Characterization of brassinazole, a triazole-type brassinosteroid biosynthesis inhibitor

Abstract

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.

Figure 1

Structure of brassinazole. In this study, brassinazole was used as a racemic mixture.

Figure 2

Effect of brassinazole (Brz) on Arabidopsis seedlings grown in the light. A, Brassinazole (5, 1, and 0.5 μm)-treated Arabidopsis (14-d-old) show dwarfism in a concentration-dependent manner. B, Brassinazole (1 μm)-treated Arabidopsis (14-d-old) show a BR-deficient mutant-like phenotype, which is rescued by the application of brassinolide (BL) (10 nm). CONT, Control.

Figure 3

Effect of brassinazole (Brz) on Arabidopsis seedlings grown in the dark. Brassinazole (1 μm)-treated Arabidopsis (7-d-old) shows BR-deficient mutant-like phenotype. A, Brassinazole-induced short hypocotyl and open cotyledon, which are rescued by brassinolide (BL) treatment (10 nm). B, Open cotyledon. C, Both uniconazole (U) (0.1 μm) and brassinazole (1 μm) induced short hypocotyl. GA₃ (1 μm) treatment rescued uniconazole-induced short hypocotyl, but not the brassinazole-induced one. CONT, Control.

Figure 4

Light microscopy of brassinazole-treated and non-treated Arabidopsis stems sectioned longitudinally and transversally. A, Cross-section of non-treated plant. B, Longitudinal section of non-treated plant. C, Cross-section of brassinazole-treated plant. Cell size is increased in different tissues. D, Longitudinal section of brassinazole-treated plant. Cell size is reduced drastically in many different tissues. Arabidopsis seedlings were grown for 10 d in the dark. Bar = 100 μm.

Figure 5

Accumulation of mRNAs for rbcS, cab, and psbA in light-grown and dark-grown brassinazole-treated and non-treated plants. Lane 1, Dark-grown non-treated plants; lane 2, dark-grown brassinazole-treated plants; lane 3, light-grown non-treated plants; lane 4, light- grown brassinazole-treated plants. Two micrograms of total RNA was loaded per lane. Bottom panel, Stained gel showing rRNAs.

Figure 6

Dark-grown 10-d-old seedlings of Arabidopsis treated with different concentrations of brassinazole (Brz) from 0 to 10 μm. Hypocotyl length decreased as the concentration of brassinazole increased. The change in length occurred between 0.1 and 0.5 μm. Data are the means ± se obtained from 20 seedlings. CONT, Control.

Figure 7

Retardation of cress seedling growth by brassinazole (Brz) or uniconazole (U) and rescue by brassinolide or GA₃. Brassinolide rescues plants treated with brassinazole but not uniconazole. GA₃ added to plants rescues those treated with uniconazole but not brassinazole. CONT, Control.

Figure 8

Brassinazole (Brz)-treated Arabidopsis hypocotyl elongation in the dark in response to applied cathasterone (CT), teasterone (TE), castasterone (CS), and brassinolide (BL). Data are the means ± se obtained from 30 seedlings. CONT, Control.