Establishment of Biosynthetic Pathways To Generate Castasterone as the Biologically Active Brassinosteroid in <i>Brachypodium distachyon</i>

Abstract

Gas chromatography-mass spectrometry (GC-MS) analysis revealed that castasterone and its biosynthetic precursors are found in <i>Brachypodium distachyon</i>. In vitro conversion experiments with crude enzyme solutions prepared from <i>B. distachyon</i> 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 <i>B. distachyon</i> that synthesize 24-methylated brassinosteroids (BRs). Biochemical analysis of BRs biosynthetic enzymes confirmed that <i>BdDET2</i>, <i>BdCYP90B1</i>, <i>BdCYP90A1</i>, <i>BdCYP90D2</i>, and <i>BdCYP85A1</i> 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 <i>B. distachyon</i>. Additionally, <i>B. distachyon</i> 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 <i>B. distachyon</i>. These results strongly suggest that the end product for brassinosteroid biosynthesis which controls the growth and development of <i>B. distachyon</i> is not brassinolide but rather castasterone.