Two genes of cytochrome P450 regulate plant height via brassinosteroid biosynthesis in Brassica napus

• In this study, we identified two cytochrome P450 genes, BnaA10.CYP90A1 and BnaC09.CYP90A1, as key regulators of plant height in Brassica napus, highlighting their role in brassinosteroid biosynthesis and plant growth modulation. • Mutations in BnaA10.CYP90A1 and BnaC09.CYP90A1 genes lead to reduced levels of bioactive brassinosteroids, significantly impairing cell elongation and causing a dwarf phenotype in rapeseed. • The single mutation of BnaA10.CYP90A1 produced semi-dwarf plants, suggesting its potential for developing new rapeseed cultivars with optimized plant height for enhanced yield and mechanical harvesting efficiency. Rapeseed is globally important as an oil crop to provide essential edible plant oils for various agricultural products. Currently, manipulating plant height with branching effectively balances biomass and yields. However, the genetic mechanisms to control plant height largely remains unknown in rapeseed. To address this gap, we isolated an extremely d warf m utant ( dm1 ) from ethyl-methanesulfonate (EMS) mutagenesis, and revealed the dwarfism is result from a significant reduction of cell length. Bulk segregant analysis (BSA) identified BnaA10 . CYP90A1 and BnaC09 . CYP90A1 as the causative genes of dm1 . Both genes encoded the proteins homologous to the Arabidopsis cytochrome P450 AtCPD/AtCYP90A1, which is cruicial for brassinosteroid (BR) biosynthesis. In concern with that, we demonstrated the reduced levels of bioactive BRs, castasterone (CS), and its precursor 6-deoxoCS in dm1 , resulting in the down-regulation of various genes for cell expansion. The reduced BR levels also caused negative feedback to promote the expression of BR biosynthetic genes in dm1 . Furthermore, we proved that the single mutation of BnaA10 . CYP90A1 gene conferred semi-dwarfism, potentially beneficial for producing ideal type of plant to improve cultivars with a balance on yield and machinery harvest through genetic modifications. Collectively, these findings highlighted the critical role of BnaCYP90A1s in BR biosynthesis and validated their influence on the regulation of plant height in rapeseed.