The plant steroid hormone brassinosteroids (BRs) are important signal mediators that regulate broad aspects of plant growth and development. With the discovery of brassinoazole (Brz), the first specific inhibitor of BR biosynthesis, several triazole-type BR biosynthesis inhibitors have been developed. In this article, we report that fenarimol (FM), a pyrimidine-type fungicide, exhibits potent inhibitory activity against BR biosynthesis. FM induces dwarfism and the open cotyledon phenotype of Arabidopsis seedlings in the dark. The IC50 value for FM to inhibit stem elongation of Arabidopsis seedlings grown in the dark was approximately 1.8 ± 0.2 μM. FM-induced dwarfism of Arabidopsis seedlings could be restored by brassinolide (BL) but not by gibberellin (GA). Assessment of the target site of FM in BR biosynthesis by feeding BR biosynthesis intermediates indicated that FM interferes with the side chain hydroxylation of BR biosynthesis from campestanol to teasterone. Determination of the binding affinity of FM to purified recombinant CYP90D1 indicated that FM induced a typical type II binding spectrum with a Kd value of approximately 0.79 μM. Quantitative real-time PCR analysis of the expression level of the BR responsive gene in Arabidopsis seedlings indicated that FM induces the BR deficiency in Arabidopsis.
To explore small molecules with ethylene-like biological activity, we conducted a triple response-based assay system for chemical library screening. Among 9600 compounds, we found N-[(1,3,5-trimethyl-1H-pyrazol-4-yl)methyl]-N-methyl-2-naphthalenesulfonamide (EH-1) displayed promising biological activity on inducing a triple response in Arabidopsis seedlings. Chemical synthesis and structure–activity relationship (SAR) analysis of EH-1 analogues with different substitution patterns on the phenyl ring structure of the sulfonamide group indicated that 3,4-dichloro-N-methyl-N-(1,3,5-trimethyl-1H-pyrazol-4-yl-methyl)benzenesulfonamide (8) exhibits the most potent biological activity. To determine the mechanism of action, we conducted RNA sequencing (RNA-Seq) analysis of the effect of EH-1 and 1-aminocyclopropane-1-carboxylate (ACC), the precursor of ethylene biosynthesis, following the quantitative real-time polymerase chain reaction (RT-PCR) confirmation. Data obtained from RNA-Seq analysis indicated that EH-1 and ACC significantly induced the expression of 39 and 48 genes, respectively (above 20 fold of control), among which five genes are up-regulated by EH-1 as well as by ACC. We also found 67 and 32 genes that are significantly down-regulated, respectively, among which seven genes are in common. For quantitative RT-PCR analysis. 12 up-regulated genes were selected from the data obtained from RNA-Seq analysis. We found a good correlation of quantitative RT-PCR analysis and RNA-Seq analysis. Based on these results, we conclude that the action mechanism of EH-1 on inducing triple response in Arabidopsis is different from that of ACC.
We carried out the biochemical evaluation of the target site of propiconazole in BR biosynthesis. Applying BR biosynthesis intermediates to Arabidopsis seedlings grown in the presence of propiconazole under dark condition, we found that the target site of propiconazole in BR biosynthesis can be identified among the C22 and C23 side chain hydroxylation steps from campestanol to teasterone. Using differential spectra techniques to determine the binding affinity of propiconazole to CYP90D1, which is responsible for C23 hydroxylation of BR, we found that propiconazole induced typical type II binding spectra in response to purified recombinant CYP90D1 and the K d value was found approximately 0.76 mM.
Twenty-seven analogues of pyrazole derivatives were synthesized and subjected to structure-activity relationship studies on inducing the triple response in Arabidopsis seedlings. We found that 3,4-Dichloro-N-methyl-N-[(1-allyl-3,5-dimethyl-1H-pyrazol-4-yl)methyl]benzenesulfonamide (C26) exhibits potent activity on inducing the triple response in Arabidopsis seedlings. C26 (10 µM) induced an exaggerated apical hook in Arabidopsis seedlings. The curvature of the hook of the Arabidopsis seedlings was found to be 300±23 degrees, while ethephon (10 µM), a prodrug of ethylene, and a non-chemically treated control were found to be 128±19 and 58±16 degrees, respectively. C26 also exhibited potent activity on reducing stem elongation. The hypocotyl length of Arabidopsis seedlings treated with C26 (10 µM) was found to be 0.25±0.02 cm, while those of ethephon-treated (10 µM) and treated controls were found to be 0.69±0.06 and 1.15±0.01 cm, respectively. C26 displayed potency inhibiting the root growth of Arabidopsis seedlings similar to that of ethephon.
Abstract:To explore new antifungal agents for rice blast control, the antifungal activity of a series of novel 1,2,4-triazole derivatives against Magnaporthe oryzae has been evaluated. The antifungal activity was determined by using in vitro mycelial growth inhibition tests. Among the 19 test compounds, we found that the compound 1-(4-phenoxymethyl-2-phenyl-[1,3]dioxolan-2-ylmethyl)-1H-1,2,4-triazole (Gj) displayed potent antifungal activity against M. oryzae. The IC 50 value was found approximately 3.8±0.5 μM and the IC 50 value of propiconazole was found to be approximately 3.7±0.2 μM, respectively. Structure-activity relationship studies on aromatic ring structures provided insight and information about the structural requirements for antifungal activity of this synthetic series against M. oryzae.
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