Overexpression of Ath-MIR160b increased the biomass while reduced the content of tanshinones in Salvia miltiorrhiza hairy roots by targeting ARFs genes

Abstract

MicroRNAs (miRNAs) have attracted increasing attention in the field of medicinal plant research. Our previous analyses indicated that the mature miR160a level was related to the accumulation of tanshinones in Salvia miltiorrhiza (S. miltiorrhiza). Therefore, the aim of this research was to reveal how the miR160a regulates the tanshinones biosynthesis. The overexpression of miR160a in hairy roots of S. miltiorrhiza down-regulated the levels of the target genes ARF10, 16, and 17, and inhibited tanshinones biosynthesis but increased hairy root biomass. In overexpression line 1, the content of four major tanshinones dropped sharply to 9.06% (dihydrotanshinone I), 12.56% (cryptotanshinone), 12.02% (tanshinone I), and 8.52% (tanshinone IIA) compared with that of the wildtype (WT). The fresh weight (FW) of hairy roots increased to 1.72-fold of the WT, and in overexpression lines 4, tanshinones were not detected. However, the FW of hairy roots in overexpression line 4 increased to 1.90-fold of the WT. We also observed the down regulated genes in the tanshinones biosynthesis pathway. In particular, the transcription level of KSL2 dropped to 3% as compared to the WT, which was consistent with the decrease in tanshinones content. We further analyzed hormone changes and found an increase in indole acetic acid accumulation, while the accumulation of salicylic acid (SA) and jasmonic acid (JA) decreased. These findings indicate that miR160a may negatively regulate the biosynthesis of tanshinones, while promote the growth of hairy roots by targeting ARF10, 16, and 17. Our findings demonstrate that miRNAs could be used as an alternative strategy to dissect the complex network of secondary metabolism in medicinal plants. MiR160a overexpression promoted the IAA level and the growth of hairy roots of Salvia miltiorrhiza, but reduced the GH3-like level and production of tanshinones by targeting ARF10, ARF16, and ARF17.