Characterization of C29-Brassinosteroids and Their Biosynthetic Precursors in Immature Seeds of Phaseolus vulgaris

Abstract

Brassinosteroids (BRs) are unique steroids which show hormonal actions in plant growth and development. The BRs can be classified into C27, C28 and C29 based on their carbon skeleton. Metabolic and molecular genetic studies revealed that the C28-BRs are generated from campesterol (CR). Recently, we demonstrated that a C27-BR, 28-norcastasterone (28-norCS) is biosynthesized from cholesterol (CHR) that carries an identical carbon skeleton to that of 28norCS. These results suggested that the C29-BRs are also biosynthesized from a phytosterol carrying the same skeleton as that of the C29-BRs. Solid evidence to support this notion, however, is not yet to be obtained. This deficiency has prompted us to investigate the presence of BRs with a C-24 ethyl group and their potent biosynthetic precursors in the immature seeds of Phaseolus vulgaris wherein 24α-ethylcholesterol, sitosterol (STR), is a major sterol, which provides a clue for possible C29-BRs biosynthesis in plants. As summarized in Table 1, the strongest peak on the total ion chromatogram (TIC) at 17.29 min gave a mass spectrum at m/z 486 (M), 471, 396, 381, 357, 255, 213, 145 and 129 whose mass spectrum and retention times on GC are identical to those of authentic 24S-ethylcholesterol (STR) trimethylsilyl (TMSi) ether, demonstrating that the compound is STR. On TIC, a peak appeared at 17.57 min showing a molecular ion at m/z 488 and prominent ions at m/z 473, 398, 383, 359, 257, 215, 147 and 131. The molecular ion and characteristic ions in the TMSi compound are 2 mass increased than those of sitosterol TMSi, suggesting that the compound is dihydro-sitosterol, most likely sitostanol (STN). As shown in Table 2, 300 MHz proton NMR of the acetylated STN compound gave the key proton resonances at δ 0.64 (Me -18, s), 0.81 (Me -19, s), 4.68 (H-3, m), 2.02 (3-OAc, s), 0.90 (Me -21, d, J = 6.6 Hz), 0.85 (Me -26, d, J = 7.2 Hz), 0.83 (Me -27, d, J = 7.2 Hz) and 0.80 (Me -29, d, J = 5.7 Hz). The signals for H-4 (δ 2.32, d) and H-6 (δ 5.38, m) in STR-acetate were not observed in the compound, however, indicating that a double bond between C-5 and C-6 in STR acetate is not present in the compound. By saturating the ring structure, most of the aforementioned proton signals from the compound are upfield shifted compared to those from STR-acetate. The downfield shifted signal for H-3 is also understandable because the absorption of H-3 (δ 4.60 m) derived from CHR-acetate is downfield shifted to δ 4.69 (m) by diminishing the double bond at C-5. Taken together, the compound is undoubtedly STN-acetate, determining that the natural structure of the compound is STN. The ethyl acetate soluble fraction (64 g) obtained from immature seeds of P. vulgaris was purified by several