Seed-Specific Expression of Arabidopsis AtCYP85A2 Produces Biologically Active Brassinosteroids Such as Castasterone and Brassinolide to Improve Grain Yield and Quality in Seeds of Brachypodium Distachyon

Brachypodium distachyon is a monocotyledonous model plant that contains castasterone (CS) but no brassinolide (BL) as the end product of brassinosteroids (BR) biosynthesis, indicating dysfunction of BL synthase, which catalyzes the conversion of CS to BL. To increase BR activity, Arabidopsis cytochrome P450 85A2 (AtCYP85A2) encoding BR 6-oxidase/BL synthase was introduced into B. distachyon with the seed-specific promoters pBSU1, pAt5g10120, and pAt5g54000. RT-PCR analysis and GUS activity revealed that AtCYP85A2 was only expressed in the seeds of the transgenic plants pBSU1-AtCYP85A2::Bd21-3, pAt5g10120-AtCYP85A2::Bd21-3, and pAt5g54000-AtCYP85A2::Bd21-3. The crude enzyme prepared from the seeds of these three transgenic plants catalyzed the conversion of 6-deoxoCS to CS. The transgenic plants showed greater specific enzyme activity than the wild-type plant for the conversion of 6-deoxoCS to CS, indicating enhanced BR 6-oxidase activity in the transgenic plants. The enzyme solution also catalyzed the conversion of CS into BL. Additionally, BL was identified from the seeds of transgenic plants, verifying that seed-specific AtCYP85A2 encodes a functional BL synthase to increase BR activity in the seeds of transgenic Brachypodium. In comparison with wild-type Brachypodium, the transgenic plants showed better growth and development during the vegetative growing stage. The flowers of the transgenic plants were remarkably larger, resulting in increments in the number, size, and height of seeds. The total starch, protein, and lipid contents in transgenic plants were higher than those in wild-type plants, indicating that seed-specific expression of AtCYP85A2 improves both grain yield and quality in B. distachyon.