Smi-miR164a positively regulates phenolic acid biosynthesis while negatively regulates tanshinone production in Salvia miltiorrhiza

Abstract

Introduction MicroRNAs (miRNAs) are key post-transcriptional regulators of plant secondary metabolism. Their primary mechanism involves silencing target genes through mRNA cleavage or translational inhibition, which is a major focus of current research in this field. However, the specific regulatory roles of individual miRNAs in coordinating different secondary metabolic pathways in medicinal plants remain largely uncharacterized. Methods This study investigated the roles of Smi-miR164a in Salvia miltiorrhiza. We generated Smi-miR164a-overexpressing (OE-miR164a) transgenic lines and performed comprehensive metabolic profiling and gene expression analysis. Results Overexpression of Smi-miR164a resulted in significant accumulation of phenolic acids, with rosmarinic acid (RA) and salvianolic acid B (SalB) levels increased by up to 2.8-fold compared to wild-type (WT). Conversely, it markedly reduced the accumulation of tanshinones, decreasing tanshinone I (T-I) and tanshinone IIA (T-IIA) to 25-68% of WT levels. Transcriptional analysis showed that expression changes in key biosynthetic genes were tightly correlated with the metabolic alterations. Genes involved in the tanshinone pathway (e.g., HMGR1, DXS2) were downregulated, whereas those in the salvianolic acid pathway (e.g., PAL1, C4H) were upregulated, consistent with the reciprocal accumulation of their corresponding metabolites. Conclusion These findings demonstrate that the Smi-miR164a module acts as a pivotal regulator, positively influencing phenolic acid biosynthesis while negatively regulating tanshinone production in S. miltiorrhiza. This gene presents a promising target for molecular breeding aimed at enhancing the yield of specific bioactive compounds.