Synthesis of 2RS,4RS-1-[2-Phenyl-4-[2-(2-trifluromethoxy-phenoxy)-ethyl]-1,3-dioxolan-2-yl-methyl]-1H-1,2,4-triazole Derivatives as Potent Inhibitors of Brassinosteroid Biosynthesis

Brassinosteroids are important phytohormones that affect many aspects of plant growth and development. In order to manipulate brassinosteroid levels in plant tissues by using specific biosynthesis inhibitors, we have carried out a systemic search for specific inhibitors of brassinosteroid biosynthesis. Synthesis of triazole derivatives based on the ketoconazole scaffold revealed a series of novel brassinosteroid biosynthesis inhibitors (the YCZ series). To explore the structure-activity relationships of this synthetic series, we now report the synthesis of new triazole derivatives with different aromatic structures at position 2 of 1,3-dioxolane skeleton. We found that the variation of aromatic substituent significantly affect the inhibitory potency. Structure-activity relationships studies indicated that 4-chlorophenyl analogue is the most potent inhibitor of BR biosynthesis with an IC50 value approximately 0.12 ± 0.04 µM, while a bulky biphenyl group exhibited a great negative effect on promoting the inhibitory potency with an IC50 larger than 10 µM

Comparison of Effect of Brassinosteroid and Gibberellin Biosynthesis Inhibitors on Growth of Rice Seedlings

Brassinosteroid (BR) and gibberellin (GA) are two predominant plant hormones that regulate plant cell elongation. Mutants disrupt the biosynthesis of these hormones and display different degrees of dwarf phenotypes in rice. Although the role of each plant hormone in promoting the longitudinal growth of plants has been extensively studied using genetic methods, their relationship is still poorly understood. In this study, we used two specific inhibitors targeting BR and GA biosynthesis to investigate the roles of BR and GA in growth of rice seedlings. Yucaizol, a specific inhibitor of BR biosynthesis, and Trinexapac-ethyl, a commercially available inhibitor of GA biosynthesis, were used. The effect of Yucaizol on rice seedlings indicated that Yucaizol significantly retarded stem elongation. The IC50 value was found to be approximately 0.8 μmol/L. Yucaizol also induced small leaf angle phenocopy in rice seedlings, similarly to BR-deficient rice, while Trinexapac-ethyl did not. When Yucaizol combined with Trinexapac-ethyl was applied to the rice plants, the mixture of these two inhibitors retarded stem elongation of rice at lower doses. Our results suggest that the use of a BR biosynthesis inhibitor combined with a GA biosynthesis inhibitor may be useful in the development of new technologies for controlling rice plant height

New Compounds Induce Brassinosteroid Deficient-like Phenotypes in Rice

Brassinosteroids (BRs) are steroidal plant hormones with potent plant growth promoting activity. Because BR-deficient mutants of rice exhibit altered plant architecture and important agronomic traits, we conducted a systemic search for specific inhibitors of BR biosynthesis to manipulate the BR levels in plant tissues. Although previous studies have been conducted with BR biosynthesis inhibitors in dicots, little is known regarding the effects of BR biosynthesis inhibition in monocot plants. In this work, we used potent inhibitors of BR biosynthesis in Arabidopsis, and we performed a hydroponic culture of rice seedlings to evaluate the effects of BR biosynthesis inhibition. Among the test compounds, we found that 1-[[2-(4-Chlorophenyl)-4-(phenoxymethyl)-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole (1) is a potent inhibitor that could induce phenotypes in rice seedlings that were similar to those observed in brassinosteroid deficient plants. The IC50 value for the retardation of plant growth in rice seedlings was approximately 1.27 ± 0.43 μM. The IC50 value for reducing the bending angle of the lamina joint was approximately 0.55 ± 0.15 μM

A Chemical Genetics Strategy That Identifies Small Molecules Which Induce the Triple Response 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

Fenarimol, a Pyrimidine-Type Fungicide, Inhibits Brassinosteroid Biosynthesis

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

YCZ-18 is a new brassinosteroid biosynthesis inhibitor.

Plant hormone brassinosteroids (BRs) are a group of polyhydroxylated steroids that play critical roles in regulating broad aspects of plant growth and development. The structural diversity of BRs is generated by the action of several groups of P450s. Brassinazole is a specific inhibitor of C-22 hydroxylase (CYP90B1) in BR biosynthesis, and the application use of brassinazole has emerged as an effective way of complementing BR-deficient mutants to elucidate the functions of BRs. In this article, we report a new triazole-type BR biosynthesis inhibitor, YCZ-18. Quantitative analysis the endogenous levels of BRs in Arabidopsis indicated that YCZ-18 significantly decreased the BR contents in plant tissues. Assessment of the binding affinity of YCZ-18to purified recombinant CYP90D1 indicated that YCZ-18 induced a typical type II binding spectrum with a Kd value of approximately 0.79 μM. Analysis of the mechanisms underlying the dwarf phenotype associated with YCZ-18 treatment of Arabidopsis indicated that the chemically induced dwarf phenotype was caused by a failure of cell elongation. Moreover, dissecting the effect of YCZ-18 on the induction or down regulation of genes responsive to BRs indicated that YCZ-18 regulated the expression of genes responsible for BRs deficiency in Arabidopsis. These findings indicate that YCZ-18 is a potent BR biosynthesis inhibitor and has a new target site, C23-hydroxylation in BR biosynthesis. Application of YCZ-18 will be a good starting point for further elucidation of the detailed mechanism of BR biosynthesis and its regulation