An Early C-22 Oxidation Branch in the Brassinosteroid Biosynthetic Pathway

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Abstract
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-5α-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. roseuscells and wild-type Arabidopsis seedlings, [2H6]22-OH-4-en-3-one, [2H6]22-OH-3-one, [2H6]6-deoxoCT, and [2H6]3-epi-6-deoxoCT were identified as metabolites of [2H6]22-OHCR, whereas the major metabolite in det2 seedlings was [2H6]22-OH-4-en-3-one. Analysis of endogenous levels of these brassinosteroids revealed thatdet2 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.