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

Abstract

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.

Acknowledgements

This work was supported by the National Natural Science Fund (82373979), the Zhejiang Natural Science Fund (LZ24H280002), and the Key Scientific and Technological Grant of Zhejiang for Breeding New Agricultural Varieties (2021C02074-3-4). We appreciate the great experimental support from the Public Platform of Medical Research Center, Academy of Chinese Medical Science, Zhejiang Chinese Medical University.

Author contributions

W.Z. and G.K. conceived and designed the study; X.W., X.X., S.G., Y.S., J.W., and Q.W. performed the research; Y.S., W.Z., X.W., L.Z., and X.Z. analyzed the data; W.Z. and X.W. wrote the paper.

Data availability

All data generated in present study are included in this published article and its supplementary data files.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

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