Propanil and swep inhibit 4-coumarate:CoA ligase activity in vitro

4-Coumarate:CoA ligase (4CL, EC 6.2.1.12) in the phenylpropanoid pathway in plants has attracted interest as a novel target for developing effective plant growth inhibitors (PGIs). In a previous study in which the 4CL inhibitory activity of 28 existing herbicides was investigated using an optimized in vitro screening assay, 4CL activity was found to be strongly inhibited by propanil and swep at 100 microM. Here, further experimental evidence is provided to substantiate the previous result. Using 4-coumaric acid as substrate, tobacco 4CL activity was inhibited by propanil or swep in a concentration-dependent manner, with 50% inhibition concentrations (I(50)) of 39.6 and 6 microM respectively. These herbicides also exhibited uncompetitive inhibition towards 4-coumaric acid. Furthermore, 4CLs from several plant species were inhibited by the herbicides within a range from 1 to 50 microM. It is proposed that these herbicides have another site of action as a result of the inhibition of 4CL in the phenylpropanoid pathway, and this enzyme represents a new target site for the development of PGI.     

Suppression of induced resistance in cucumber through disruption of the flavonoid pathway

ABSTRACT In this study, cucumber plants (Cucumis sativus) expressing induced resistance against powdery mildew (caused by Podosphaera xanthii) were infiltrated with inhibitors of cinnamate 4-hydroxylase, 4-coumarate:CoA ligase (4CL), and chalcone synthase (CHS) to evaluate the role of flavonoid phytoalexin production in induced disease resistance. Light and transmission electron microscopy demonstrated ultrastructural changes in inhibited plants, and biochemical analyses determined levels of CHS and beta-glucosidase enzyme activity and 4CL protein accumulation. Our results showed that elicited plants displayed a high level of induced resistance. In contrast, down regulation of CHS, a key enzyme of the flavonoid pathway, resulted in nearly complete suppression of induced resistance, and microscopy confirmed the development of healthy fungal haustoria within these plants. Inhibition of 4CL ligase, an enzyme largely responsible for channeling phenylpropanoid metabolites into the lignin pathway, had little effect on induced disease resistance. Biochemical analyses revealed similar levels of 4CL protein accumulation for all treatments, suggesting no alterations of nontargeted functions within inhibited plants. Collectively, the results of this study support the idea that induced resistance in cucumber is largely correlated with rapid de novo biosynthesis of flavonoid phytoalexin compounds.     

MEAM1和MED两个烟粉虱隐种取食对甘蓝转录组影响的差异

为绿色防控烟粉虱Bemisia tabaci提供新策略,采用转录组分析MEAM1和MED两个烟粉虱隐种取食后甘蓝差异基因表达情况,并选取差异表达基因最多的甘蓝苯丙烷类通路上PAL2、C4H、4CL1和CHI四个基因进行实时荧光定量PCR测定。结果显示,与对照相比,MEAM1和MED烟粉虱隐种取食后甘蓝差异表达基因数分别为693个和1 030个。GO功能注释和KEGG富集分析显示,差异表达基因主要集中在苯丙烷类生物合成、萜类挥发物合成、芥子油苷合成、类黄酮生物合成和植物激素信号传导途径等与甘蓝抗虫物质合成相关的通路上。虽然MEAM1与MED两个烟粉虱隐种均能显著激活植物激素信号传导途径和苯丙烷类合成通路调控的酚类物质合成途径,但MED烟粉虱隐种与甘蓝互作的差异表达基因数高于MEAM1烟粉虱隐种。MEAM1烟粉虱隐种取食甘蓝12 h后,甘蓝苯丙烷通路上游PAL2、C4H和4CL1三个基因均显著上调,而MED烟粉虱隐种取食后,甘蓝苯丙烷通路上PAL2、C4H、4CL1和CHI四个基因均变化不显著,这4个差异基因的表达模式与测序结果一致,表明测序结果的可信度高。     

Effect of naringenin on the growth and lignin biosynthesis of gramineous plants

Lignin biosynthesis is essential for plant growth. 4‐Coumarate CoA ligase (4‐CL, EC6.2.1.12) is involved in the monolignol synthesis and occupies a key role in regulating carbon flow into the phenylpropanoid metabolism pathway. Naringenin, one of the metabolites in this pathway, is known as a potent in vitro inhibitor of 4‐CL. The growth of rice (Oryza sativa L. cv. Koshihikari), maize (Zea mays L. cv. Yellow corn) and Echinochloa oryzicola Vasing seedlings at the 2nd leaf stage was inhibited after continuous root application with 0.1 mmol L^?1 naringenin for 1 week, although naringenin did not kill these gramineous plants. The highest inhibition of fresh weight increase was observed in maize, followed by rice and E. oryzicola. The symptoms in these plants were root browning, delay of leaf/root development and shoot dwarfing. Naringenin treatment increased the contents of 4‐CL substrates, cinnamic acid, 4‐coumaric acid, caffeic acid and ferulic acid from 1.2 to 7.2 times and from 1.2 to 3.5 times in shoots and roots, respectively, except for ferulic acid in E. oryzicola roots. It also caused a slight decrease of the lignin content and alteration of lignin constitutions in rice plants. These results suggested that the monolignol pathways after 4‐CL towards lignin has the possibility to be the novel action sites of plant growth retardants, although further investigations are needed to clarify the mode of action.     

Glyphosate-induced metabolic changes in susceptible and glyphosate-resistant soybean (Glycine max L.) roots

Glyphosate (N-(phosphonomethyl)glycine) blocks the shikimate pathway, reducing the biosynthesis of aromatic amino acids, followed by the arrest interruption of protein production and a general metabolic disruption of the phenylpropanoid pathway. Glyphosate-resistance is conferred to soybean by incorporating a gene encoding a glyphosate-insensitive enzyme (CP4-EPSP synthase) that acts in the shikimate pathway. This paper evaluates the metabolic effects caused by this herbicide on the shikimate (shikimate dehydrogenase activity and shikimate content) and phenylpropanoid (phenylalanine ammonia-lyase activity, phenolic and lignin contents) pathways in BRS-133 (susceptible) and BRS-245RR (resistant) soybean (Glycine max L.) roots. In general, the results showed that in susceptible roots (1) glyphosate affects the shikimate pathway (massive shikimate accumulation and enhanced shikimate dehydrogenase activity) and the phenylpropanoid pathway (increase in PAL activity, production of benzoate derivatives and decrease of lignin) and (2) the metabolic disruption contributes to the production of p-hydroxybenzoate and vanillate, which likely originate from shikimate and/or cinnamate and their derivatives. No such changes were observed in the genetically modified soybean consistent with its resistance to glyphosate.     

Selective growth suppression of five annual plant species by chalcone and naringenin correlates with the total amount of 4-coumarate: coenzyme A ligase

The 4-coumarate: coenzyme A ligase (4CL) is one of the key enzymes in the biosynthesis of lignin monomers. It has been demonstrated that the 4CL is a new potential target site for developing effective plant growth inhibitors. Although previous studies demonstrate that chalcone and naringenin differentially suppress the growth of several annual plant species, we show here that the compounds can inhibit the 4CL enzyme activity in the plants. The enzyme was extracted and partially purified from the leaf tissues of two tolerant plants (wheat and soybean) and three susceptible plants (tomato, barnyard grass, and common chickweed). A maximal 29-fold purification of the enzyme, with a yield of 32% (tomato), was achieved by a six-step procedure, including anion-exchange column chromatography. Naringenin strongly inhibited the 4CL specific activity in wheat, soybean and barnyard grass, whereas chalcone showed the highest inhibitory effect in common chickweed. A good correlation was observed between the level of growth suppression by the compounds and the total 4CL amount in the plants. These results suggest that the inhibitor treatment at the same concentration could not inactivate the entire 4CL enzyme produced in the tolerant plants. Taken together, these results highlight the possibility of the 4CL as a new action site of growth suppression.     

Molecular cloning and characterization of the cinnamate 4‐hydroxylase gene from Eupatorium adenophorum

Cinnamate‐4‐hydroxylase (CA4H), a cytochrome P450‐dependent monooxygenase, plays crucial roles in phenylpropanoid metabolism and plant defense. Previously, the authors showed that the expression of CA4H was induced in response to an allelopathic substance in Eupatorium adenophorum. Here, the full‐length cDNA of EaCA4H was cloned by using rapid amplification of cDNA ends. The 1518 bp open reading frame of EaCA4H was deduced to encode a protein of 505 amino acid residues. Like other CA4H proteins, the predicted EaCA4H polypeptides contained conserved domains of cytochrome P450. A Southern blot analysis indicated that at least five copies of EaCA4H exists in the genome of E. adenophorum. Subcellular localization revealed nuclear‐localized EaCA4H–green fluorescent protein fusion protein in onion epidermal cells. Heterologous silencing of endogenous CA4H in tobacco by a conserved EaCA4H fragment resulted in reduced expressions of key enzymatic genes and the production of downstream flavonoids in the phenylpropanoid pathway. Intriguingly, similar effects were observed in transgenic tobacco plants overexpressing EaCA4H. Altogether, the results indicate that the disturbed expression of CA4H in plants leads to relatively low expression levels of key enzymatic genes and the accumulation of the flavonoids that are involved in phenylpropanoid metabolism.     

Potential physical and chemical barriers to infection by the burrowing nematode Radopholus similis in roots of susceptible and resistant banana (Musa spp.)

Resistance in banana roots against the burrowing nematode Radopholus similis has been correlated in the past with the phenylpropanoid pathway of secondary metabolism, but quantitative chemical analyses to support histological data are lacking. Therefore, healthy and infected roots of two susceptible (Grande naine and Obino l'ewai) and three resistant cultivars (Yangambi km5, Pisang jari buaya and Calcutta 4) were extracted and chemically analysed for their lignin content and phenylpropanoid profile using a quantitative lignin assay, high performance liquid chromatography and liquid chromatography/mass spectrometry. Through histochemical staining phenylpropanoids were localized in root tissue. Compared to the susceptible cultivars, the resistant cultivars had constitutively significantly higher levels of lignin in the vascular bundle and cell-wall bound ferulic acid esters in the cortex. Infection-induced lignification was observed in the vascular bundles of all cultivars. The catecholamine dopamine was identified as a major metabolite in banana roots. Levels varied from 2·8 to 8·4 mg per g root fresh weight and were significantly higher in the resistant cultivars. Other compounds, tentatively identified as anthocyanidin-related, were present in high quantities and may, besides dopamine, make up the substrates for polyphenol oxidation products in necrotic tissue.     

Chalcone suppresses lignin biosynthesis in illuminated soybean cells

Lignin and its related metabolites play critical roles in plant growth and development. Thus, lignin biosynthesis has attracted interest as a novel target site of plant growth inhibitors. Chalcone has been shown to not only inhibit lignin biosynthesis in plants, but also to suppress the growth of many annual plant species. In order to know the direct effect of chalcone on plant metabolism, the effects of chalcone on the activities of key enzymes in lignin biosynthesis and on the related metabolites were clarified with a time‐course study by using light‐induced suspension cultures of soybean cells. The fresh weight and packed cell volume of the soybean cells were inhibited after 8 h of chalcone treatment. The activities of phenylalanine ammonia lyase (EC 4.3.1.24) and 4‐coumarate: coenzyme A ligase (4CL; EC 6.2.1.12) were largely inhibited 4 h after the treatment with 0.15 mmol L^?1 chalcone. Unlike these two enzymes, the activity of cinnamyl alcohol dehydrogenase (EC 1.1.1.195) was not inhibited until 16 h after the chalcone treatment. The content of the 4CL substrates and lignin in the soybean cells became relatively lower than the control under the light condition within 4 h and 8 h after the chalcone treatment, respectively. These results suggest that the growth suppression of soybean cells is positively associated with the inhibition of lignin biosynthesis by exogenous chalcone.     

棉疫病菌90kD胞外蛋白激发子诱导烟草过敏性反应的研究

 就棉疫病菌90 kD胞外蛋白激发子诱导烟草过敏反应(HR)过程中细胞死亡和防卫反应酶系活性变化及病程相关蛋白PR5的诱导进行研究。结果是,以10 nmol/L激发子溶液注射处理W38烟草叶片,HR枯斑周围5 mm宽组织在UV光下呈现蓝色荧光,对处理部位进行Evans blue染色测定结果是至20 h处理部位细胞全部死亡;激发子可诱导烟草防卫反应中苯丙氨酸解氨酶(PAL)的活性提高;可快速诱导PR5基因的转录。上述结果表明90 kD蛋白激发子可诱发烟草的细胞死亡、苯丙烷代谢和PR基因的表达等多条信号途径。     

In search of new herbicidal inhibitors of the non‐mevalonate pathway

The first five steps of the non‐mevalonate pathway have been tested in high‐throughput screening (HTS) campaigns, using enzymes of plant origin. Hit rates were in general relatively low, which could be attributed to the high polarity and charged nature of substrates and active sites of these enzymes. Still, for all the enzymes, apart from IspF (2‐methylerythritol 2,4‐cyclodiphosphate synthase), inhibitors could be identified with activities below 100 μM, and these were followed up to identify structure–activity relationships (SARs). For the enzyme IspD (2C‐methyl‐D‐erythritol 4‐phosphate cytidyltransferase), inhibitors with IC₅₀ down to 35 nM were identified that also showed herbicidal activity.     

乙酰乳酸合成酶及其抑制剂研究新进展

乙酰乳酸合成酶(AHAS)是支链氨基酸生物合成途径中的一个关键酶,是绿色除草剂的重要作用靶标。由于此生物合成过程只存在于植物和微生物体内,因此该类抑制剂对哺乳动物具有生物安全性。近年来,随着AHAS三维结构的阐明,人们不仅深入了解了已有抑制剂的作用机制,并且依此设计开发了一些新型的抑制剂,拓展了其在抑菌活性方面的生物学功能。文章对近年来AHAS及其抑制剂的最新研究进展进行了综述,重点就AHAS的酶学特征、结构特征及结合方式,以AHAS为靶标的新颖除草活性化合物的设计开发以及AHAS抑制剂的抗菌生物活性研究进展等问题详细进行了总结,以期为设计开发靶向AHAS的新型除草剂或抗菌药物提供参考。     

Differential Metal Binding Interactions of the Imidazolinones Revealed by NMR and UV Spectroscopy

NMR and UV spectroscopy and molecular modeling methods were applied to probe the interaction of the two imidazolinones, imazethapyr (5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid) and its structural isomer CL 303,135 (5-ethyl-3-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)picolinic acid), with metal ions. Both the imidazolinones inhibit the enzyme acetohydroxyacid synthase (AHAS) in vitro. However, while imazethapyr is a herbicide that is used widely in agriculture, CL303,135 does not exhibit herbicidal activity. Imazethapyr and CL303,135 exhibited considerable differences in their interactions with metals. In the metal complex of imazethapyr, the carboxyl moiety binds strongly and the pyridine nitrogen binds weakly with metals. In the case of CL303,135, both the pyridine nitrogen and the carboxyl group that are positioned ortho to each other participated strongly in the binding and were found to act together as a strong bidentate ligand to a metal ion. Both of the imidazolinones form predominantly 2:1 complexes with multivalent metal ions. However, imazethapyr binds two orders-of-magnitude more weakly (1·0×10⁹ M ^-2) with metal ions compared to CL303,135 (1·7×10¹¹ M ^-2). The interactions of the model compounds, nicotinic acid and picolinic acid, with metals were examined similarly. It was concluded that the strong affinity of CL303,135 for metals compared to imazethapyr may affect its absorption from soil into plants, or its translocation in plants, thereby explaining the differences in herbicidal activity of imazethapyr and CL303,135. © 1997 SCI.     

Inhibition of insect cytochrome P-450 by some metyrapone analogues and compounds containing a cyclopropylamine moiety and their evaluation as inhibitors of juvenile hormone biosynthesis

Two metyrapone analogues, 2-(l-imidazolyl)-2-methyl-l-phenyl-l-pro-panone (A-phenyl-B-imidazolyl-metyrapone; III) and 2-methyl-l-phenyl-2-{1,2,4-triazol-l-yl)-l -propanone (A-phenyl-B-triazolyl-meiyrapone; IV) as well as two cyclopropylamine derivatives. N-cyclopropyl-4-icrt-butylbenzylamine (V) and N-cyclopropyl-4-(3,7-dimethyl-7-methoxy-octyloxy)benzamide (cyclopropylamine acylated with a JH analogue acid of known structure; VI) were synthesized and evaluated in biological assays for JH biosynthesis on cockroach, Diploptera punctata corpora allata and egg growth in adult cockroach as well as for mixed function oxidase activities, i.e. epoxidation of aldrin to dieldrin and O-demethylation of 7-methoxy-4-methylcoumarin to 7-hydroxy-4-methylcoumarin on microsomes from housefly, Musca domestica, abdomen and from cockroach midgut. Compound VI was a good in-vitro inhibitor of JH biosynthesis, but it had significantly lower activities in the assays for inhibition of microsomal cytochrome P-450. Compound IV and metyrapone had moderate activity as inhibitors of oocyte growth. Compounds III, IV and V were more potent inhibitors of housefly aldrin epoxidation than metyrapone and they inhibited the enzyme activity by almost 100% at 02mM, while in cockroach midgut microsome assay metyrapone was more potent than these three compounds.     

Purification and Characterization of 4-Hydroxyphenylpyruvate Dioxygenase from Maize

The proposed target enzyme for benzoylcyclohexanedione herbicides, 4-hydroxyphenylpyruvate dioxygenase (HPPD) was purified from etiolated maize seedlings with a purification factor of 105. Enzyme activity was measured by detection of carbon dioxide formed from radiolabelled substrate. The enzyme has a pH optimum of 7·3 and an apparent molecular mass of 43 kDa, similar to that of the mammalian liver enzyme. Activity needs the presence of a reducing system glutathione/dichlorophenol indophenol or ascorbate and catalase. Surprisingly, a commercial catalase preparation of low specific activity—generally used for the enzyme assay—showed HPPD activity which was separable from the catalase activity on a gel filtration column. According to kinetic studies with purified maize HPPD, experimental herbicides from the family mentioned were strong competitive inhibitors of the plant enzyme in nanomolar range withK_i values of 5 and 15 nM for 2-(2-nitro-4-chlorobenzoyl)-5-(2-methoxyethyl) cyclohexane- 1,3-dione and 2-(2-chloro-4-methanesulfonylbenzoyl)- cyclohexane-1,3-dione (SC-0051; sulcotrione), respectively.     

A fungal elicitor enhances the resistance of tomato fruit to Fusarium oxysporum infection by activating the phenylpropanoid metabolic pathway

Fungi infection in fruits is an important factor in postharvest losses. The effect of a treatment with a fungal elicitor on the response of tomato fruit to Fusarium oxysporum infection and changes in the phenylpropanoid metabolic pathway was studied. Fungal elicitor retarded for 3 days the development of the Fusarium rot development in tomato, at concentration of 2 g?l ^?1 (B2-F treatment). This treatment also induced a 3.11- and 6.03-fold increase of caffeic and chlorogenic acids, respectively, as compared with the control. Furthermore, the flavonoids naringenin-7-O-glucoside, rutin and kaempferol-3-O-glucoside, showed a greater abundance in tomato under the B2-F treatment after 6 days at 20°C. It is concluded that the fungal elicitor reduced the development of Fusarium rot by inducing the biosynthesis of metabolites from the phenylpropanoid metabolic pathway which forms part of the defense response in tomato fruit.     

The carboxyterminal processing protease of D1 protein: expression, purification and enzymology of the recombinant and native spinach proteins

The carboxyterminal processing protease of D1 protein (CtpA) is predicted to be an excellent target for a general broad-spectrum herbicide. The gene for spinach CtpA has been expressed in Escherichia coli. The expressed protein that was found mainly in inclusion bodies has been purified and refolded on a nickel-chelate column. Active recombinant CtpA was recovered. Two assays for CtpA activity were developed, a medium-throughput HPLC assay using a fluorescent substrate and a high-throughput assay based on fluorescence polarization capable of application in a high-throughput 96-well plate format. This high-throughput assay was developed to screen chemistry for CtpA inhibitors. Native spinach CtpA was partially purified and the native and recombinant enzymes were compared kinetically for their K(m) and V(max) values using different peptide substrates. Native CtpA partially purified from spinach was shown to have similar kinetic properties to recombinant CtpA. Antibodies developed against the recombinant protein were used to estimate the in planta abundance of the native enzyme in spinach. Since only a small proportion of the recombinant protein is refolded during isolation and it appears that only a small proportion of this enzyme is active, size-exclusion chromatography and light scattering experiments were performed on rCtpA in order to gain insight into its structure and the reasons why most of the protein is not active. The use of rCtpA to screen for herbicidal compounds and the more general question of how good a herbicide target the enzyme is are discussed.     

Identification of novel juvenile-hormone signaling activators via high-throughput screening with a chemical library

Juvenile hormone (JH) is an insect-specific hormone that regulates molting and metamorphosis. Hence, JH signaling inhibitors (JHSIs) and activators (JHSAs) can be used as effective insect growth regulators (IGRs) for pest management. In our previous study, we established a high-throughput screening (HTS) system for exploration of novel JHSIs and JHSAs using a Bombyx mori cell line (BmN_JF&AR cells) and succeeded in identifying novel JHSIs from a chemical library. Here, we searched for novel JHSAs using this system. The four-step HTS yielded 10 compounds as candidate JHSAs; some of these compounds showed novel basic structures, whereas the others were composed of a 4-phenoxyphenoxymethyl skeleton, the basic structure of several existing JH analogs (pyriproxyfen and fenoxycarb). Topical application of seven compounds to B. mori larvae significantly prolonged the larval period, suggesting that the identified JHSAs may be promising IGRs targeting the JH signaling pathway.     

新型吡唑-喹唑啉二酮类对羟基苯丙酮酸双加氧酶抑制剂的设计、合成及生物活性

对羟基苯丙酮酸双加氧酶 (HPPD) 抑制剂近年来因其高活性和低抗性风险成为了除草剂领域的研究热点。喹唑啉二酮被证明是一类具有潜力的骨架结构,为了继续发挥该骨架的优势,在前期工作的基础上,结合已有的构效关系研究结果,设计并合成了30个新型含喹唑啉二酮结构的吡唑类HPPD抑制剂,其结构均经过了高分辨质谱 (HRMS)、核磁共振氢谱 (1H NMR) 和碳谱 (13C NMR) 的表征。酶水平和活体活性测试结果表明,大部分目标化合物展现出了与对照药剂喹草酮相当甚至更优异的酶抑制活性。温室除草活性测试结果表明,目标化合物对6种供试杂草均有一定的生长抑制作用,特别是化合物 9-28 ,在有效成分150 g/hm2剂量下,对6种杂草的防治效果均在80%以上,其中对稗草和马唐的防治效果达100%。最后,培养了代表性化合物 9-28 与拟南芥HPPD的复合物晶体结构,从分子层面说明了该类抑制剂与靶标的结合模式,也为后续该类抑制剂的开发提供了一定的分子基础和设计思路。     

Differential effects of phenylalanine ammonia lyase, cinnamyl alcohol dehydrogenase, and energetic metabolism inhibition on resistance of appropriate host and nonhost cereal-rust interactions

ABSTRACT Effects of phenylpropanoid and energetic metabolism inhibition on resistance were studied during appropriate host and nonhost cereal-rust interactions. In the appropriate barley-Puccinia hordei interaction, phenylalanine ammonia lyase (PAL) and cinnamyl alcohol dehydrogenase (CAD) inhibition reduced penetration resistance in two genotypes, suggesting a role for phenolics and lignins in resistance. Interestingly, penetration resistance of the barley genotype 17.5.16 was not affected by phenylpropanoid biosynthesis but penetration resistance was almost completely inhibited by D-mannose, which reduces the energy available in plant host cells. This suggests a parallel in the cellular basis of penetration resistance between 17.5.16 rust and mlo barleys powdery mildew interaction. Results revealed differing patterns of programmed cell death (PCD) in appropriate versus nonhost rust interactions. PAL and CAD inhibitors reduced PCD (hypersensitivity) in appropriate interactions. Conversely, they had no effect in PCD of wheat to P. hordei; whereas D-mannose dramatically reduced nonhost resistance and allowed colony establishment. The differential effects of inhibitors in the expression of the different resistances and the commonalities with the cereal-powdery mildew interaction is analyzed and discussed.