Molecular mechanism of <i>SmMYB53</i> activates the expression of <i>SmCYP71D375</i>, thereby modulating tanshinone accumulation in <i>Salvia miltiorrhiza</i>

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