Biosynthetic pathways of brassinolide in Arabidopsis

Our previous studies on the endogenous brassinosteroids (BRs) in Arabidopsi s have provided suggestive evidence for the operation of the early C6-oxida tion and the late CG-oxidation pathways, leading to brassinolide (BL) in Ar abidopsis. However, to date the in vivo operation of these pathways has not been fully confirmed in this species. This paper describes metabolic studi es using deuterium-labeled BRs in wild-type and BR-insensitive mutant (bri1 ) seedlings to establish the intermediates of the biosynthetic pathway of B L in Arabidopsis. The first evidence for the conversion of campestanol to 6 -deoxocathasterone and the conversion of 6-deoxocathasterone to 6-deoxoteas terone is provided. The later biosynthetic steps (6-deoxoteasterone --> 3-d ehydro-6-deoxoteasterone --> 6-deoxotyphasterol --> 6-deoxocastasterone --> 6 alpha-hydroxy-castasterone --> castasterone --> BL) were demonstrated by stepwise metabolic experiments. Therefore, these studies complete the docu mentation of the late Cb-oxidation pathway. The biosynthetic sequence invol ved in the early C6-oxidation pathway (teasterone --> 8-dehydroteasterone - -> typhasterol --> castasterone --> BL) was also demonstrated. These result s 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 typhaster ol, and increased metabolic flow in BR-insensitive mutants.