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Comparative Study
. 2002 Oct;130(2):930-9.
doi: 10.1104/pp.008722.

An early C-22 oxidation branch in the brassinosteroid biosynthetic pathway

Shozo Fujioka  1 , Suguru Takatsuto, Shigeo Yoshida
Affiliations
Comparative Study

An early C-22 oxidation branch in the brassinosteroid biosynthetic pathway

Shozo Fujioka et al. Plant Physiol. 2002 Oct.

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-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.

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Figures

Figure 1
Figure 1
Structures of BRs identified in cultured C. roseus cells and Arabidopsis seedlings.
Figure 2
Figure 2
Metabolism of [2H6]22-OHCR in cultured C. roseus cells (V208) and Arabidopsis seedlings (wild type and det2 mutant). Five, 10, or 20 μg of [2H6] 22-OHCR was fed to cultured cells or seedlings for 2 d. Each value shows the amounts (μg) of unmetabolized substrate and metabolites detected. nd, Not detected (below detection limit).
Figure 3
Figure 3
Effect of 22-OHCR, (22S,24R)-22-hydroxy-ergost-4-en-3β-ol (22-OH-4-en-3β-ol), (22S,24R)-22-hydroxy-ergost-4-en-3-one (22-OH-4-en-3-one), 22-OH-3-one, 6-deoxoCT, and brassinolide (BL) on hypocotyl elongation in dark-grown det2 mutant seedlings. Each data point represents the mean of 15 replicates ± se. Hypocotyl length of the wild type (Columbia) without BR treatment was 14.4 ± 0.4 mm, whereas that of the det2 mutant was 4.4 ± 0.4 mm.
Figure 4
Figure 4
Effect of 22-OHCR, (22S,24R)-22-hydroxy-ergost-4-en-3β-ol (22-OH-4-en-3β-ol), 22-OH-4-en-3-one, 22-OH-3-one, 6-deoxoCT, and brassinolide (BL) on hypocotyl elongation in light-grown det2 mutant seedlings. Each data point represents the mean of 15 replicates ± se. Hypocotyl length of the wild type (Columbia) without BR treatment was 1.6 ± 0.1 mm, whereas that of the det2 mutant was 0.8 ± 0.04 mm.
Figure 5
Figure 5
Effect of 28-nor-22-OHCR, 28-nor-22-OH-4-en-3-one, 28-nor-22-OH-3-one, 28-nor-6-deoxoCT, and brassinolide (BL) on hypocotyl elongation in dark-grown det2 mutant seedlings. Each data point represents the mean of 15 replicates ± se. Hypocotyl length of the wild type (Columbia) without BR treatment was 12.3 ± 0.4 mm, whereas that of the det2 mutant was 4.4 ± 0.1 mm.
Figure 6
Figure 6
Proposed biosynthetic pathway for brassinolide and endogenous levels of BRs in wild-type and det2 Arabidopsis seedlings. The endogenous levels are shown under the names of each compound (ng g−1 fresh weight). nd, Not detected (below detection limit).
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