Enantioselectivity of protoporphyrinogen oxidase-inhibiting herbicides

Protoporphyrinogen oxidase (Protox) was inhibited stereoselectively by three pairs of enantiomers belonging to diphenyl ether (DPE) and pyrazole phenyl ether (PPE) herbicide classes. The (R) enantiomers were 10- to 44-fold more active than the (S) enantiomers as inhibitors of Protox from barley etioplasts. Similarly, the (R) enantiomers caused green barley tissue to accumulate greater amounts of porphyrins and caused greater tissue damage than the (S) enantiomers. The (R) enantiomers competed more successfully with [ 14 C]acifluorfen than the (S) enantiomers for the binding sites on Protox. In the PPE class, the in-vitro and in-vivo activity differences were wider in the isopropyl pairs than in the n-propyl pairs. The DPE enantiomers were tested on ten dicotyledonous and six monocotyledonous weed species and ten crops for weed control and crop safety. In general, neither enantiomer had pre-emergence activity on monocotyledons, but the (R) enantiomer provided some monocotyledonous weed control when applied post-emergence. On dicotyledons, the (R) enantiomer provided excellent pre-emergence control, whereas the (S) enantiomer was inactive. The (R) enantiomer caused no injury to corn, cotton, peanuts, rice, sorghum, or soybeans applied pre-emergence, but it severely injured crops when applied post-emergence. There was a positive correlation between the activities of the compounds at the molecular, cellular and whole plant levels. The only molecular property differences found to account for differences in activity between members of chiral pairs were steric parameters.     

Quinone activation of protoporphyrinogen oxidase of barley plastids

Protoporphyrinogen oxidase catalyzing the oxidation of protoporphyrinogen to protoporphyrin is the target enzyme for several highly active herbicides. The plastidic plant enzyme under normal conditions uses oxygen as electron acceptor. Duroquinone, however, can be an alternative electron acceptor of protoporphyrinogen oxidase of barley plastids. In this respect the enzyme from the plastids may be an evolutionary intermediate between bacterial enzymes coupled to ubiquinone and mammalian mitochondrial enzymes coupled to oxygen.     

新型原卟啉原氧化酶抑制剂Y11049的作用特性

为明确新型原卟啉原氧化酶(PPO)抑制剂Y11049[化学名称为2-((6-氟-5-(3-甲基-2,6-二氧代-4-三氟甲基-3,6-二氢嘧啶-1(2H)-基)苯并[d]噻唑-2-基)硫代)丙酸乙酯]的作用特性,本研究以同类型药剂苯嘧磺草胺为对照,选择对Y11049敏感的几种阔叶杂草为测试靶标,采用室内生物测定法,分别...     

Pesticides group symposium aromatic ether herbicides

The following are summaries of papers presented at a meeting of the Pesticides Group on 13 March 1984 at the Society of Chemical Industry, 14 Belgrave Square, London SW1X 8PS. The papers so presented are entirely the responsibility of the authors and do not reflect the views of the Editorial Board of Pesticide Science.     

Synthesis and herbicidal activity of novel heterocyclic protoporphyrinogen oxidase inhibitors

Protox inhibitors are applied as foliar sprays, thus causing very rapid cellular collapse and desiccation of many troublesome weeds in the presence of light. In many respects, they appear to be ideal herbicides, because they act rapidly and do not harm mammals under normal conditions. The main limitation to their widespread adoption is that few crops are naturally resistant to them. Crop tolerance has mainly been pursued with the synthesis of the cyclic imide classes containing 5- and 6-membered heterocycles, including pyrazole, pyridazine, 1,2,4-triazine, triazolinone and trifluoromethyluracil derivatives. Because of their structural novelties and biological performance, active investigations on heterocyclic protox inhibitors have been carried out in our laboratories and we have found 3-arylpyrroles to be a new class of light-activated, membrane-disrupting herbicides. They are active on both grass and broadleaf weeds at low rates. The synthesis and structure-activity relationships are presented.     

微生物降解二苯醚类除草剂的研究进展

本文概述了降解二苯醚类除草剂的微生物种类、降解机理及影响微生物降解二苯醚类除草剂的因素,指出利用微生物修复土壤中二苯醚类除草剂残留是一个有效的手段; 同时指出二苯醚类除草剂微生物降解过程中起关键作用的酶及基因有待于进一步研究。     

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.     

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.     

Formation of nitrite in plants treated with herbicides that inhibit photosynthesis

Avena sativa and Sinapis alba plants were sprayed with selected herbicides and time was allowed for the herbicides to penetrate into the plants. The plants were then illuminated in a nitrogen atmosphere for 60 min. Nitrite (up to 600 nmol/g fresh weight) was found in plants treated with herbicides that inhibit photosynthesis; these plants died a few hours later. Nitrite (up to 408 nmol/g fresh weight) was found in plants treated with herbicides that uncouple oxidative phosphorylation, but here the highest values were obtained after incubation in air. No nitrite was found in control plants incubated in nitrogen or in plants treated with photosynthesis-inhibiting herbicides and incubated in air. Experiments with plants either grown on different nitrogen sources or adapted to different light intensities rule out the possibility that the damage observed in the plants could be caused by nitrite. Possible reasons for the herbicidally induced damage are discussed.     

Expression of human protoporphyrinogen oxidase in transgenic rice induces both a photodynamic response and oxyfluorfen resistance

A human protoporphyrinogen oxidase (Protox) coding sequence under the control of a ubiquitin promoter was introduced into rice to determine whether transgenic rice overexpressing the human Protox gene exhibits resistance against a peroxidizing herbicide. The transgenic rice lines (H3, H4, H5, H6, H9, and H10) transcribed the human Protox mRNA, whereas hybridizing RNA band was not detected in wild-type rice, indicating that the human Protox gene had been successfully transmitted into transgenic rice plants. The transgenic lines H9 and H10 showed growth retardation and light-dependent formation of necrotic lesions. Compared with wild-type rice plants, rice with a human Protox gene had increased Protox activity and content of the photosensitizer protoporphyrin IX, and reduced chlorophyll. The photosynthetic efficiency in lines H9 and H10, as indicated by F_v/F_m, was not different from that of wild type. The two transgenic lines had decreased levels of antheraxanthin, lutein, and β-carotene and similar levels of neoxanthin and violaxanthin as compared with wild-type plants. The staining activities of catalase, peroxidase, superoxide dismutase, and glutathione reductase were higher in transgenic lines than in wild type. Line H9 germinated in the presence of 20 μM oxyfluorfen, whereas 2 μM oxyfluorfen inhibited the germination of wild-type seeds. Thus, the transgenic rice plants exhibited enhanced resistance to oxyfluorfen.     

Use of Myxococcus xanthus protoporphyrinogen oxidase as a selectable marker for transformation of rice

Protoporphyrinogen oxidase (PPO) is the target enzyme of peroxidizing herbicides. The overexpression of Myxococcus xanthus PPO (Mx PPO) confers a high level of herbicide resistance in rice. Among the peroxidizing herbicides, butafenacil has an efficiency ∼1000-fold that of oxadiazon, as judged by calli susceptibility tests upon herbicide treatment. Butafenacil (0.1 μM) was used to select transgenic rice plants expressing Mx PPO under the control of the constitutive maize ubiquitin promoter. The ectopic expression of the Mx PPO transgene was investigated in the T₀ generation by Northern blot and Western blot analysis. The T₀ transgenic plants expressing the Mx PPO gene were resistant to butafenacil based on in vitro leaf disk and in vivo foliar spray tests.     

Mechanisms of action and structure activity relations of herbicides that inhibit photosynthesis

A survey and chemical classification are given for the most important herbicidal inhibitors of photosynthesis. The photochemical reactions involved in photosynthesis are reviewed. The herbicides discussed here all interfere with photosynthetic electron transport in the light reactions. Comments are made on the redox catalysts and the electron transport chain. The mode of action of the herbicides due to inhibition of the light reactions I and II or of photophosphorylation are described. Structure activity correlations according to the regression analysis (Hansch-approach) are discussed with examples in the classes of acetanilides, benzimidazoles and triazinones. The correlation studies in the series of the Hill-reaction inhibitors have led to a model for the essential structural elements. Some work on the problem of selectivity and its importance is reviewed.     

Insect metabolism of a phenyl epoxygeranyl ether juvenoid and related compounds

1-(4′-Ethylphenoxy)-3,7-dimethyl-6,7-epoxy-trans-2-octene (the ethyl-epoxide), a potent insect morphogenetic agent, is converted to 6,7-diol and other derivatives in living cockroaches and mealworms. Enzyme preparations of these organisms, and of houseflies and several other insect species, also carry out these hydration and/or oxidation reactions. In addition, housefly microsomes epoxidize the ethyl-epoxide to a diepoxide. The diepoxide and diol are then converted by microsomes to at least six cyclic diols, probably via an epoxy-diol intermediate, the major ones being the cis- and trans-tetrahydrofurandiol derivatives. The metabolites formed by these reactions have little or no morphogenetic activity in Tenebrio assays. Attempts to find potent inhibitors for housefly epoxide hydratases were unsuccessful. The corresponding ethylphenyl geranyl ether is epoxidized by housefly microsomes, forming the more morphogenetically active ethyl-epoxide, but the major reaction is oxidation on the geranyl moiety to an unidentified olefinic carboxylic acid. The chemical modifications needed for improved stability and morphogenetic activity in this juvenoid series depend on the insect species and strain and the relative activities of their enzymes involved in various inactivation pathways.     

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.     

Modifying Myxococcus xanthus protoporphyrinogen oxidase to plant codon usage and high level of oxyfluorfen resistance in transgenic rice

Protoporphyrinogen oxidase (Protox) of Myxococcus xanthus (Mx Protox) is a 49-kDa membrane protein that catalyzes conversion of protoporphyrinogen IX (Protogen IX) into protoporphyrin IX (Proto IX). Upon heterologous expression in transgenic rice plants, Mx Protox is dually targeted into plastids and mitochondria, increasing resistance against the herbicidal Protox inhibitor oxyfluorfen. Here, we describe the chemical synthesis of the Mx Protox gene by assembling several small synthetic DNA fragments derived by ligation-PCR. Codon usage in the resulting 1416-bp gene was modified to correspond to that of the Arabidopsis Protox gene, a change that resulted in a decrease in G+C content from 71 to 49%. The modified Mx Protox gene was used to generate transgenic rice plants via Agrobacterium-mediated transformation. Integration, expression, and inheritance of the transgenes were demonstrated by Southern, Northern, and Western blot analyses. In plants transformed with the modified, low G+C-content Mx Protox gene, levels of Protox expression and enzyme activity were low compared to the levels observed for plants transformed with the native Mx Protox gene. Nonetheless, like the native gene, the modified gene conferred a high level of resistance to the herbicide oxyfluorfen in a seedling growth test.     

A tobacco plastidal transit sequence cannot override the dual targeting capacity of Myxococcus xanthus protoporphyrinogen oxidase in transgenic rice

The effect of a plastidal transit sequence in Myxococcus xanthus protoporphyrinogen oxidase (Protox) on gene targeting ability was investigated by generating transgenic rice that overexpressed M. xanthus Protox with the additional plastidal transit sequence (TTS line). In transgenic lines TTS3 and TTS4, the Protox antibody cross-reacted with the mature M. xanthus Protox protein of 50 kDa. In an in vitro import system using the M. xanthus Protox gene with the plastidal transit sequence, M. xanthus protein was detected in both chloroplasts and mitochondria, confirming that it was targeted into both organelles, as in transgenic rice line, M4, that overexpressed M. xanthus Protox lacking the plastidal transit sequence. A prominent increase in chloroplastic and mitochondrial Protox activity was observed in TTS3 and TTS4 relative to the wild type. However, the increase was lower than that in transgenic line M4. Seeds from all transgenic lines (TTS3, TTS4, and M4) were able to germinate when treated with up to 500 μM of the Protox-inhibiting herbicide, oxyfluorfen, whereas seeds from the wild type failed to germinate even when treated at levels as low as 1 μM. After foliar application of oxyfluorfen, TTS3 and TTS4 exhibited a reduced Protox activity, however, it was much greater than uninhibited Protox activity of wild type. The great increase in conductivity was followed by the great accumulation of photodynamic protoporphyrin IX only in oxyfluorfen-treated wild-type plants, not in oxyfluorfen-treated TTS lines. The presence of the plastidal transit sequence neither excludes the intrinsic ability of subcellular translocation of M. xanthus Protox nor changes herbicide resistance in TTS lines.