Cytochrome P450 710A1/A2 as Brassinosteroid C24-Desaturases to Connect C27- and C28-Brassinosteroids Biosynthesis in Arabidopsis thaliana

MBP-fused Arabidopsis CYP710A1 and CYP710A2 were obtained from E. coli, and their biochemical functions were examined to characterize the C24-desaturase(s) involved in converting 28-norcastaterone (28-norCS) into dolichosterone (DS). When 28-norCS was used as a substrate, GC–MS analysis revealed that 28-norCS was converted into dehydro-28-norCS, most likely due to Δ24-28-norCS exposure to NADPH-CYP reductase. When only MBP-CYP710A1 and MBP-CYP710A2 were used as co-enzyme sources, MBP-fused sterolmethyltransferase 1 (MBP-SMT1) successfully catalyzed the C24-methylation of 28-norCS to DS, verifying that CYP710A1 and CYP710A2 act as C24-desaturases for SMT1-mediated 28-norCS to DS C24-methylation intermediated by Δ24-28-norCS. During seedling growth, primary root lengths in cyp710a1 and cyp710a2 were significantly attenuated than those in the wild-type. The abated primary root growth in cyp710a1 and cyp710a2 was restored by exogenously applied dolichosterone and castasterone, indicating that cyp710a1 and cyp710a2 are brassinosteroid-deficient mutants from the disrupted conversion of 28-norCS to DS and CS in Arabidopsis. In addition, CYP710A1 and CYP710A2 expressions were downregulated in bes1-D and bzr1-D. Chromatin immune precipitation assay revealed that BES1 and BZR1 directly bind to E-box and BRRE sequences on CYP710A1 and CYP710A2 promotor regions, indicating that CYP710A1 and CYP710A2 expressions are feedback regulated by brassinosteroid signaling via BES1 and BZR1 in Arabidopsis. Conclusively, CYP710A1 and CYP710A2 serve as C24-desaturases to connect C27- to C28-brassinosteroids biosynthesis, which is strictly regulated by brassinosteroid signaling to maintain homeostasis for regulating Arabidopsis growth and development.