Effects of structural analogs of brassinosteroids on the recovery of growth inhibition by a specific brassinosteriod biosynthesis inhibitor

A brassinosteroid inhibitor (Brz2001) was used to block the growth of roots, hypocotyls, and epicotyls of soybean seedlings, producing a dwarf phenotype. The application of 24-epibrassinolide completely reversed the inhibitory effects of Brz2001. Two other growth-promoting brassinosteroid analogs, MH5 and BB6, partially overcame the Brz2001-induced growth defects. The growth inhibition of Brz2001-treated seedlings was more effectively reversed by MH5 than by BB6, which may be due to the structural differences between the two compounds. These results indicate that the studied analogs may show brassinosteroid-like activity and therefore may have some practical use instead of brassinolide or its analogues.     

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.     

Characterization of brassinazole, a triazole-type brassinosteroid biosynthesis inhibitor

Screening for brassinosteroid (BR) biosynthesis inhibitors was performed to find chemicals that induce dwarfism in Arabidopsis, mutants that resembled BR biosynthesis mutants that can be rescued by BR. Through this screening experiment, the compound brassinazole was selected as the most potent chemical. In dark-grown Arabidopsis, brassinazole-induced morphological changes were nearly restored to those of wild type by treatment with brassinolide. The structure of brassinazole is similar to pacrobutrazol, a gibberellin biosynthesis inhibitor. However, in assays with cress (Lepidium sativum) plants, brassinazole-treated plants did not show recovery after the addition of gibberellin but showed good recovery after the addition of brassinolide. These data demonstrate that brassinazole is a specific BR biosynthesis inhibitor. Brassinazole-treated cress also showed dwarfism, with altered leaf morphology, including the downward curling and dark green color typical of Arabidopsis BR-deficient mutants, and this dwarfism was reversed by the application of 10 nM brassinolide. This result suggests that BRs are essential for plant growth, and that brassinazole can be used to clarify the function of BRs in plants as a complement to BR-deficient mutants. The brassinazole action site was also investigated by feeding BR biosynthesis intermediates to cress grown in the light.     

Molecular cloning and characterization of a novel brassinolide enhanced gene OsBLE1 in Oryza sativa seedlings.

Brassinosteroids (BRs) are plant steroids essential for normal growth and development. To gain insight into the molecular mechanism by which BRs regulate the growth and development of plants, it is necessary to identify and analyze more genes that are regulated by BRs. A novel brassinolide (BL)-enhanced gene designated OsBLE1, which was originally identified by using rice (Oryza sativa L.) cDNA microarray, was cloned and characterized. Its cDNA is 598 bp long, encoding a predicted polypeptide with 81 amino acid residues. Northern blots analysis revealed that OsBLE1 expression began to increase at 6 h and reached its maximum at 12 h after BL treatment. OsBLE1 expression was most responsive to BL in lamina joint in rice seedlings; besides, IAA and GA3 also enhanced its expression. OsBLE1 expressed mainly in active tissues such as vascular bundles and root primordial. Transgenic rice expressing antisense OsBLE1 exhibits various degrees of repressed growth. Results suggest that OsBLE1 might be involved in BL-regulated growth processes in rice seedlings.     

Physiological Roles of Brassinosteroids in Early Growth of <Emphasis Type="Italic">Arabidopsis:</Emphasis> Brassinosteroids Have a Synergistic Relationship with Gibberellin as well as Auxin in Light-Grown Hypocotyl Elongation

We examined the physiological effects of brassinosteroids (BRs) on early growth of Arabidopsis. Brassinazole (Brz), a BR biosynthesis inhibitor, was used to elucidate the significance of endogenous BRs. It inhibited growth of roots, hypocotyls, and cotyledonous leaf blades dose-dependently and independent of light conditions. This fact suggests that endogenous BRs are necessary for normal growth of individual organs of Arabidopsis in both photomorphogenetic and skotomorphogenetic programs. Exogenous brassinolide (BL) promoted hypocotyl elongation remarkably in light-grown seedlings. Cytological observation disclosed that BL-induced hypocotyl elongation was achieved through cell enlargement rather than cell division. Furthermore, a serial experiment with hormone inhibitors showed that BL induced hypocotyl elongation not through gibberellin and auxin actions. However, a synergistic relationship of BL with gibberellin A₃ (GA₃) and indole-3-acetic acid (IAA) was observed on elongation growth in light-grown hypocotyls, even though gibberellins have been reported to be additive to BR action in other plants. Taken together, our results show that BRs play an important role in the juvenile growth of Arabidopsis; moreover, BRs act on light-grown hypocotyl elongation independent of, but cooperatively with, gibberellins and auxin.     

AXR1 is involved in BR-mediated elongation and SAUR-AC1 gene expression in Arabidopsis

Limited information is available concerning the interactions between the brassinosteroid (BR) and auxin signaling pathways. The expression pattern of the SAUR-AC1 gene, an early auxin-inducible gene in Arabidopsis, was studied in response to brassinolide (BL), in the presence of a BR-biosynthesis inhibitor, in a BR-deficient mutant, and in combination with auxin. The results suggested that the SAUR-AC1 gene is regulated by BRs independently of auxin levels, and that it is important in BR-mediated elongation. The axr1 (auxin insensitive 1) mutant was less sensitive to BL-induced elongation and BL-induced SAUR-AC1 expression, suggesting that a ubiquitin ligase-mediated system is involved in BR-mediated elongation.     

Influence of the<Emphasis Type="Italic">SMT2</Emphasis> knock-out on hypocotyl elongation in<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Inhibitors are very important in the study of hormone function. Brasinazole (Brz) is a specific inhibitor of brassinosteroids (BRs) biosynthesis. To expand our knowledge of the molecular mechanisms of plant steroid signaling, we performed genetic screening using medium containing Brz under dark conditions. Mutants insensitive to Brz developlonger hypocotyls than their wild type counterparts. We isolatedabz453 as a Brz insensitive mutant. TAIL-PCR and the segregation ratio of T2 plants indicated a single T-DNA insertion at the 24-Sterol C-methyltransferase (SMT2) gene in theabz453 mutant. Recapitulation for putative FCP serine phosphatase (FSP), the gene neighboringSMT2, indicated no significant phenotypes, but theSMT2 anti-sense (SMT2-AS) line developed longer hypocotyls than the wild type in medium containing Brz. Additionally, theSMT2-AS line displayed similar phenotypes to theabz453 line in soil including enhanced growth and smaller silique. Theabz453 andSMT2-AS mutants showed phenotypes similar to those of wild type in medium containing benzylaminopurine, pacrobutrazol and ACC (precursor for ethylene) under dark conditions. However, when brassinolide (BL) dose response was observed, theabz453 andSMT2-AS lines showed higher sensitivity than wild type. Theabz453/det2 andabz453/bri1-119 double mutants showed enhanced growth compared to thedet2 andbri1-119 line under both dark and light conditions. Specially, in dark conditions double mutants displayed nearly 2- and 1.5-fold longer hypocotyls thandet2 andbri1-119 plants. Brz insensitivity to theSMT2 knock-out mutant and phenotypes of double mutants indicate that not only do BRI1 and DET2 influence the BRs response, as evidenced by hypocotyl elongation, but another sterol derived signals may also be affected in mutants, suggesting that another pathway is involved in hypocotyl elongation due to SMT2.     

Suppression of Wolffia arrhiza growth by brassinazole, an inhibitor of brassinosteroid biosynthesis and its restoration by endogenous 24-epibrassinolide

The effect of the brassinosteroid (BR) 24-epibrassinolide (epiBL; 10(-13)-10(-6)M) on growth and levels of chlorophylls, carotenoids, sugars and protein in Wolffia arrhiza after 7 days of cultivation is reported. Application of epiBL to W. arrhiza cultures stimulates the growth and increases the content of photosynthetic pigments, sugar and protein. The greatest effect of epiBL is observed at a concentration of 10(-9)M. We tested the action of Brz2001, a specific BR biosynthesis inhibitor, in the range of 10(-6)-10(-4)M. Addition of Brz2001 to W. arrhiza cultures inhibits their growth after 7 days of cultivation. The inhibition of growth could be reversed by the addition of epiBL. Moreover, there was not complete recovery to the level of control, especially at 5 x 10(-5)-10(-4)M Brz2001. The effects of treatment with 10(-9)M epiBL mixed with a mevalonate pathway inhibitor (mevinolin), or a 2-methylerythritol 4-phosphate pathway inhibitor (clomazone), were also investigated. Mevinolin did not inhibit growth of W. arrhiza after 7 days of cultivation. However, clomazone did. Addition of epiBL overcame this inhibition. These results suggest that the mevalonate pathway may not function well in W. arrhiza and that biosynthesis of BRs through the non-mevalonate pathway in W. arrhiza could be possible.     

The brassinosteroid growth response in pea is not mediated by changes in gibberellin content

The objective of this study was to increase our understanding of the relationship between brassinosteroids (BRs) and gibberellins (GAs) by examining the effects of BR deficiency on the GA biosynthesis pathway in several tissue types of pea (Pisum sativum L.). It was suggested recently that, in Arabidopsis, BRs act as positive regulators of GA 20-oxidation, a key step in GA biosynthesis [Bouquin et al. (2001) Plant Physiol 127:450–458]. However, this may not be the case in pea as GA₂₀ levels were consistently higher in all shoot tissues of BR-deficient (lk and lkb) and BR-response (lka) mutants. The application of brassinolide (BL) to lkb plants reduced GA₂₀ levels, and metabolism studies revealed a reduced conversion of GA₁₉ to GA₂₀ in epi-BL-treated lkb plants. These results indicate that BRs actually negatively regulate GA₂₀ levels in pea. Although GA₂₀ levels are affected by BR levels, this does not result in consistent changes in the level of the bioactive GA, GA₁. Therefore, even though a clear interaction exists between endogenous BR levels and the level of GA₂₀, this interaction may not be biologically significant. In addition to the effect of BRs on GA levels, the effect of altered GA₁ levels on endogenous BR levels was examined. There was no significant difference in BR levels between the GA mutants and the wild type (wt), indicating that altered GA₁ levels have no effect on BR levels in pea. It appears that the BR growth response is not mediated by changes in bioactive GA levels, thus providing further evidence that BRs are important regulators of stem elongation.     

Effects of brassinazole,an inhibitor of brassinosteroid biosynthesis,on light- and dark-grown<Emphasis Type="Italic"> Chlorella vulgaris</Emphasis>

Treatment of cultured Chlorella vulgaris Beijerinck cells with 0.1–10 M brassinazole (Brz2001), an inhibitor of brassinosteroid (BR) biosynthesis, inhibits their growth during the first 48 h of cultivation in the light. This inhibition is prevented by the co-application of BR. This result suggests that the presence of endogenous BRs during the initial steps of the C. vulgaris cell cycle is indispensable for their normal growth in the light. In darkness, a treatment with 10 nM brassinolide (BL) promotes growth through the first 24 h of culture, but during the following 24 h the cells undergo complete stagnation. Treatment of dark-grown cells with either Brz2001 alone, or a mixture of 10 nM BL and 0.1/10 M Brz2001, also stimulates their growth. The effects of treatment with 10 nM BL mixed with 0.1–10 M of a mevalonate-pathway inhibitor (mevinolin), or a non-mevalonate-pathway inhibitor (clomazone), were also investigated. Mevinolin at these concentrations did not inhibit growth of C. vulgaris; however, clomazone did. Addition of BL overcame the inhibition. These results suggest that the mevalonate pathway does not function in C. vulgaris, and that the non-mevalonate pathway for isopentenyl diphosphate biosynthesis is responsible for the synthesis of one of the primary precursors in BR biosynthesis.Abbreviations Brz Brassinazole - BL Brassinolide - BR Brassinosteroid - Clo Clomazone - DMAPP Dimethylallyl diphosphate - IPP Isopentenyl diphosphate - MVA Mevalonic acid - Mev Mevinoline     

Physiology and molecular mode of action of brassinosteroids

Brassinosteroids (BRs) comprise a group of polyhydroxysteroids, which show close structural similarity to steroid hormones from arthropods and mammals. BRs are now accepted as a new class of phytohormones due to their ubiquitous occurrence in plants, their highly effective elicitation of various responses and the identification of mutants defective in BR-biosynthesis or -response. Important steps of BR-biosynthesis were elucidated with precursor-feeding experiments and by the analysis of BR-biosynthesis-deficient mutants. The altered phenotypes of these mutants, particularly in Arabidopsis, revealed the essential nature of BRs for normal growth and development. A major role of BRs is the positive regulation of cell expansion. Furthermore, BRs modulate plant responses to biotic and abiotic stresses and to other phytohormones, and influence differentiation processes of cells and tissues. BR-insensitive mutants such as bri1 hold the potential for uncovering BR-signalling pathway(s) at the molecular level. The identification of BR-regulated genes demonstrates a genetic basis for BR mode of action with reference to their multiple effects. This review focuses on the relevance of BRs to the control of various physiological processes, BR-signalling and underlying molecular mechanisms by considering known mutants.     

Loss-of-function of a rice brassinosteroid biosynthetic enzyme,C-6 oxidase,prevents the organized arrangement and polar elongation of cells in the leaves and stem

Molecular genetic and physiological studies on brassinosteroid (BR)-related mutants of dicot plants have revealed that BRs play important roles in normal plant growth and development. However, little is known about the function of BR in monocots (grasses), except for the phenotypic analysis of a rice mutant partially insensitive to BR signaling. To investigate the function of BR in monocots, we identified and characterized BR-deficient mutants of rice, BR-deficient dwarf1 (brd1). The brd1 mutants showed a range of abnormalities in organ development and growth, the most striking of which were defects in the elongation of the stem and leaves. Light microscopic observations revealed that this abnormality was primarily owing to a failure in the organization and polar elongation of the leaf and stem cells. The accumulation profile of BR compounds in the brd1 mutants suggested that these plants may be deficient in the activity of BR C-6 oxidase. Therefore, we cloned a rice gene, OsDWARF, which has a high sequence similarity to the tomato C-6 oxidase gene, DWARF. Introduction of the wild-type OsDWARF gene into brd1 rescued the abnormal phenotype of the mutants. The OsDWARF gene was expressed at a low level in all of the examined tissues, with preferential expression in the leaf sheath, and the expression was negatively regulated by brassinolide treatment. On the basis of these findings, we discuss the biological function of BRs in rice plants.     

Brassinosteroid-insensitive dwarf mutants of Arabidopsis accumulate brassinosteroids

Seven dwarf mutants resembling brassinosteroid (BR)-biosynthetic dwarfs were isolated that did not respond significantly to the application of exogenous BRs. Genetic and molecular analyses revealed that these were novel alleles of BRI1 (Brassinosteroid-Insensitive 1), which encodes a receptor kinase that may act as a receptor for BRs or be involved in downstream signaling. The results of morphological and molecular analyses indicated that these represent a range of alleles from weak to null. The endogenous BRs were examined from 5-week-old plants of a null allele (bri1-4) and two weak alleles (bri1-5 and bri1-6). Previous analysis of endogenous BRs in several BR-biosynthetic dwarf mutants revealed that active BRs are deficient in these mutants. However, bri1-4 plants accumulated very high levels of brassinolide, castasterone, and typhasterol (57-, 128-, and 33-fold higher, respectively, than those of wild-type plants). Weaker alleles (bri1-5 and bri1-6) also accumulated considerable levels of brassinolide, castasterone, and typhasterol, but less than the null allele (bri1-4). The levels of 6-deoxoBRs in bri1 mutants were comparable to that of wild type. The accumulation of biologically active BRs may result from the inability to utilize these active BRs, the inability to regulate BR biosynthesis in bri1 mutants, or both. Therefore, BRI1 is required for the homeostasis of endogenous BR levels.     

Brassinosteroids promote photosynthesis and growth by enhancing activation of Rubisco and expression of photosynthetic genes in <Emphasis Type="Italic">Cucumis sativus</Emphasis>

Brassinosteroids (BRs) are a new group of plant growth substances that promote plant growth and productivity. We showed in this study that improved growth of cucumber (Cucumis sativus) plants after treatment with 24-epibrassinolide (EBR), an active BR, was associated with increased CO₂ assimilation and quantum yield of PSII (Φ_PSII). Treatment of brassinazole (Brz), a specific inhibitor for BR biosynthesis, reduced plant growth and at the same time decreased CO₂ assimilation and Φ_PSII. Thus, the growth-promoting activity of BRs can be, at least partly, attributed to enhanced plant photosynthesis. To understand how BRs enhance photosynthesis, we have analyzed the effects of EBR and Brz on a number of photosynthetic parameters and their affecting factors, including the contents and activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Northern and Western blotting demonstrated that EBR upregulated, while Brz downregulated, the expressions of rbcL, rbcS and other photosynthetic genes. In addition, EBR had a positive effect on the activation of Rubisco based on increased maximum Rubisco carboxylation rates (V _c,max), total Rubisco activity and, to a greater extent, initial Rubisco activity. The accumulation patterns of Rubisco activase (RCA) based on immunogold-labeling experiments suggested a role of RCA in BR-regulated activation state of Rubisco. Enhanced expression of genes encoding other Calvin cycle genes after EBR treatment may also play a positive role in RuBP regeneration (J _max), thereby increasing maximum carboxylation rate of Rubisco (V _c,max). Thus, BRs promote photosynthesis and growth by positively regulating synthesis and activation of a variety of photosynthetic enzymes including Rubisco in cucumber.     

Brassinazole, an inhibitor of brassinosteroid biosynthesis, inhibits development of secondary xylem in cress plants (Lepidium sativum)

Brassinazole (Brz) is a specific brassinosteroid biosynthesis inhibitor. Cress plants (Lepidium sativum) grown in medium containing Brz exhibited a slight predominance of phloem differentiation at the expense of xylem differentiation and remarkable inhibition of the development of secondary xylem. This result indicates that brassinosteroids function in xylem development in vivo.     

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.     

Brassinosteroid action in flowering plants: a Darwinian perspective

The year 2012 marks the 150th anniversary of the publication of Charles Darwin's first botanical book, on the fertilization of orchids (1862), wherein he described pollen grains and outlined his evolutionary principles with respect to plant research. Five decades later, the growth-promoting effect of extracts of Orchid pollen on coleoptile elongation was documented. These studies led to the discovery of a new class of phytohormones, the brassinosteroids (BRs) that were isolated from rapeseed (Brassica napus) pollen. These growth-promoting steroids, which regulate height, fertility, and seed-filling in crop plants such as rice (Oryza sativa), also induce stress- and disease resistance in green algae and angiosperms. The origin and current status of BR-research is described here, with reference to BR-action and -signal transduction, and it is shown that modern high-yield rice varieties with erect leaves are deficient in endogenous BRs. Since brassinosteroids induce pathogen resistance in rice plants and hence can suppress rice blast- and bacterial blight-diseases, genetic manipulation of BR-biosynthesis or -perception may be a means to increase crop production. Basic research on BR activity in plants, such as Arabidopsis and rice, has the potential to increase crop yields further as part of a 21th century 'green biotech-revolution' that can be traced back to Darwin's classical breeding experiments. It is concluded that 'Nothing in brassinosteroid research makes sense except in the light of Darwinian evolution' and the value of basic science is highlighted, with reference to the genetic engineering of better food crops that may become resistant to a variety of plant diseases.