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Biosynthetic Pathways of Brassinolide in Arabidopsis1
Our previous studies on the endogenous brassinosteroids (BRs) in Arabidopsis have provided suggestive evidence for the operation of the early C6-oxidation and the late C6-oxidation pathways, leading to brassinolide (BL) in Arabidopsis. However, to date the in vivo operation of these pathways has not been fully confirmed in this species. This paper describes metabolic studies using deuterium-labeled BRs in wild-type and BR-insensitive mutant (bril) seedlings to establish the intermediates of the biosynthetic pathway of BL in Arabidopsis. The first evidence for the conversion of campestanol to 6-deoxocathasterone and the conversion of 6-deoxocathasterone to 6-deoxoteasterone is provided. The later biosynthetic steps (6-deoxoteasterone - 3-dehydro-6-deoxoteasterone - 6-deoxotyphasterol - 6-deoxocastasterone -- 6a-hydroxycastasterone - castasterone -- BL) were demonstrated by stepwise metabolic experiments. Therefore, these studies complete the documentation of the late C6-oxidation pathway. The biosynthetic sequence involved in the early C6-oxidation pathway (teasterone - 3-dehydroteasterone - typhasterol - castasterone - BL) was also demonstrated. These results show that both the early and late C6-oxidation pathways are functional in Arabidopsis. In addition we report two new observations: the presence of a new branch in the pathway, C6 oxidation of 6-deoxotyphasterol to typhasterol, and increased metabolic flow in BR-insensitive mutants.
Brassinosteroids (BRs) are now recognized as a major hormone controlling plant growth and development (Yokota, 1997; Altmann, 1998; Clouse and Feldmann, 1999). Up to now more than 40 BRs have been fully characterized (Fujioka, 1999). The biosynthetic pathways of brassinolide (BL), the most active BR, have been elucidated using cultured cells of Catharanthus roseus. Through extensive metabolic studies, parallel branched pathways for BL, namely the early C6-oxidation and late C6-oxidation pathways, have been proposed (Fujioka and Sakurai, 1997a, 1997b). The first reaction toward BL is the conversion of campesterol (CR) to campestanol (CN), and then CN is converted to castasterone (CS) through either the early C6-oxidation pathway or the late C6oxidation pathway. Finally CS is converted to BL, the most active BR. The natural occurrence of most BR intermediates was demonstrated in cultured cells of C. roseus. In addition most of the steps were defined by feeding deuterium-labeled substrates followed by identification of the metabolites using gas chromatography-mass spectrometry (GC-MS). However, some steps remained to be validated. In fact the first...