An in vitro screening assay to discover novel inhibitors of 4-coumarate:CoA ligase

4-Coumarate:CoA ligase (4CL, EC 6.2.1.12) exists only in plants and plays an important role in the phenylpropanoid pathway. Identification of inhibitors targeting 4CL provides a novel approach for developing effective plant growth inhibitors (PGIs). The full-length gene of tobacco 4CL (Nt4CL1) was cloned and expressed in Escherichia coli Cast & Chalm. The recombinant 4CL protein was extracted and purified by several purification steps including gel-filtration and anion-exchange chromatography. 4CL activity assay was miniaturized and optimized using a 96-well microplate and a reader. Among 28 existing herbicides, propanil and swep strongly inhibited in vitro 4CL enzyme activity, and they were selected for further studies. The process of this assay can be developed into a high-throughput screening system of PGI targeting 4CL in the phenylpropanoid pathway.     

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.     

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.     

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.     

Contribution of militarine and dactylorhin A to the plant growth‐inhibitory activity of a weed‐suppressing orchid,Bletilla striata

The methanol extract of Bletilla striata, an ornamental orchid in eastern Asia, exhibited plant growth‐inhibitory activity. It was purified by continuous chromatography, based on the inhibitory activity against the growth of lettuce seedlings, resulting in two glycosidic compounds, militarine and dactylorhin A. The EC₅₀ values of militarine and dactylorhin A against the radicle elongation of the lettuce seedlings were 0.28 and 0.88 mmol L^?1, respectively. The amount of militarine and dactylorhin A in the methanol extract of the aerial part of B. striata was calculated to be 5.6 and 7.5 mg g^?1 fresh weight, respectively. The inhibitory activity of militarine and its content in the methanol extract revealed that the plant growth‐inhibitory activity of the extract of B. striata was mainly related to militarine. The inhibitory activity of militarine against the growth of Italian ryegrass and timothy were of the same level as that of lettuce.     

Root‐associated microbes in sustainable agriculture: models,metabolites and mechanisms

Since the discovery of penicillin in 1928 and throughout the ‘age of antibiotics’ from the 1940s until the 1980s, the detection of novel antibiotics was restricted by lack of knowledge about the distribution and ecology of antibiotic producers in nature. The discovery that a phenazine compound produced by Pseudomonas bacteria could suppress soilborne plant pathogens, and its recovery from rhizosphere soil in 1990, provided the first incontrovertible evidence that natural metabolites could control plant pathogens in the environment and opened a new era in biological control by root‐associated rhizobacteria. More recently, the advent of genomics, the availability of highly sensitive bioanalytical instrumentation, and the discovery of protective endophytes have accelerated progress toward overcoming many of the impediments that until now have limited the exploitation of beneficial plant‐associated microbes to enhance agricultural sustainability. Here, we present key developments that have established the importance of these microbes in the control of pathogens, discuss concepts resulting from the exploration of classical model systems, and highlight advances emerging from ongoing investigations. © 2019 Society of Chemical Industry     

普城沙雷菌A21-4根际定殖能力及其对辣椒生长和诱导抗病性的影响

 本文探讨了辣椒疫病生防菌普城沙雷菌A21-4在辣椒根际的定殖能力及其对辣椒的促生长和诱导抗病性影响。以10⁸cfu·mL^-1A21-4菌液处理辣椒苗,A21-4能够有效地定殖在辣椒根际土壤和辣椒根部,移栽第30 d在根际土壤和辣椒根部均保持10⁶cfu·g^-1以上定殖密度。辣椒根际土壤中A21-4的定殖密度和病原菌存在与否无显著差异,而辣椒根部A21-4的定殖密度在病原菌存在时显著高于没有病原菌的;A21-4处理有效促进了辣椒地上部和根部的各项生育指标,同时,显著提高了辣椒叶绿素含量和根系活力。A21-4处理的辣椒苗叶绿素含量和根系活力比对照各提高86.1%和481.8%;经A21-4处理后,辣椒根部和叶部的SOD、POD和PAL活性明显提高,辣椒根部的SOD、POD和PAL活性峰值分别比对照提高44.7%、64.2%和77.0%,辣椒叶部SOD、POD和PAL活性峰值分别比对照提高27.9%、134.9%和87.0%;此外,A21-4浸根处理还能够提高辣椒叶部对辣椒疫霉菌的抗性。     

2,4-D and MCPA and their derivatives: Effect on the activity of membrane erythrocytes acetylcholinesterase (in vitro)

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA), and their derivatives: phenol, 2,4-dichlorophenol (2,4-DCP), 2,4-dimethylphenol (2,4-DMP), and catechol on the activity of acetylcholinesterase (AChE, EC 3.1.1.7) in human erythrocytes was studied. Phenol, MCPA, and 2,4-DMP did not significantly change AChE activity in human erythrocytes (in vitro). Decrease of AChE activity was observed under the highest applied dose of 2,4-D—500 and 1000 ppm. Decrease of AChE activity exposed to 2,4-DCP and catechol was noted and depended on the doses of applied compounds. The relationship between activities and substrate concentrations (curves) was analyzed for reactions of acetylcholinesterase. Catalytic constants K_m and V_max were calculated from the Michaelis curve. Statistically significant decrease of V_max and K_m was observed in the activity of AChE incubated with 2,4-DCP and catechol, revealing mixed inhibition type of AChE inhibition (this compound may affect not only on enzyme but also on complex ES as well). 2,4-D decreases V_max but do not change K_m value, what reveals non-competitive type of AChE inhibition by this compounds. Non-competitive inhibition does not depend on the substrate concentrations but only on the inhibitor concentration and its K_i value, characterizes the affinity of inhibitors towards enzyme. In conclusion, changes of AChE activity upon 2,4-D, 2,4-DCP, and catechol are the consequences of direct interactions between compounds and the enzyme and indirect via membrane modification and increase of Reactive Oxygen Species.     

Assessment of species specificity of moulting accelerating compounds in Lepidoptera: comparison of activity between Bombyx mori and Spodoptera littoralis by in vitro reporter and in vivo toxicity assays

BACKGROUND: Dibenzoylhydrazine analogues have been developed successfully as a new group of insect growth regulators, called ecdysone agonists or moulting accelerating compounds. A notable feature is their high activity against lepidopteran insects, raising the question as to whether species‐specific analogues can be isolated. In this study, the specificity of ecdysone agonists was addressed through a comparative analysis in two important lepidopterans, the silkworm Bombyx mori L. and the cotton leafworm Spodoptera littoralis (Boisd.). RESULTS: When collections of non‐steroidal ecdysone agonists containing different mother structures (dibenzoylhydrazine, acylaminoketone, tetrahydroquinoline) were tested, in vitro reporter assays showed minor differences using cell lines derived from both species. However, when compounds with high ecdysone agonist activity were examined in toxicity assays, larvicidal activity differed considerably. Of note was the identification of three dibenzoylhydrazine analogues with > 100‐fold higher activity against Bombyx than against Spodoptera larvae. CONCLUSION: The present study demonstrated that species‐specific ecdysone‐agonist‐based insecticides can be developed, but their species specificity is not based on differences in the activation of the ecdysone receptor but rather on unidentified in vivo parameters such as permeability of the cuticle, uptake/excretion by the gut or metabolic detoxification. Copyright © 2010 Society of Chemical Industry