The organic occurrence of 22-hydroxylated steroids in cultured cells and in Arabidopsis seedlings was investigated. demonstrated that a few of these substances take place in plant life naturally. Many BRs with one hydroxyl group in the medial side string have already been found in plants. The first compound recognized was cathasterone, which was found in cultured cells of (Fujioka et al., 1995), and later 6-deoxoCT and 3-cells and Arabidopsis seedlings. We then examined [2H6]22-OHCR metabolism in wild-type (Columbia) and mutant Arabidopsis seedlings and in cultured cells. Furthermore, we examined the rescue effects of 22-hydroxylated steroids around the mutant. Here, we provide several lines of evidence for a new subpathway via early C-22 oxidation, and for a new blocked step in the mutant. RESULTS Identification of Novel BRs in Cultured Cells and Arabidopsis Seedlings Cultured cells (V208) in log phase were used to identify 22-hydroxylated steroids. The cells were extracted with methanol, subjected to solvent partitioning and several chromatographic steps, and finally purified by HPLC. Purified fractions were analyzed by GC-MS after conversion to a trimethylsilyl derivative. By direct comparison with authentic specimens, the LY3009104 price natural occurrence of many 22-hydroxylated steroids was definitely established (Table ?(TableI;I; Fig. ?Fig.1).1). These steroids were 6-deoxoCT, 3-cells. Although authentic 3-cells, based on a comparison with mass spectral data and the retention occasions of closely related compounds such as 6-deoxoCT, 3-cells and Arabidopsis seedlings. In wild-type Arabidopsis seedlings, 22-OHCR, 22-OH-3-one, 6-deoxoCT, 3-cells (data not shown). In the mutant, 22-OHCR, 22-OH-4-en-3-one, 28-Cells To investigate the metabolic relationship of these BRs, we examined the metabolism of [2H6]22-OHCR in cultured cells. Ten micrograms of [2H6]22-OHCR was fed to cultured cells, and the cells were incubated for 2 d. The culture was extracted with methanol, and the extract was purified by HPLC. The purified fractions were converted to trimethylsilyl derivatives and analyzed by GC-MS. The results are summarized in Physique ?Physique2.2. [2H6]22- OH-4-en-3-one (0.3 g), [2H6]22-OH-3-one (3.1 g), [2H6]6-deoxoCT (1.5 g), and [2H6]3-cells. Open in a separate window Physique 2 Metabolism of [2H6]22-OHCR in cultured cells (V208) and Arabidopsis seedlings (wild type and mutant). Five, 10, or 20 g of [2H6] 22-OHCR was fed to cultured cells or seedlings for 2 d. Each value shows the amounts (g) of unmetabolized substrate and metabolites detected. nd, Not detected (below detection limit). Metabolism of [2H6]6-deoxoCT in Cultured Cells The metabolism of [2H6]6-deoxoCT was investigated in cultured cells. Ten micrograms of [2H6]6-deoxoCT was fed to the cells, and they were incubated for 2 d. The culture was extracted with methanol, and the extract was purified by HPLC. The purified fractions were converted to trimethylsilyl derivatives and analyzed by GC-MS. [2H6]3-mutant Arabidopsis seedlings. Ten micrograms of [2H6]22-OHCR was fed to seedlings produced in one-half-strength Murashige-Skoog medium, and they were incubated for 2 d. The culture was extracted with methanol, and the extract was purified by HPLC. The purified fractions were converted to trimethylsilyl derivatives and analyzed by GC-MS. In wild-type seedlings, [2H6]22-OH-4-en-3-one (0.7 g, 7%), [2H6]22-OH-3-one (0.5 g, 5%), [2H6]6-deoxoCT (0.8 g, 8%), and [2H6]3-seedlings (Fig. ?(Fig.2).2). Neither [2H6]22-OH-3-one nor [2H6]3-seedlings, comparable results were obtained (Fig. ?(Fig.2).2). These results strongly suggest HNRNPA1L2 that the biosynthetic sequence: 22-OHCR 22-OH-4-en-3-one 22-OH-3-one 6-deoxoCT also operates in Arabidopsis seedlings. In addition, these metabolic studies indicate that this mutant is defective in the conversion of 22-OH-4-en-3-one to 22-OH-3-one and in the conversion of 4-en-3-one to 3-one that leads to brassinolide biosynthesis (Fujioka et al., 1997; Noguchi et al., 1999). The metabolism of [2H6]6-deoxoCT (10 g) was investigated in wild-type seedlings. Although most of the substrate remained unmetabolized (6.3 g), [2H6]3-Assay If the pathway proposed in this scholarly study operates LY3009104 price in Arabidopsis, we’d expect 22-OH-3-1 and its own downstream materials to recovery the mutant towards the wild-type phenotype. To check this simple idea, we examined the result of 22-hydroxylated steroids in hypocotyl elongation in in both dark and light circumstances. Although 22-OH-4-en-3-ol had not been discovered within this scholarly research, we also analyzed the effect of the steroid just as one precursor of 22-OH-4-en-3-one. At night, 22-OHCR, 22-OH-4-en-3-ol, and 22-OH-4-en-3-one didn’t recovery hypocotyl elongation in plant life, a single program of 500 ng of 22-OH-3-one LY3009104 price or 6-deoxoCT towards the capture apex rescued mutant phenotypes in the light. Two times after application, the petiole duration was elongated, and a week later the entire morphology of mutant plant life was almost similar to that from the wild-type handles (data not proven). On the other hand, equivalent application of 22-OH-4-en-3-1 or 22-OHCR didn’t rescue the mutant phenotype. Thus, in afterwards developmental levels also,.