Asymmetric synthesis and effect of absolute stereochemistry of YCZ-2013, a brassinosteroid biosynthesis inhibitor

The four stereoisomers of 2RS,4RS-1-[[2-(2,4-dichlorophenyl)-4-(2-(2-propenyloxy)phenoxymethyl)-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole (YCZ-2013), a novel brassinosteroid biosynthesis inhibitor, were prepared. The diastereomers of 2RS,4R-5 and 2RS,4S-5 were prepared by using the corresponding optically pure R and S toluene-4-sulfonic acid 2,3-dihydroxypropyl ester (R-4,S-4). The enatiomerically and diastereomerically pure acetonide (5) was obtained by a method involving diastereoselective crystallisation of the tosylate salt, followed by re-equilibration with the mother liquor and chromatography. The optical purity of four target compounds (YCZ-2013) was confirmed by chiral high-performance liquid chromatography (HPLC) and NMR. The effects of these stereoisomers on Arabidopsis stem elongation indicated that the cis isomers of 2S,4R-YCZ-2013 and 2R,4S-YCZ-2013 exhibited potent inhibitory activity with IC₅₀ values of approximately 24 ± 3 and 24 ± 2 nM, respectively. The IC₅₀ values of the trans isomers of 2S,4S-YCZ-2013 and 2R,4R-YCZ-2013 are approximately 1510 ± 50 and 3900 ± 332 nM, respectively. Co-application of brassinolide (10 nM), the most potent BR, and GA₃ (1 μM) to Arabidopsis seedlings grown in the dark with 2R,4S-YCZ-2013 and 2S,4R-YCZ-2013 revealed that brassinolide recovered the induced dwarfism of Arabidopsis seedlings, whereas GA₃ showed no effect.     

Ligand Perception,Activation, and Early Signaling of Plant Steroid Receptor Brassinosteroid Insensitive

Leucine-rich repeat receptor-like kinases （LRR-RLKs） belong to a large group of cell surface proteins involved in many aspects of plant development and environmental responses in both monocots and dicots. Brassinosteroid insensitive 1 （BRI1）, a member of the LRR X subfamily, was first identified through several forward genetic screenings for mutants insensitive to brassinosteroids （BRs）, which are a class of plant-specific steroid hormones. Since its identification, BRI1 and its homologs had been proved as receptors perceiving BRs and initiating BR signaling. The co-receptor BRIl-associated kinase 1 and its homologs, and other BRI1 interacting proteins such as its inhibitor BRI1 kinase inhibitor I （BKI1） were identified by genetic andbiochemical approaches. The detailed mechanisms of BR perception by BRI1 and the activation of BRI1 receptor complex have also been elucidated. Moreover, several mechanisms for termination of the activated BRI1 signaling were also discovered. In this review, we will focus on the recent advances on the mechanism of BRI1 phosphorylation and activation, the regulation of its receptor complex, the structure basis of BRI1 ectodomain and BR recognition, its direct substrates, and the termination of the activated BRI1 receptor complex.     

Isolation and characterization of brassinosteroids from algal cultures of Chlorella vulgaris Beijerinck (Trebouxiophyceae)

The brassinosteroids (BRs) occur ubiquitously in the plant kingdom. The occurrence of BRs has been demonstrated in almost every part of higher plants, such as pollen, flower buds, fruits, seeds, vascular cambium, leaves, shoots and roots. In this study, BRs were isolated and identified in the culture of wild-type Chlorella vulgaris. Seven BRs, including teasterone, typhasterol, 6-deoxoteasterone, 6-deoxotyphasterol, 6-deoxocastasterone, castasterone and brassinolide, were identified by GC–MS. All compounds belong to the BR biosynthetic pathway. The results suggest that early and late C6 oxidation pathways are operating in C. vulgaris. This study represents the first isolation of BRs from C. vulgaris cultures.     

甘蓝型油菜的BR响应及BnBZL2基因的功能分析

对甘蓝型油菜(Brassica napus L.)与拟南芥(Arabidopsis thaliana L.)中保守的油菜素甾醇(Brassinosteroids,BR)信号相关基因进行对比分析,并以甘蓝型油菜品种‘沪油15’为材料,对BR信号通路相关同源基因进行了组织表达分析。结果显示,BR合成基因与信号组分在花和幼嫩种子中表达量更高;低浓度BR处理可以促进幼苗根的生长,高浓度BR处理则起抑制作用; BR合成抑制剂(Brassinozole,BRZ)处理可抑制黑暗条件下幼苗下胚轴的伸长; BR处理可以降低BR合成基因的表达水平,而BRZ处理则相反,表明甘蓝型油菜中BR信号增加能反馈抑制BR的合成。烟草(Nicotiana tabacum L.)瞬时表达实验结果发现,与拟南芥BZR1基因同源的甘蓝型油菜BnBZL2编码蛋白定位在细胞质和细胞核中,BR处理可增加BnBZL2的核定位。蛋白质免疫印迹检测结果显示,BR处理可增加去磷酸化BnBZL2的比例。本研究进一步模拟了拟南芥bzr1-1D功能获得性突变体对BnBZL2蛋白进行点突变(BnBZL2*),并构建载体转化拟南芥,黑暗条件下转基因植株幼苗对BRZ处理不敏感,提示BnBZL2*可提高转基因植株的BR信号水平。本研究结果表明甘蓝型油菜中存在与拟南芥相似且保守的BR信号通路和调控机制。     

Brassinosteroid signaling and application in rice

Combined approaches with genetics, biochemistry, and proteomics studies have greatly advanced our understanding of brassinosteroid (BR) signaling in Arabidopsis. However, in rice, a model plant of monocot and as well an important crop plant, BR signaling is not as well characterized as in Arabidopsis. Recent studies by forward and reverse genetics have identified a number of either conserved or specific components of rice BR signaling pathway, bringing new ideas into BR signaling regulation mechanisms. Genetic manipulation of BR level or BR sensitivity to improve rice yield has established the great significance of BR research achievements.     

Brassinolide influences the regeneration of adventitious shoots from cultured leaf discs of tobacco

When several concentrations of brassinolide (BL) were added to a shoot induction medium (SIM) that contained only BA, redifferentiation of adventitious shoots from tobacco leaf discs was unaffected at low BL levels (10^-10~10^-8 M), but was inhibited at higher concentrations. In comparison, when BL was applied without BA, only cell expansion occurred and no shoots formed. The determination time for shoot formation was shortened at low BL concentrations, but their formation was postponed (i.e., time was lengthened) at higher concentrations. Elongation of shoots incubated for 30 d was unaffected at low BL concentrations, but was inhibited as that amount increased.NTH1, a tobacco homeobox gene that is expressed in the central zone of the tobacco shoot apex, showed greater expression levels in the SIM over time, and its expression was stronger in media treated with low concentrations of BL compared with the SIM control at the same time point. Expression ofNTH1 was postponed at higher BL concentrations. In conclusion, at low concentrations, brassinolide has no effect on shoot formation. However, it inhibits their formation at high concentrations when cytokinin is included in the media.     

Brassinosteroid-Mediated Stress Responses

Brassinosteroids (BRs) are a group of naturally occurring plant steroidal compounds with wide-ranging biological activity that offer the unique possibility of increasing crop yields through both changing plant metabolism and protecting plants from environmental stresses. In recent years, genetic and biochemical studies have established an essential role for BRs in plant development, and on this basis BRs have been given the stature of a phytohormone. A remarkable feature of BRs is their potential to increase resistance in plants to a wide spectrum of stresses, such as low and high temperatures, drought, high salt, and pathogen attack. Despite this, only a few studies aimed at understanding the mechanism by which BRs promote stress resistance have been undertaken. Studies of the BR signaling pathway and BR gene-regulating properties indicate that there is cross-talk between BRs and other hormones, including those with established roles in plant defense responses such as abscisic acid, jasmonic acid, and ethylene. Recent studies aimed at understanding how BRs modulate stress responses suggest that complex molecular changes underlie BR-induced stress tolerance in plants. Analyses of these changes should generate exciting results in the future and clarify whether the ability of BRs to increase plant resistance to a range of stresses lies in the complex interactions of BRs with other hormones. Future studies should also elucidate if BRI1, an essential component of the BR receptor, directly participates in stress response signaling through interactions with ligands and proteins involved in plant defense responses.     

Brassinosteroids, microtubules and cell elongation in Arabidopsis thaliana. I. Molecular, cellular and physiological characterization of the Arabidopsis bul1 mutant, defective in the Δ7-sterol-C5-desaturation step leading to brassinosteroid biosynthesis

Although cell elongation is a basic function of plant morphogenesis, many of the molecular events involved in this process are still unknown. In this work an extremely dwarf mutant, originally named bul, was used to study one of the main processes of plant development, cell elongation. Genetic analyses revealed that the BUL locus was linked to the nga172 marker on chromosome 3. Recently, after mapping the new dwf7 mutation of Arabidopsis, which is allelic to ste1, it was reported that dwf7 is also linked to the same marker. Sterol analyses of the bul1-1 mutant indicated that bul1-1 is defective in the Δ⁷-sterol-C5-desaturation step leading to brassinosteroid biosynthesis. Considering these findings, we designated our bul mutant as bul1-1/dwf7-3/ste1-4. The bul1-1 mutant was characterized by a very dwarf phenotype, with delayed development and reduced fertility. The mutant leaves had a dark-green colour, which was probably due to continuous stomatal closure. The bul1-1 mutant showed a partially de-etiolated phenotype in the dark. Cellular characterization and rescue experiments with brassinosteroids demonstrated the involvement of the BUL1-1 protein in brassinosteroid-dependent plant growth processes. Received: 28 April 2000 / Accepted: 6 October 2000     

A proteomics study of the mung bean epicotyl regulated by brassinosteroids under conditions of chilling stress

Mung bean CYP90A2 is a putative brassinosteroid (BR) synthetic gene that shares 77% identity with the Arabidopsis CPD gene. It was strongly suppressed by chilling stress. This implies that exogenous treatment with BR could allow the plant to recover from the inhibited growth caused by chilling. In this study, we used proteomics to investigate whether the mung bean epicotyl can be regulated by brassinosteroids under conditions of chilling stress. Mung bean epicotyls whose growth was initially suppressed by chilling partly recovered their ability to elongate after treatment with 24-epibrassinolde; 17 proteins down-regulated by this chilling were re-up-regulated. These up-regulated proteins are involved in methionine assimilation, ATP synthesis, cell wall construction and the stress response. This is consistent with the re-up-regulation of methionine synthase and S-adenosyl-L-methionine synthetase, since chilling-inhibited mung bean epicotyl elongation could be partially recovered by exogenous treatment with DL-methionine. This is the first proteome established for the mung bean species. The regulatory relationship between brassinosteroids and chilling conditions was investigated, and possible mechanisms are discussed herein.