Gas chromatography–mass
spectrometry
(GC–MS) analysis revealed that castasterone and its biosynthetic
precursors are found in Brachypodium distachyon. In vitro conversion experiments with crude enzyme solutions prepared
from B. distachyon demonstrated the
presence of the following biosynthetic sequences: campesterol →
campesta-4-en-3-one → campesta-3-one → campestanol →
6-deoxocathasterone → 6-deoxoteasterone → teasterone
↔ 3-dehydroteasterone ↔ typhasterol → castasterone.
campesterol → 22-hydroxycampesterol → 22-hydroxy-campesta-4-en-3-one
→ 22-hydroxy-campesta-3-one → 6-deoxo-3-dehydroteasterone
→ 3-dehydroteasterone. 6-deoxoteasterone ↔ 6-deoxo-3-dehydroteasterone
↔ 6-deoxotyphasterol → 6-deoxocastasterone →
castasterone. This shows that there are campestanol-dependent and
campestanol-independent pathway in B. distachyon that synthesize 24-methylated brassinosteroids (BRs). Biochemical
analysis of BRs biosynthetic enzymes confirmed that BdDET2, BdCYP90B1, BdCYP90A1, BdCYP90D2, and BdCYP85A1 are orthologous
to BR 5α-reductase, BR C-22 hydroxylase, BR C-3 oxidase, BR
C-23 hydroxylase, and BR C-6 oxidase, respectively. Brassinolide was
not identified in B. distachyon. Additionally, B. distachyon crude enzyme solutions could not catalyze
the conversion of castasterone to brassinolide, and the gene encoding
an ortholog of CYP85A2 (a brassinolide synthase) was not found in B. distachyon. These results strongly suggest that
the end product for brassinosteroid biosynthesis which controls the
growth and development of B. distachyon is not brassinolide but rather castasterone.