An early C-22 oxidation branch in the brassinosteroid biosynthetic pathway.
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.
Fujioka
Shozo
S
RIKEN (The Institute of Physical and Chemical Research), Wako-shi, Saitama 351-0198, Japan. sfujioka@postman.riken.go.jp
Takatsuto
Suguru
S
Yoshida
Shigeo
S
D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D029681:Arabidopsis Proteins; D060406:Brassinosteroids; D030008:Catharanthus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002777:Cholestanols / Q000737:chemistry / Q000378:metabolism*; D003903:Deuterium; D004305:Dose-Response Relationship, Drug; D008401:Gas Chromatography-Mass Spectrometry; D018546:Hypocotyl / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D015394:Molecular Structure; D009154:Mutation; D010084:Oxidation-Reduction; D010840:Phytosterols / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D018520:Plant Shoots / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D012697:Serine Endopeptidases; D013260:Steroids, Heterocyclic / Q000737:chemistry / Q000378:metabolism*
D029681:Arabidopsis Proteins; D060406:Brassinosteroids; D002777:Cholestanols; D010840:Phytosterols; D013260:Steroids, Heterocyclic; D003903:Deuterium; D012697:Serine Endopeptidases; C023623:brassinolide