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.     

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     

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.     

Inhibition of brassinosteroid biosynthesis by either a dwarf4 mutation or a brassinosteroid biosynthesis inhibitor rescues defects in tropic responses of hypocotyls in the arabidopsis mutant nonphototropic hypocotyl 4

The nonphototropic hypocotyl 4 (nph4)/auxin response factor 7 (arf7) mutant of Arabidopsis (Arabidopsis thaliana) is insensitive to auxin and has defects in hypocotyl tropism, hook formation, differential leaf growth, and lateral root formation. To understand an auxin-signaling pathway through NPH4, we carried out screening of suppressor mutants of nph4-103 and obtained a dwarf suppressor mutant, suppressor of nph4 (snp2). snp2 had short hypocotyls in the dark condition and dark green and round leaves, short petioles, and more lateral shoots than the wild type in the light condition. The snp2 phenotypes were rescued by adding brassinolide to the growth medium in both light and dark conditions. Genetic mapping, sequence analysis, and a complementation test indicated that snp2 was a weak allele of DWARF4 (DWF4), which functions in brassinosteroid (BR) biosynthesis. snp2, which was renamed dwf4-101, exhibited photo- and gravitropisms of hypocotyls similar to those of the wild type with a slightly faster response in gravitropism. dwf4-101 almost completely suppressed defects in both tropisms of nph4-103 hypocotyls and completely suppressed hyponastic growth of nph4-103 leaves. Treatment with brassinazole, an inhibitor of BR biosynthesis, also partially rescued the tropic defects in nph4-103. Hypocotyls of nph4-103 were auxin insensitive, whereas hypocotyls of dwf4-101 were more sensitive than those of the wild type. dwf4-101 nph4-103 hypocotyls were as sensitive as those of dwf4-101. Auxin inducibility of massugu 2 (MSG2)/IAA19 gene expression was reduced in nph4-103. mRNA level of MSG2 was reduced in dwf4-101 and dwf4-101 nph4-103, but both mutants exhibited greater auxin inducibility of MSG2 than the wild type. Taken together, dwf4-101 was epistatic to nph4-103. These results strongly suggest that BR deficiency suppresses nph4-103 defects in tropic responses of hypocotyls and differential growth of leaves and that BR negatively regulates tropic responses.     

Characterization of gravitropic inflorescence bending in brassinosteroid biosynthesis and signaling Arabidopsis mutants

The interaction between the plant hormones, brassinosteroids and auxins has been documented in various processes using a variety of plants and plant parts. In this study, detached inflorescences from brassinosteroid biosynthesis and signaling Arabidopsis mutants were evaluated for their gravitropic bending in response to epibrassinolide (EBR) and indole-3-acetic acid (IAA). EBR supplied to the base of detached inflorescences stimulated gravitropic bending in all BR biosynthetic mutants but there was no effect on the BR signaling mutant or wild type plants. When IAA was supplied to the base of BR mutant inflorescences both natural and EBR-induced gravitropic bending was inhibited. Treatment with the auxin inhibitors also decreased both natural and EBR-induced gravitropic bending. No gravitropic bending was observed when the apical tips of BR mutant inflorescences were removed. IAA treatment to the tips of decapitated BR mutant inflorescences restored gravitropic bending to values observed in the inflorescences with an apical tip, however, EBR applied to the tip had no effect. When decapitated inflorescences from BR mutants were treated with IAA to the base and either gel, EBR or IAA was applied to the tip; there was no gravitropic bending. These results show that brassinosteroids have a role in the gravitropic bending response in Arabidopsis and mutants serve to uncover this hidden contributor.     

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.     

Treatment of dark-grown Arabidopsis thaliana with a brassinosteroid-biosynthesis inhibitor, brassinazole, induces some characteristics of light-grown plants

When a brassinosteroid biosynthesis inhibitor, brassinazole (Brz), was applied at concentrations ranging from 0.1 to 2 μM, Arabidopsis thaliana (L.) Heynh seedlings grown in the dark exhibited morphological features of light-grown plants, i.e. short hypocotyls, expanded cotyledons, and true leaves, in a dose-dependent manner. Control (non Brz-treated) seedlings grown in the dark for 40 d did not develop leaf primordia. However, treatment with the lowest concentration of Brz induced the development of leaf buds, although it hardly induced any short hypocotyls, and treatment with the highest concentration of Brz induced both short hypocotyls and leaves. Labeling experiments with the thymidine analogue 5-bromo-2′-deoxyuridine revealed that amplification of cell nuclei and organellar nucleoids is activated in the shoot apical meristems of dark-grown Brz-treated seedlings. These results suggest that Brz-treatment induces development of true leaves. Furthermore, condensation and scattering of plastid nucleoids, which is known to occur during the differentiation of etioplasts into chloroplasts, was observed in the plastids of dark-grown Brz-treated cotyledons. In addition, high levels of ribulose-1,5-bisphosphate carboxylase-oxygenase proteins accumulated in the plastids of the cotyledons. Electron microscopy showed that the plastids were etioplasts with a prolamellar body and few thylakoid membranes. These results suggest that Brz treatment in the dark induces the initial steps of plastid differentiation, which occur prior to the development of thylakoid membranes. This is a novel presumed function of brassinosteroids. These cytological changes seen in Brz-treated Arabidopsis were exactly the same as those seen in a brassinosteroid-biosynthesis-deficient mutant, det2, supporting the hypothesis that Brz has no side-effects except inhibiting brassinosteroid biosynthesis, and should prove a useful tool in clarifying the role of brassinosteroids. Received: 10 February 2000 / Accepted: 11 April 2000     

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.     

A specific brassinosteroid biosynthesis inhibitor, Brz2001: evaluation of its effects on Arabidopsis, cress, tobacco, and rice

Brassinazole is the only known specific brassinosteroid (BR)-biosynthesis inhibitor, and it has been shown to be useful for elucidating the function of BRs. In the course of a structure-activity relationship study of brassinazole, we found a more specific BR-biosynthesis inhibitor, Brz2001. This new inhibitor induced similar morphological changes to those seen in brassinazole-treated plants, including Arabidopsis thaliana (L.) Heynh., Nicotiana tabacum L., and Lepidium sativum L. These changes included dwarfism with altered leaf morphology, including downward curling and dark-green color, and the changes were reversed by brassinolide. Although the structure of Brz2001 is similar to that of uniconazole, a gibberellin-biosynthesis inhibitor, Brz2001-treated plants showed almost no recovery with the addition of gibberellic acid (GA3). Comparison of the responses of both brassinazole- and Brz2001-treated cress to brassinolide and GA3 suggested that Brz2001 is a more specific BR-biosynthesis inhibitor than brassinazole. Unlike the results just described, Brz2001-treated rice did not show any morphological changes. This suggests that the roles of BRs in rice may be different from those in the dicotyledonous plants examined in this study. Brz2001 can be used to clarify the function of BRs in dicots as a complement to BR-deficient mutants, and to elucidate the different roles of BRs in monocots and dicots.     

New lead compounds for brassinosteroid biosynthesis inhibitors

The first brassinosteroid biosynthesis inhibitor is reported. Among newly synthesized triazole derivatives, 4-(4-chlorophenyl)-2-phenyl-3-(1,2,4-triazoyl)butan-2-ol (6) was found to inhibit the growth of cress seedlings, and this inhibition was recovered by the treatment of brassinolide, suggesting that compound 6 primarily inhibits brassinosteroid biosynthesis.     

Isolation and characterization of a rice dwarf mutant with a defect in brassinosteroid biosynthesis

We have isolated a new recessive dwarf mutant of rice (Oryza sativa L. cv Nipponbare). Under normal growth conditions, the mutant has very short leaf sheaths; has short, curled, and frizzled leaf blades; has few tillers; and is sterile. Longitudinal sections of the leaf sheaths revealed that the cell length along the longitudinal axis is reduced, which explains the short leaf sheaths. Transverse sections of the leaf blades revealed enlargement of the motor cells along the dorsal-ventral axis, which explains the curled and frizzled leaf blades. In addition, the number of crown roots was smaller and the growth of branch roots was weaker than those in the wild-type plant. Because exogenously supplied brassinolide considerably restored the normal phenotypes, we designated the mutant brassinosteroid-dependent 1 (brd1). Further, under darkness, brd1 showed constitutive photomorphogenesis. Quantitative analyses of endogenous sterols and brassinosteroids (BRs) indicated that BR-6-oxidase, a BR biosynthesis enzyme, would be defective. In fact, a 0.2-kb deletion was detected in the genomic region of OsBR6ox (a rice BR-6-oxidase gene) in the brd1 mutant. These results indicate that BRs are involved in many morphological and physiological processes in rice, including the elongation and unrolling of leaves, development of tillers, skotomorphogenesis, root differentiation, and reproductive growth, and that the defect of BR-6-oxidase caused the brd1 phenotype.     

Translocation of paclobutrazol, a gibberellin biosynthesis inhibitor, in apple seedlings

The [(2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1- yl)-pentan-3-ol] (paclobutrazol, PP333) measured in apple seedlings (`York Imperial' Malus domestica Borkh) was confirmed by gas chromatography-mass spectrometry. Data showed that paclobutrazol was taken up through roots and transported primarily in the xylem through the stems and accumulated in leaves. No detectable basipetal movement of paclobutrazol in apple seedlings was found.     

Brz220 interacts with DWF4, a cytochrome P450 monooxygenase in brassinosteroid biosynthesis, and exerts biological activity

Arabidopsis thaliana (Arabidopsis) treated with the four stereoisomers of Brz220 (2RS, 4RS-1-[4-propyl-2-(4-trifluoromethylphenyl)-1, 3-dioxane-2-ylmethyl]-1H-1, 2, 4-triazole) showed a dwarf phenotype like brassinosteroid (BR) biosynthesis mutants that were rescued by treatment of BRs. The target sites of each Brz220 stereoisomer were investigated by treatment of Arabidopsis with BRs in the dark. The results suggest that the stereoisomers block the 22-hydroxylation step in BR biosynthesis. This step is catalyzed by DWF4, an Arabidopsis cytochrome P450 identified as a steroid 22-hydroxylase. The enzyme was expressed in E. coli, and the binding affinity of the stereoisomers to recombinant DWF4 was analyzed. The results indicate that in these stereoisomers there exists a positive correlation between binding affinity to DWF4 and inhibition of Arabidopsis hypocotyl growth. In this context, we concluded that DWF4 is the target site of Brz220 in Arabidopsis.     

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.