Genome-wide transcriptional profiling for elucidating the effects of brassinosteroids on Glycine max during early vegetative development.

Soybean is a widely grown grain legume and one of the most important economic crop species. Brassinosteroids play a crucial role in plant vegetative growth and reproductive development. However, it remains unclear how BRs regulate the developmental processes in soybean, and the molecular mechanism underlying soybean early development is largely unexplored. In this study, we first characterized how soybean early vegetative growth was specifically regulated by the BR biosynthesis inhibitor propiconazole; this characterization included shortened root and shoot lengths, reduced leaf area, and decreased chlorophyll content. In addition, the growth inhibition induced by Pcz could be rescued by exogenous brassinolide application. The RNA-seq technique was employed to investigate the BR regulatory networks during soybean early vegetative development. Identification and analysis of differentially expressed genes indicated that BRs orchestrate a wide range of cellular activities and biological processes in soybean under various BR concentrations. The regulatory networks between BRs and multiple hormones or stress-related pathways were investigated. The results provide a comprehensive view of the physiological functions of BRs and new insights into the molecular mechanisms at the transcriptional level of BR regulation of soybean early development.

Structural insights into a key step of brassinosteroid biosynthesis and its inhibition.

Brassinosteroids (BRs) are essential plant steroid hormones that regulate plant growth and development1. The most potent BR, brassinolide, is produced by addition of many oxygen atoms to campesterol by several cytochrome P450 monooxygenases (CYPs). CYP90B1 (also known as DWF4) catalyses the 22(S)-hydroxylation of campesterol and is the first and rate-limiting enzyme at the branch point of the biosynthetic pathway from sterols to BRs2. Here we show the crystal structure of Arabidopsis thaliana CYP90B1 complexed with cholesterol as a substrate. The substrate-binding conformation explains the stereoselective introduction of a hydroxy group at the 22S position, facilitating hydrogen bonding of brassinolide with the BR receptor3-5. We also determined the crystal structures of CYP90B1 complexed with uniconazole6,7 or brassinazole8, which inhibit BR biosynthesis. The two inhibitors are structurally similar; however, their binding conformations are unexpectedly different. The shape and volume of the active site pocket varies depending on which inhibitor or substrate is bound. These crystal structures of plant CYPs that function as membrane-anchored enzymes and exhibit structural plasticity can inform design of novel inhibitors targeting plant membrane-bound CYPs, including those involved in BR biosynthesis, which could then be used as plant growth regulators and agrochemicals.

In silico exploration for agonists/antagonists of brassinolide.

Brassinolide (BL) is a plant steroid hormone that is necessary for stem elongation and cell division. To date more than 70 steroidal BL-like compounds, which are collectively named as brassinosteroids, have been identified. However, non-steroidal compounds that mimic BL have not been reported yet, which can be used as plant growth regulators. Twenty-two non-steroidal compounds were screened from the database containing about 5 million compound structures using a pharmacophore-based in silico screening method. The crystal structure (PDB: 4LSX) of the BL receptor was used to generate a pharmacophore model required for in silico screening. Among 22 hit compounds, 15 compounds that are thought to be physicochemically acceptable were submitted to the in vivo rice lamina inclination assay. Although no compound showed BL like activity, three compounds were detected as BL antagonist. The most potent compound was an ester derivative of 1,4-diphenlenedimethanol and isoxazole-4-carboxylic acid, and the other two compounds contain 2-phenylfuran and pyrimidin-2(1H)-one moieties bridged by an ethenyl substructure. The 50% effective doses (ED50) for the antagonistic activity were in a range of 0.6-5nmol per plant. The inhibition of the lamina inclination by the most potent agonist was recovered by the co-application of BL in a dose-dependent manner.

Synthesis and biological evaluation of novel azole derivatives as selective potent inhibitors of brassinosteroid biosynthesis.

Brassinosteroids (BRs) are phytohormones that control several important agronomic traits, such as flowering, plant architecture, seed yield, and stress tolerance. To manipulate the BR levels in plant tissues using specific inhibitors of BR biosynthesis, a series of novel azole derivatives were synthesized and their inhibitory activity on BR biosynthesis was investigated. Structure-activity relationship studies revealed that 2RS, 4RS-1-[4-(2-allyloxyphenoxymethyl)-2-(4-chlorophenyl)-[1,3]dioxolan-2-ylmethyl]-1H-[1,2,4]triazole (G(2)) is a highly selective inhibitor of BR biosynthesis, with an IC(50) value of approximately 46 ± 2 nM, which is the most potent BR biosynthesis inhibitor observed to date. Use of gibberellin (GA) biosynthesis mutants and BR signaling mutants to analyze the mechanism of action of this synthetic series indicated that the primary site of action is BR biosynthesis. Experiments feeding BR biosynthesis intermediates to chemically treated Arabidopsis seedlings suggested that the target sites of this synthetic series are CYP90s, which are responsible for the C-22 and/or C-23 hydroxylation of campesterol.

Phytosulfokine control of growth occurs in the epidermis, is likely to be non-cell autonomous and is dependent on brassinosteroids.

Phytosulfokine (PSK) is a secreted disulfated pentapeptide that controls root and shoot growth. The ubiquitous expression of PSK precursor and of the LRR receptor kinase genes in Arabidopsis raised the question of whether PSK acts as an autocrine growth factor in planta. Expression of PSKR1 under the control of tissue- and cell type-specific promoters in a receptor null background strongly suggests that PSK is a non-cell autonomous signal that controls growth through localized activity in the epidermis. pskr1-3 pskr2-1 seedlings had shorter roots and hypocotyls than the wild type, whereas 35S: PSKR1 or 35S: PSKR2 seedlings were larger, indicating that receptor abundance limits growth in planta. The preferential expression of PSKR1 in the epidermis of CER6: PSKR1 pskr1-3 pskr2-1 seedlings was sufficient to promote wild-type growth. Moreover, in GL2:PSKR1 pskr1-3 pskr2-1 seedlings that express PSKR1 in atrichoblasts of the root epidermis, root growth was restored to wild-type levels. In pskr1-3 pskr2-1 seedlings, trichoblasts and atrichoblasts were shorter than in the wild type. Trichoblasts of GL2:PSKR1 pskr1-3 pskr2-1 seedlings, which are unable to sense PSK, nonetheless had acquired wild-type length, suggesting that PSK acts as a non-cell autonomous signal. Inhibition of brassinosteroid (BR) biosynthesis with brassinazole (BZ) caused a loss of responsiveness to PSK in wild-type, tpst-1 (tyrosylprotein sulfotransferase-1), PSKR1ox12 and CER6:PSKR1-3-1 seedlings, as did the genetic knock-out of BR synthesis in det2-1 and of BR perception in bri1-9, suggesting that BR mediates PSK-dependent growth. Quantitative PCR analysis of BR-related genes in wild-type, pskr1-3 pskr2-1, PSKR1ox and tpst-1 seedlings showed largely unchanged transcript levels of BR biosynthesis genes.

Propiconazole is a specific and accessible brassinosteroid (BR) biosynthesis inhibitor for Arabidopsis and maize.

Brassinosteroids (BRs) are steroidal hormones that play pivotal roles during plant development. In addition to the characterization of BR deficient mutants, specific BR biosynthesis inhibitors played an essential role in the elucidation of BR function in plants. However, high costs and limited availability of common BR biosynthetic inhibitors constrain their key advantage as a species-independent tool to investigate BR function. We studied propiconazole (Pcz) as an alternative to the BR inhibitor brassinazole (Brz). Arabidopsis seedlings treated with Pcz phenocopied BR biosynthetic mutants. The steady state mRNA levels of BR, but not gibberellic acid (GA), regulated genes increased proportional to the concentrations of Pcz. Moreover, root inhibition and Pcz-induced expression of BR biosynthetic genes were rescued by 24epi-brassinolide, but not by GA(3) co-applications. Maize seedlings treated with Pcz showed impaired mesocotyl, coleoptile, and true leaf elongation. Interestingly, the genetic background strongly impacted the tissue specific sensitivity towards Pcz. Based on these findings we conclude that Pcz is a potent and specific inhibitor of BR biosynthesis and an alternative to Brz. The reduced cost and increased availability of Pcz, compared to Brz, opens new possibilities to study BR function in larger crop species.

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.

Boosting crop yields with plant steroids.

Plant sterols and steroid hormones, the brassinosteroids (BRs), are compounds that exert a wide range of biological activities. They are essential for plant growth, reproduction, and responses to various abiotic and biotic stresses. Given the importance of sterols and BRs in these processes, engineering their biosynthetic and signaling pathways offers exciting potentials for enhancing crop yield. In this review, we focus on how alterations in components of sterol and BR metabolism and signaling or application of exogenous steroids and steroid inhibitors affect traits of agronomic importance. We also discuss areas for future research and identify the fine-tuning modulation of endogenous BR content as a promising strategy for crop improvement.

Synthesis of novel brassinosteroid biosynthesis inhibitors based on the ketoconazole scaffold.

Brassinosteroids (BRs) are steroidal plant hormones that control several important agronomic traits such as plant architecture, seed yield, and stress tolerance. Inhibitors that target BR biosynthesis are candidate plant growth regulators. We synthesized novel triazole derivatives, based on the ketoconazole scaffold, that function as inhibitors of BR biosynthesis. The biological activity of the test compounds was evaluated by determining their ability to induce dwarfism in Arabidopsis seedlings grown in the dark. The chemically induced dwarfism of Arabidopsis seedlings was further evaluated by a rescue experiment using the co-application of brassinolide and/or gibberellins (GA). The structure-activity relationship studies revealed a potent BR biosynthesis inhibitor, 2RS, 4RS-1-{2-(4-chlorophenyl)-4-[2-(2-ethoxyphenyl)-ethyl]-1,3-dioxolan-2-ylmethyl}-1H-1,2,4-triazole (7m), with an IC(50) value of 0.10±0.03 µM for retardation of Arabidopsis seedling stem elongation. The compound-induced hypocotyl dwarfism was counteracted by the co-application of 10nM brassinolide, but not 1 µM GA(3), which produced seedlings that resembled BR-deficient mutants. This result suggests that 7m is a potent and specific inhibitor of BR biosynthesis.