C-23 Hydroxylation by Arabidopsis CYP90C1 and CYP90D1 Reveals a Novel Shortcut in Brassinosteroid Biosynthesis

Null Mutations Caused by T-DNA Insertions in the CYP90C1 and CYP90D1 Genes.

(A) Exon-intron structures of the CYP90C1 and CYP90D1 genes. Bars represent exons, and numbers indicate exon sizes in nucleotide base pairs. Arrowheads show T-DNA insertion sites in the cyp90c1 and cyp90d1 mutant alleles.

(B) RT-PCR transcript analysis of the CYP90C1, CYP90D1, and UBQ10 transcripts in wild-type Col-0, cyp90c1, cyp90d1, and cyp90c1 cyp90d1 seedlings.

Morphology of the CYP90C1- and CYP90D1-Deficient Mutants.

(A) to (H) Seven-day-old etiolated ([A] to [D]) and light-grown ([E] to [H]) seedlings. Wild-type Col-0 ([A] and [E]), cyp90c1 ([B] and [F]) cyp90d1 ([C] and [G]), and cyp90c1 cyp90d1 ([D] and [H]). Bar = 5 mm.

(I) to (M) One-month-old wild-type Col-0 (I), cyp90c1 (J), cyp90d1 (K), and cyp90c1 cyp90d1 ([L] and [M]) plants. Bars = 30 mm in (I) to (L) and 5 mm in (M).

(N) to (Q) Flowers of wild-type (N), cyp90c1 (O), cyp90d1 (P), and cyp90c1 cyp90d1 (Q) plants. Bar = 5 mm.

GC-MS Analysis of BR Metabolites Produced by Heterologously Expressed CYP90C1 and CYP90D1.

Selected ion chromatograms of the reaction products generated by CYP90C1 and CYP90D1 from 6-deoxoCT and CT, respectively.

Rescue of Hypocotyl and Cotyledon Elongation in the cyp90c1 cyp90d1 Mutant by BRs.

Hypocotyl elongation of etiolated seedlings (A) and cotyledon elongation of light-grown seedlings (B) treated by intermediates (100 nM) of the early and late C-6 oxidation pathways. Hypocotyl elongation of etiolated seedlings (C) and cotyledon elongation of light-grown seedlings (D) treated by intermediates (100 nM) of the early C-22 oxidation pathway. Seedlings of det2 were used as control. Ctr, control; 22-OHCR, 22-hydroxycampesterol; 3-epi-6-deoxoCT, 3-epi-6-deoxocathasterone; 22,23-diOHCR, 22,23-dihydroxycampesterol. Panels show representative experiments. Values are averages of 50 seedlings; error bars indicate se.