Fujioka Shozo, Takatsuto Suguru, Yoshida Shigeo
RIKEN (The Institute of Physical and Chemical Research), Wako-shi, Saitama 351-0198, Japan.
Plant Physiol. 2002 Oct;130(2):930-9. doi: 10.1104/pp.008722.
The natural occurrence of 22-hydroxylated steroids in cultured Catharanthus roseus cells and in Arabidopsis seedlings was investigated. Using full-scan gas chromatography-mass spectrometry analysis, (22S)-22-hydroxycampesterol (22-OHCR), (22S,24R)-22-hydroxyergost-4-en-3-one (22-OH-4-en-3-one), (22S,24R)-22-hydroxy-5alpha-ergostan-3-one (22-OH-3-one), 6-deoxocathasterone (6-deoxoCT), 3-epi-6-deoxoCT, 28-nor-22-OHCR, 28-nor-22-OH-4-en-3-one, 28-nor-22-OH-3-one, 28-nor-6-deoxoCT, and 3-epi-28-nor-6-deoxoCT were identified. Metabolic experiments with deuterium-labeled 22-OHCR were performed in cultured C. roseus cells and Arabidopsis seedlings (wild type and det2), and the metabolites were analyzed by gas chromatography-mass spectrometry. In both C. roseus cells and wild-type Arabidopsis seedlings, [(2)H(6)]22-OH-4-en-3-one, [(2)H(6)]22-OH-3-one, [(2)H(6)]6-deoxoCT, and [(2)H(6)]3-epi-6-deoxoCT were identified as metabolites of [(2)H(6)]22-OHCR, whereas the major metabolite in det2 seedlings was [(2)H(6)]22-OH-4-en-3-one. Analysis of endogenous levels of these brassinosteroids revealed that det2 accumulates 22-OH-4-en-3-one. The levels of downstream compounds were remarkably reduced compared with the wild type. Exogenously applied 22-OH-3-one and 6-deoxoCT were found to rescue det2 mutant phenotypes, whereas 22-OHCR and 22-OH-4-en-3-one did not. These results substantiate the existence of a new subpathway (22-OHCR --> 22-OH-4-en-3-one --> 22-OH-3-one --> 6-deoxoCT) and reveal that the det2 mutant is defective in the conversion of 22-OH-4-en-3-one to 22-OH-3-one, which leads to brassinolide biosynthesis.
研究了22-羟基化甾体化合物在长春花培养细胞和拟南芥幼苗中的天然存在情况。通过全扫描气相色谱-质谱分析，鉴定出了(22S)-22-羟基胆固醇(22-OHCR)、(22S,24R)-22-羟基麦角甾-4-烯-3-酮(22-OH-4-en-3-one)、(22S,24R)-22-羟基-5α-麦角甾烷-3-酮(22-OH-3-one)、6-脱氧菜甾酮(6-deoxoCT)、3-表-6-脱氧菜甾酮、28-降-22-OHCR、28-降-22-OH-4-en-3-one、28-降-22-OH-3-one、28-降-6-脱氧菜甾酮和3-表-28-降-6-脱氧菜甾酮。在长春花培养细胞和拟南芥幼苗（野生型和det2）中进行了用氘标记的22-OHCR的代谢实验，并通过气相色谱-质谱分析代谢产物。在长春花细胞和野生型拟南芥幼苗中，[(2)H(6)]22-OH-4-en-3-one、[(2)H(6)]22-OH-3-one、[(2)H(6)]6-脱氧菜甾酮和[(2)H(6)]3-表-6-脱氧菜甾酮被鉴定为[(2)H(6)]22-OHCR的代谢产物，而在det2幼苗中的主要代谢产物是[(2)H(6)]22-OH-4-en-3-one。对这些油菜素甾体化合物内源性水平的分析表明，det2积累了22-OH-4-en-3-one。与野生型相比，下游化合物的水平显著降低。发现外源施加的22-OH-3-one和6-脱氧菜甾酮可挽救det2突变体表型，而22-OHCR和22-OH-4-en-3-one则不能。这些结果证实了一条新的子途径(22-OHCR --> 22-OH-4-en-3-one --> 22-OH-3-one --> 6-脱氧菜甾酮)的存在，并揭示det2突变体在22-OH-4-en-3-one向22-OH-3-one的转化中存在缺陷，而这一转化是油菜素内酯生物合成所必需的。
