AH 2.429 and 2.431 were less effective and not effective in all tissues in causing herbicidal damage and PPIX accumulation. AH 2.432 was ineffective in tissue section assays. Mg-PPIX levels were not significantly affected by any of the compounds. Protochlorophyllide levels were decreased by AH 2.430 and 2.431 in barley and increased by AH 2.429, 2.431, and 2.432 in cucumber. A positive relationship was found between herbicidal activity and the amount of PPIX that was caused to accumulate by each compound. All of the compounds inhibited Protox activity. Positive correlations were found between herbicidal activity in planta over a 300-fold range and in vitro Protox inhibition and the amount of PPIX caused to accumulate in vivo. These data support the view that the pyrazole phenyl ethers exert their herbicidal activity entirely through inhibition of Protox.     

Resistance pattern and antioxidant enzyme profiles of protoporphyrinogen oxidase (PROTOX) inhibitor-resistant transgenic rice

We quantified the resistance levels of transgenic rice plants, expressing Myxococcus xanthus protoporphyrinogen oxidase (PROTOX) in chloroplasts and mitochondria, to PROTOX inhibitors, acifluorfen, oxyfluorfen, carfentrazone-ethyl, and oxadiazon. We also determined whether active oxygen species-scavenging enzymes are involved in the resistance mechanism of transgenic rice. The transgenic rice line M4 was about >200-fold more resistant to oxyfluorfen than the wild-type (WT). M4 was also resistant to acifluorfen, carfentrazone-ethyl, and oxadiazon, but did not show multiple resistance to imazapyr and paraquat, which have different target sites. Acifluorfen, oxyfluorfen, carfentrazone-ethyl, and oxadiazon reduced the chlorophyll content in leaves of WT, but had minimal or no effect on M4. The PROTOX inhibitors also caused significant lipid peroxidation in the treated leaves of WT rice. However, the malondialdehyde production in M4 was not affected by these herbicides. The WT rice had higher activities of superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase than M4 after treatment with PROTOX inhibitors. A similar response was observed in all cases of antioxidant isozyme profiles analyzed. However, the induction in antioxidant activity in WT was not enough to overcome the toxic effects of a PROTOX inhibitor so the plant eventually died.     

Inhibition of carotenogenesis by substituted diphenyl ethers of the m-phenoxybenzamide type

Diphenyl ethers exhibit different modes of action according to their chemical constitution. Diphenyl ethers of the m-phenoxybenzamide type, which were found to be effective on carotenogenesis resulting in an accumulation of colorless carotenoid precursors, mostly phytoene, indicative of inhibition of desaturation, are discussed. As seen with other carotenoid biosynthesis inhibitors, a concurrent loss of chlorophyll was observed as a secondary effect caused by the absence of protective carotenoids. In contrast to peroxidative p-nitrodiphenyl ethers like oxyfluorfen (2-chloro-4-trifluoromethylphenyl-3′-ethoxy-4′-nitrophenyl ether), the m-phenoxybenzamides assayed showed the same phytotoxic mode of action in the dark as observed when using heterotrophic Scenedesmus cultures. As expected, chlorophylls were not affected. The decrease of carotenoids was not due to their degradation but to inhibited carotenogenesis. Examination of carotenoid fractions show that the m-phenoxybenzamides, e.g., 3-(2,5-dimethylphenoxy)-N-ethylbenzamide, used here act similarly to 2-phenylpyridazinones like norflurazon [4-chloro-5-methylamino-2-(2-trifluoromethylphenyl)-pyridazin-3(2H)one]. All these inhibitors strongly decrease the α- and β-carotene content, while xanthophyll content is not lowered as much.     

The herbicide aclonifen: The complex theoretical bases of sunflower tolerance

Aclonifen is a diphenylether herbicide in which one ring is unsubstituted and the other is NH₂-1, Cl-2, NO₂-6 substituted. This substitution, especially NH₂-1, is a particular feature in the diphenylether herbicidal family. In contrast with other herbicides of the same family (acifluorfen, oxyfluorfen, bifenox) this compound is not only acting through a phytotoxic protoporphyrin IX accumulation but also through an inhibition of carotenoid biosynthesis. Both biochemical targets are associated to chloroplast structuration in young seedlings under light and the pre-emergence or preplant treatment is consequently an appropriate agronomic strategy. Numerous weeds such as Alopecurus sp., Sinapis sp., Brassica sp., Chenopodium sp. are very sensitive to this herbicide but sunflower culture appears very tolerant. Our results suggest that this tolerance has a complex origin including (1) low root uptake, (2) conjugation in the roots probably with glutathione and (3) low xylem transfer from root to shoot. The low root uptake is associated to aclonifen crystallization on the root surface at high concentrations. The combination of this physico-chemical effect with a sufficient root conjugation rate can explain both the very low amounts of aclonifen or derivatives found in the aerial parts and the strong sunflower tolerance.     

Resistance of a soybean cell line to oxyfluorfen by overproduction of mitochondrial protoporphyrinogen oxidase

The diphenyl ether herbicide oxyfluorfen (2-chloro-4-trifluoromethylphenyl 3-ethoxy-4-nitrophenyl ether) inhibits protoporphyrinogen oxidase (Protox) which catalyzes the oxidation of protoporphyrinogen IX (Protogen) to protoporphyrin IX (Proto IX), the last step of the common pathway to chlorophyll and haeme biosynthesis. We have selected an oxyfluorfen-resistant soybean cell line by stepwise selection methods, and the resistance mechanism has been investigated. No growth inhibition was observed in resistant cells at a concentration of 10(-7) M oxyfluorfen, a concentration at which normal cells did not survive. While the degree of inhibition of total extractable Protox by oxyfluorfen was the same in both cell types, the enzyme activity in the mitochondrial fraction from non-treated resistant cells was about nine-fold higher than that from normal cells. Northern analysis of mitochondrial Protox revealed that the concentration of mitochondrial Protox mRNA was much higher in resistant cells than that in normal cells. There were no differences in the absorption and metabolic breakdown of oxyfluorfen. The growth of resistant cells was also insensitive to oxadiazon [5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2-(3H)- one], the other chemical class of Protox inhibitor. Therefore, the resistance of the selected soybean cell line to oxyfluorfen is probably mainly due to the overproduction of mitochondrial Protox.     

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.     

The role of light in bentazon toxicity to cocklebur: Physiology and ultrastructure

Leaves from intact 10 to 14-day-old common cocklebur (Xanthium pensylvanicum Wallr.) plants were treated with 0.01 to 1 kg/ha of 3-isopropyl-1H-2,1,3-benzothiadiazin-(4)3H-one 2,2-dioxide (bentazon), and exposed to 21 to 86 klux. At intervals from 30 min to 38 hr, primary leaves were fixed for electron microscopic examination. Also, immediately after application of the herbicide, treated plants were placed in an assimilation chamber and net CO₂ exchange was measured.Light was required for necrosis to develop in bentazon-treated leaves; the higher the illuminance, the faster necrosis developed. At low levels of illuminance (21 to 36 klux), the chloroplasts became spherical and aggregated in the cells before the occurrence of general membrane rupture and the subsequent development of necrosis. However, at 86 klux, chloroplast shape and distribution did not change before membrane rupture. In both control and treated leaves that were placed in darkness, chloroplasts became spherical and aggregated. Therefore, the changes in shape and distribution of chloroplasts were not considered a toxic response.In all cases, cessation of photosynthesis preceded cytological changes. Photosynthesis was arrested more rapidly as the dose of bentazon increased. Regardless of the length of time required to stop photosynthesis, necrosis developed about 7 hr after photosynthesis was arrested when plants were grown under 86 klux. These data are consistent with the hypothesis that photo-induced toxic by-products result from stopping photosynthesis.