Molecular mechanism of SmMYB53 activates the expression of SmCYP71D375 thereby modulating the tanshinones accumulation in salvia miltiorrhiza

Abstract

Tanshinones are the bioactive diterpenoid chemicals of the herb Salvia miltiorrhiza with the characteristic furan D-ring. As a newly identified downstream enzyme, SmCYP71D375 catalyzes hydroxylation at 14,16-ether (hetero) cyclization to form the furan D-ring from the precursor of phenolic abietane-type diterpenoids widely exists in the Lamiaceae plants. But its transcriptional regulatory network with SmCYP71D375 as the direct target gene, remains unclear. In present study, the promoter of SmCYP71D375 is employed to be the bait to mine the upstream regulatory protein using the cDNA yeast library of S. miltiorrhiza. An R2R3-MYB transcription factor, SmMYB53, was identified. Overexpressing SmMYB53 in transgenic hairy roots upregulate the SmCYP71D375 expression thereby accelerating the tanshinones accumulation. Whereas, the tanshinones accumulation is inhibited in SmMYB53-RNAi transgenic hairy root lines. To dissect its regulatory network of SmMYB53, SmbZIP51 was captured using SmMYB53 as the bait to prey its potential interacting proteins in the cDNA yeast library. Yeast two-hybrid, GST pull-down and bimolecular fluorescence complementation assays were independently introduced to verify their interaction between the proteins of SmMYB53 and SmbZIP51. We further verified that the upregulation of SmCYP71D375 activated by SmMYB53 would be inhibited by their interaction of SmMYB53 and SmbZIP51. The present findings uncover the molecular regulatory network underlying SmCYP71D375 as the direct target to regulate tanshinones biosynthesis and offer the basis for the genetic improvement of medicinal substances biosynthesis in S. miltiorrhiza.