Brassinosteroids (BRs) are steroid hormones that regulate the growth and development of plants. Detailed study of the biosynthesis of brassinolide, a C₂₈BR, revealed that two parallel routes, the early and late C-6 oxidation pathways, are connected at multiple steps and also are linked to the early C-22 oxidation pathway. Thus, BR biosynthetic pathways are highly networked. Furthermore, the biosynthesis of C₂₇BRs was shown to proceed in a similar way to that of C₂₈BRs. Information on enzymes and genes involved in the BR biosynthesis, as well as their regulation, has been obtained using BR-deficient and BR-insensitive mutants. In addition, the biosynthesis of sterols, which were recently recognized not only as precursors of BRs and membrane constituents, but also as modulators of plant development, is discussed. Various metabolic reactions of BRs including epimerization, oxidation, and conjugation are also summarized.

Previously, we have shown that the Arabidopsis det2(deetiolated2) mutant is defective in the biosynthesis of brassinosteroids (BR) and that DET2 (a steroid 5α-reductase) acts early in the proposed BR biosynthetic pathway. In this paper we present further biochemical characterization of det2. We have undertaken metabolic experiments with ²H-labeled substrates of intermediates involved in the formation of campestanol from campesterol, and quantitative analysis of intermediates in Arabidopsis wild type anddet2. The results of these studies indicate the early operating steps of BR biosynthesis as: campesterol → 4-en-3β-ol → 4-en-3-one → 3-one → campestanol in Arabidopsis, withdet2 deficient in the conversion of 4-en-3-one to 3-one. We have also detected these intermediates in the formation of campestanol from campesterol and their metabolic conversions using cultured cells of Catharanthus roseus. These studies confirmed the biosynthetic sequence of events from campesterol to campestanol as was found in Arabidopsis. As such, the originally proposed biosynthetic pathway should be modified.

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.

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 (bri1) 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 → 6α-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.