Tanshinones are bioactive diterpenoid chemicals of the herb Salvia miltiorrhiza 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 SmCYP71D375 as the direct target gene, remains unclear. In the present study, the promoter of SmCYP71D375 was employed as the bait to mine the upstream regulatory protein using the cDNA yeast library of S. miltiorrhiza. An R2R3-MYB transcription factor gene, SmMYB53, was identified. Overexpressing SmMYB53 in transgenic hairy roots upregulated SmCYP71D375 expression, thereby accelerating tanshinone accumulation, whereas tanshinone accumulation was inhibited in SmMYB53-RNAi transgenic hairy root lines. To dissect the regulatory network of SmMYB53, SmbZIP51 was captured using SmMYB53 as the bait to prey for its potential interacting proteins in the cDNA yeast library. Yeast two-hybrid, glutathione S-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 SmCYP71D375 activated by SmMYB53 would be inhibited by the interaction of SmMYB53 and SmbZIP51. The present findings uncover the molecular regulatory network underlying SmCYP71D375 as the direct target regulating tanshinone biosynthesis and offer a basis for the genetic improvement of medicinal substance biosynthesis in S. miltiorrhiza.

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