农药中色素合成抑制剂的研究和应用进展

光合作用在植物的生长过程中起着至关重要的作用,以光合色素生物合成过程中的酶作为靶标,是研发除草剂的一个重要方向和热点。其中原卟啉原氧化酶(PPO),八氢番茄红素去饱和酶(PDS),ζ-胡萝卜素去饱和酶(ZDS),对羟苯基丙酮酸双氧化酶(HPPD)等作为除草剂靶酶非常成功。本文综述了近年来农药中色素合成抑制剂的作用机制及最新应用进展,并展望未来的发展趋势。     

Inhibition profile of trifludimoxazin towards PPO2 target site mutations

BACKGROUND

Target site resistance to herbicides that inhibit protoporphyrinogen IX oxidase (PPO; EC 1.3.3.4) has been described mainly in broadleaf weeds based on mutations in the gene designated protoporphyrinogen oxidase 2 (PPO2) and in one monocot weed species in protoporphyrinogen oxidase 1 (PPO1). To control PPO target site resistant weeds in future it is important to design new PPO-inhibiting herbicides that can control problematic weeds expressing mutant PPO enzymes. In this study, we assessed the efficacy of a new triazinone-type inhibitor, trifludimoxazin, to inhibit PPO2 enzymes carrying target site mutations in comparison with three widely used PPO-inhibiting herbicides.

RESULTS

Mutated Amaranthus spp. PPO2 enzymes were expressed in Escherichia coli, purified and measured biochemically for activity and inhibition kinetics, and used for complementation experiments in an E. coli hemG mutant that lacks the corresponding microbial PPO gene function. In addition, we used ectopic expression in Arabidopsis and structural PPO protein modeling to support the enzyme inhibition study. The generated data strongly suggest that trifludimoxazin is a strong inhibitor both at the enzyme level and in transgenics Arabidopsis ectopically expressing PPO2 target site mutations.

CONCLUSION

Trifludimoxazin is a potent PPO-inhibiting herbicide that inhibits various PPO2 enzymes carrying target site mutations and could be used as a chemical-based control strategy to mitigate the widespread occurrence of PPO target site resistance as well as weeds that have evolved resistance to other herbicide mode of actions. © 2022 BASF SE and The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

靶标为原卟啉原氧化酶的高效除草剂品种

原卟啉原ＩＸ氧化酶是血红素和叶绿素生物合成中的关键酶，是过氧化型除草剂的分子靶标，当植物用二苯醚类，酞酰亚胺以及一些吡啶衍生物等除草剂处理后，造成原卟啉原ＩＸ积累，膜脂质破坏，最终细胞死亡，同ＡＬＳ抑制剂一样，作用靶标为原卟啉原化酶的除草剂，具有用药量低，活性高，杀草谱广，对哺乳动物低毒，对环境影响较小等良好特性，本文介绍了作用靶标为原卟啉原氧化酶的代表性品种。     

Photodynamic herbicides VIII. Mandatory requirement of light for the induction of protoporphyrin IX accumulation in acifluorfen-treated cucumber

It is shown that continuous illumination is mandatory for the induction of tetrapyrrole accumulation in acifluorfen-sodium-treated plants and for the photosensitization of tetrapyrrole-dependent photodynamic damage. At low concentrations of acifluorfen-sodium (up to 20 μM), photoporphyrin IX appears to be the major light-induced tetrapyrrole that accumulates in the treated plants. At higher concentrations of acifluorfen-sodium, monovinyl chlorophyllide a accumulates in addition to protoporphyrin IX. In the light, the development of photodynamic injury appears to be directly related to the accumulation of the light-induced tetrapyrroles. For example when acifluorfen-sodium-treated plants are returned to darkness, or are treated with tetrapyrrole biosynthesis inhibitors, tetrapyrrole accumulation and photodynamic injury come to a halt. In-vivo and in-organello studies failed, however, to support the commonly held hypothesis that the induction of tetrapyrrole accumulation in the light, in acifluorfen-sodium-treated plants, is only dependent on the inhibition of protoporphyrinogen oxidase. Indeed, when plastids capable of very high rates of tetrapyrrole biosynthesis and accumulation were incubated with δ-aminolevulinic acid and acifluorfen-sodium, either in darkness or in the light, a severe inhibition of protoporphyrin IX and total terapyrrole formation was observed. Althoung these results are compatible with the inhibition of tetrapyrrole formation by acifluorfensodium at the level of protoporphyrinogen oxidase, they indicate that, in addition to that inhibition, other cuellular processes are probably involved in the light-dependent accumulation of protoporphyrin IX in acifluorfensodium-treated plants.     

Synthesis,mechanism of action,and QSAR of herbicidal 3-substituted-2-aryl-4,5,6,7-tetrahydroindazoles

A factorially designed set of 3-substituted 2-aryl-4,5,6,7-tetrahydroindazoles was synthesized and their herbicidal properties were evaluated using regression analysis. For optimal activity, the substituent at position 3 should be neither strongly hydrophobic nor hydrophilic and should have a small minimum radius. A study of the mechanism of action indicated that the tetrahydroindazoles are inhibitors of the enzyme protoporphyrinogen oxidase.     

原卟啉原氧化酶抑制剂类除草剂研究进展

概述了原卟啉原氧化酶抑制剂类除草剂作用机理、结构-活性关系研究进展以及新品种开发情况。     

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.     

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.     

Saline soil enzyme activities of four plant communities in Sangong River basin of Xinjiang,China

Soil enzyme activity plays an important role in the conversion of soil organic carbon into inorganic carbon,which is significant for the global carbon cycle.In this study,we investigated the soil enzyme activities of two ligninolytic enzymes(peroxidase and polyphenol oxidase)and five non-ligninolytic enzymes(α-1,4-glucosidase(AG);β-1,4-glucosidase(BG);N-acetyl-β-glucosaminidase(NAG);β-D-cellobiosidase(CBH);andβ-xylosidase(BXYL))in four plant communities of the Sangong River basin in Fukang,North Xinjiang,China.The four typical plant communities were dominated by Haloxylon ammodendron,Reaumuria soongonica,Salsola passerina,and Tamarix rarmosissima,respectively,with saline soils of varied alkalinity.The results showed that the soil peroxidase activity decreased seasonally.The activities of the five non-ligninolytic enzymes decreased with increasing soil depths,while those of the two ligninolytic enzymes did not show such a trend.In the four plant communities,BG had the highest activity among the five non-ligninolytic enzymes,and the activities of the two ligninolytic enzymes were higher than those of the four non-ligninolytic ones(AG,NAG,CBH,and BXYL).The community of H.ammodendron displayed the highest activity with respect to the two ligninolytic enzymes in most cases,but no significant differences were found among the four plant communities.The geometric mean of soil enzyme activities of the four plant communities was validated through an independently performed principal component analysis(PCA),which indicated that different plant communities had different soil enzyme activities.The correlation analysis showed that soil polyphenol oxidase activity was significantly positively correlated with the activities of the five non-ligninolytic enzymes.The soil pH value was positively correlated with the activities of all soil enzymes except peroxidase.Soil microbial carbon content also showed a significant positive correlation(P0.01)with the activities of all soil enzymes except polyphenol oxidase.The results suggested that the H.ammodendron community has the highest ability to utilize soil organic carbon,and glucoside could be the most extensively utilized non-ligninolytic carbon source in the saline soil of arid areas in Xinjiang.     

Herbicide safety relative to common targets in plants and mammals

Most modern herbicides have low mammalian toxicity. One of the reasons for this safety is that the target site for the herbicides is not often present in mammals. There are approximately 20 mechanisms of action that have been elucidated for herbicides. Of these, some do share common target sites with mammals. The mechanisms include formation of free radicals, protoporphyrinogen oxidase (PROTOX), glutamine synthetase (GS) and 4-hydroxyphenylpyruvate dioxygenase (HPPD). PROTOX, HPPD and GS inhibitors have been shown to inhibit these enzymes in both plants and mammals and there are measurable effects in mammalian systems. However, the consequences of inhibiting a common target site in plants can be quite different than in animals. What may be a lethal event in plants, eg inhibition of HPPD, can have a beneficial effect in mammals, eg treatment for tyrosinemia type I. These chemicals also have low mammalian toxicity due to rapid metabolism and/or excretion of the herbicide from mammalian systems.     

Functional diversity within the mitochondrial electron transport chain of plant pathogenic fungi

Mitochondrial transfer of electrons from NAD(P)H or FADH₂ to the terminal electron acceptor, oxygen, follows a highly complex scheme, involving numerous redox components. Whilst electron transfer has been extensively studied over past decades in mammalian, plant and some fungal species, relatively little is known about the respiratory chain of phytopathogenic fungi. The recent identification of the electron transport chain as a viable target for effective control of fungal infections has contributed to a significantly increased research effort into this area of fungal biochemistry. A striking feature of the electron transport chain within phytopathogenic fungi is the presence of components not found in the classical (mammalian) chain. Recent research has suggested the presence of a plant‐like ‘alternative’ oxidase, internal and external NAD(P)H dehydrogenases, and cyanide‐insensitive cytochrome c oxidases. In this mini‐review on electron transport in phytopathogenic fungi, the current status of research into the function and expression characteristics of these ‘alternative’ redox centres is discussed. © 2000 Society of Chemical Industry     

Morphological and histochemical investigation of the response of Olea europaea leaves to fungal attack by Spilocaea oleagina

This study investigated the response of Olea europaea (cv. Conservolea) leaves to attack by the fungal pathogen Spilocaea oleagina. Cryostat and semithin sections of healthy and S. oleagina‐infected olive leaves were analysed histochemically for polyphenol oxidase (PPO) activity and tested for programmed cell death (PCD) induction by means of terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL). At all stages of infection, the fungus remained localized between the external and internal layers of cuticle without crossing the pectocellulosic layer. No PCD phenomena could be detected in plant cells at any stage of the disease. However, extensive degeneration of palisade parenchyma cells was observed in advanced infections, with massive loss of cytoplasmic contents and disappearance of cell compartments. Polyphenol oxidases are enzymes that, in olive, oxidize o‐diphenols (principally oleuropein and rutin) to produce o‐diquinones and melanins, substances that are toxic to many pathogens. No significant increase in overall PPO activity was found in infected leaves; on the contrary, enzyme activity was gradually lost as infection progressed, most probably due to degradation of plastids within mesophyll cells, in which such enzymes are normally confined. Only a limited local PPO activation occurred in a few upper epidermal cells of the leaf, indicating a feeble induction of a plant response.     

Effects of a new inhibitor K-23 on electron transport in photosystem II of higher plants

The effects of inhibitor K-23 on variable fluorescence, oxygen evolution and DCIP photoreduction were investigated. K-23 promotes the oxygen evolution and DCIP photoreduction at low concentration and inhibits them at relatively high concentrations, while an efficient inhibition at low concentration is found in variable fluorescence. These data further confirm that the inhibitor K-23 action is based on its redox interaction rather than quenching effect. Addition of DPC could not restore the DCIP photoreduction activity. It is suggested that the inhibitory site is at the acceptor side. Using ferricyanide as electron acceptor, the effect of K-23 and DCMU on the oxygen evolution of trypsin-treated thylakoids was investigated. It is found that oxygen evolution of trypsin-treated thylakoids was insensitive to DCMU, whereas became more sensitive to K-23 and also the promotion of K-23 at low concentration disappeared. This strongly indicates that trypsin treatment modified the binding site of K-23 and increased its accessibility to K-23 target site. From the comparison of K-23 with DCMU, we conclude that the binding site of K-23 is different from that of DCMU even though they both bind at the acceptor side.     

Metabolism of juvenile hormone I by microsomal oxidase,esterase, and epoxide hydrase of Musca domestica and some comparisons with Phormia regina and Sarcophaga bullata

House fly (Musca domestica L.) microsomes prepared from larvae, pupae, or adults contain three enzyme system which can metabolize juvenile hormone I: an esterase, an oxidase, and epoxide hydrase. The presence of the oxidase is indicated by the increased metabolism when microsomes are supplemented with NADPH and by the occurrence of additional metabolites tentatively identified as products arising from oxidation of the 6, 7 double bond. Additional evidence of the activity of the oxidase system is the increased metabolism of juvenile hormone I by the NADPH-dependent system from phenobarbital-induced insects, by inhibition of the oxidation by piperonyl butoxide and carbon monoxide, and by the greater metabolism of the hormone by microsomes from insecticide-resistant (high oxidase) strains. In vivo studies of house fly adults treated with ³H-labeled juvenile hormone I reveal a pattern of metabolism similar to that seen during NADPH-supplemented in vitro metabolism. The three enzymes have somewhat different patterns of activity during the larval stage of the house fly, juvenile hormone esterase and epoxide hydrase beginning at a high level of activity in the young larvae while the juvenile hormone oxidase is low at this stage. In the late larval stage all three enzymes show increased activity followed by declines during the pupal stage and further increases in the adult stage. Comparison of in vitro enzyme levels of the house fly, flesh fly (Sarcophaga bullata Parker), and blow fly [Phormia regina (Meigen)] showed that, although the enzymes were present in the latter two species, their activity on a per insect basis was considerably less than that of the house fly.     

亚洲玉米螟几丁质酶的双靶向天然产物抑制剂及其抑制机理

多个几丁质酶可协同催化昆虫表皮几丁质的水解,在昆虫蜕皮发育过程中发挥不可或缺的作用,是潜在的绿色杀虫剂作用分子靶标。本文通过对1 680种天然产物进行高通量筛选,获得了3个靶向亚洲玉米螟Ostrinia furnacalis来源的几丁质酶OfChtI和OfChi-h的双靶标抑制剂：漆树酸、邻苯二甲酸二(2-乙基己基)酯(DEHP)和紫菀酮。它们对OfChtI的抑制常数(Ki)分别为0.57、0.53和3.95μmol/L;对OfChi-h的抑制常数分别为0.48、1.42和27.33μmol/L。分子对接结果表明,三者均通过疏水堆积作用结合在靶标酶的底物结合位点上。此外,漆树酸的1位羧基氧原子作为氢键受体,与OfChtI的Arg274和OfChi-h的Arg439侧链胍基氢原子形成氢键。DEHP的羰基和烷氧基氧原子作为氢键受体,与OfChtI的Arg274侧链胍基氢原子形成氢键;DEHP的烷氧基氧原子作为氢键受体,与OfChi-h的Arg439侧链胍基氢原子形成氢键。杀虫活性测定结果表明,在2 mmol/L浓度下,漆树酸和DEHP对亚洲玉米螟幼虫的致死率为33.3%,紫菀酮没有表现出明...     

水花生茎水浸液对萝卜幼苗土壤酶活性及土壤养分的影响

反硝化酶、纤维素酶、蛋白酶、多酚氧化酶等4种土壤酶活性均有随着水花生茎水浸液浓度增加而呈增大的趋势,其中影响最大的是多酚氧化酶,水花生茎水浸液10 mol/L时,其活性较对照增加了15.3%;土壤有机质含量、有效磷含量及速效钾含量均随着水花生茎水浸液浓度的增加而呈降低的趋势,而土壤硝态氮含量却呈增加的趋势;4种土壤酶活性均与土壤有机质含量、有效磷含量及速效钾含量呈极显著负相关,而与土壤硝态氮含量呈显著正相关(反硝化酶)或极显著正相关(其他3种酶)。     

Chlorophyll fluorescence as a marker for herbicide mechanisms of action

Photosynthesis is the single most important source of O₂ and organic chemical energy necessary to support all non-autotrophic life forms. Plants compartmentalize this elaborate biochemical process within chloroplasts in order to safely harness the power of solar energy and convert it into usable chemical units. Stresses (biotic or abiotic) that challenge the integrity of the plant cell are likely to affect photosynthesis and alter chlorophyll fluorescence. A simple three-step assay was developed to test selected herbicides representative of the known herbicide mechanisms of action and a number of natural phytotoxins to determine their effect on photosynthesis as measured by chlorophyll fluorescence. The most active compounds were those interacting directly with photosynthesis (inhibitors of photosystem I and II), those inhibiting carotenoid synthesis, and those with mechanisms of action generating reactive oxygen species and lipid peroxidation (uncouplers and inhibitors of protoporphyrinogen oxidase). Other active compounds targeted lipids (very-long-chain fatty acid synthase and removal of cuticular waxes). Therefore, induced chlorophyll fluorescence is a good biomarker to help identify certain herbicide modes of action and their dependence on light for bioactivity.     

An analogue of piperonyl butoxide facilitates the characterisation of metabolic resistance

BACKGROUND: Previous work has demonstrated that piperonyl butoxide (PBO) not only inhibits microsomal oxidases but also resistance‐associated esterases. The ability to inhibit both major metabolic resistance enzymes makes it an ideal synergist to enhance xenobiotics but negates the ability to differentiate which enzyme group is responsible for conferring resistance. RESULTS: This study examines an analogue that retains the ability to inhibit esterases but is restricted in its ability to act on microsomal oxidases, thus allowing an informed decision on resistance enzymes to be made when used in conjunction with the parent molecule. CONCLUSION: Using examples of resistant insects with well‐characterised resistance mechanisms, a combination of PBO and analogue allows identification of the metabolic mechanism responsible for conferring resistance. The relative potency of PBO as both an esterase inhibitor and an oxidase inhibitor is also discussed. Copyright © 2008 Society of Chemical Industry     

Role of the posterior vacuole in Spraguea lophii (Microsporidia) spore hatching

Microsporidia constitute a large group of obligate intracellular protozoan parasites that inject themselves into host cells via the extrusion apparatus of the infective spore stage. Although the injection process is poorly understood, its energy source is thought to reside in the posterior vacuole that swells significantly during spore firing. Here we report the presence and localisation of the key peroxisomal enzymes catalase and acyl-CoA oxidase (ACOX) within the posterior vacuole of Spraguea lophii (Doflein, 1898) spores. Western blot analyses show that these enzymes discharge out of the spore and end up in the medium external to the extruded sporoplasms. The presence of a catalase enzyme system in the Microsporidia was first made evident by the detection of significant levels of molecular oxygen in the medium containing discharging spores in the presence of hydrogen peroxide. Catalase was visualised in inactive, activated, and discharged spores using alkaline diaminobenzidine (DAB) on glutaraldehyde-fixed cells. The position of these enzymes within the extrusion apparatus before and during spore discharge support the Lom and Vávra model that postulates discharge occurs by an eversion process. In addition to these enzymes, spores of S. lophii contain another characteristic peroxisomal component, the very long chain fatty acid (VLCFA) nervonic acid. A sizeable decrease in nervonic acid levels occurs during and after spore discharge. These data indicate that nervonic acid is discharged from the spore into the external medium during firing along with the catalase and ACOX enzymes.