Jasmonic acid (JA) signaling pathway plays an important role in tanshinone and phenolic acid biosynthesis in Salvia miltiorrhiza. However, the specific regulatory mechanism remains largely unclear. Previous work showed that a JASMONATE ZIM-domain (JAZ), SmJAZ9, acted as a repressor of tanshinone production in S. miltiorrhiza. In this study, we revealed that SmJAZ9 reduced both phenolic acid accumulation and related biosynthetic gene expression, confirming that SmJAZ9 also negatively affected phenolic acid biosynthesis. Then, we identified a novel MYB transcription factor,SmMYB76, which interacted with SmJAZ9. SmMYB76 repressed phenolic acid biosynthesis by directly downregulating SmPAL1, Sm4CL2, and SmRAS1. Further investigation demonstrated that JA mediated phenolic acids biosynthesis via SmJAZ9-SmMYB76 complex. Taken together, these findings state the molecular mechanism that SmJAZ9-SmMYB76 regulated phenolic acid biosynthesis at the transcriptional and protein levels, which provided new insights into JA signaling pathway regulating plant metabolism.

Plant U-box (PUB) proteins are ubiquitin ligases (E3) involved in multiple biological processes and in response to plant stress. However, the various aspects of the genome and the differences in functions between the U-box E3 (UBE3) ubiquitin ligases remain quite obscure in Salvia miltiorrhiza. The 60 UBE3 genes in the S. miltiorrhiza genome were recognized in the present study. The phylogenetic analysis, gene structure, motifs, promoters, and physical and chemical properties of the genes were also examined. Based on the phylogenetic relationship, the 60 UBE3 genes were categorized under six different groups. The U-box domain was highly conserved across the family of UBE3 genes. Analysis of the cis-acting element revealed that the UBE3 genes might play an important role in a variety of biological processes, including a reaction to the abscisic acid (ABA) treatment. To investigate this hypothesis, an ABA treatment was developed for the hairy roots of S. miltiorrhiza. Thirteen out of the UBE3 genes significantly increased after the ABA treatment. The co-expression network revealed that nine UBE3 genes might be associated with phenolic acids or tanshinone biosynthesis. The findings of the present study brought fresh and new understanding to the participation of the UBE3 gene family in plants, specifically in their biological responses mediated by the ABA. In S. miltiorrhiza, this gene family may be crucial during the ABA treatment. Significantly, the results of this study contribute novel information to the understanding of the ubiquitin ligase gene and its role in plant growth.

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Lipid-soluble tanshinone is one of the main bioactive substances in the medicinal plant Salvia miltiorrhiza, and its medicinal demand is growing rapidly. Yeast extract (YE) modulates the tanshinone biosynthesis, but the underlying regulatory network remains obscure. In this study, a YE-responsive transcriptional factor Scarecrow1 (SCR1) was identified in S. miltiorrhiza from the YE-induced transcriptome dataset. SmSCR1 is located in the nucleus. Overexpression of SmSCR1 in S. miltiorrhiza roots resulted in a significantly higher accumulation of tanshinone than the control, with the highest 1.49-fold increase. We also detected upregulation of tanshinone biosynthetic genes, SmSCR1 and SmHMGR1, and distinct alteration of growth and development of the hairy roots in the overexpression lines compared to the control. An inverse phenotype was observed in SmSCR1-SRDX suppression expression lines. We found that SmSCR1 can bind to the promoter of SmCPS1 to induce its expression. This study provides new insight into the regulatory mechanism on the growth and development of hairy roots, tanshinone accumulation, and the metabolic engineering of bioactive compounds in S. miltiorrhiza.