SmbHLH53 is relevant to jasmonate signaling and plays dual roles in regulating the genes for enzymes in the pathway for salvianolic acid B biosynthesis in Salvia miltiorrhiza

Basic helix-loop-helix (bHLH) transcription factors play essential roles in myriad regulatory processes, including secondary metabolism. In this study with Salvia miltiorrhiza, we isolated and characterized SmbHLH53, which encodes a bHLH family member. Expression of this gene was significantly induced by wounding and multiple hormones, including methyl jasmonic acid; transcript levels were highest in the leaves and roots. Phylogenetic analysis indicated that SmbHLH53 clusters with AtbHLH17 and AtbHLH13, two negative regulators of jasmonate (JA) responses, and is localized in the nucleus and cell membrane. Yeast two-hybrid and bimolecular fluorescent complementation assays indicated that SmbHLH53 forms a homodimer as well as a heterodimer with SmbHLH37. It also interacts with both SmJAZs1/3/8 and SmMYC2, the core members of the JA signal pathway. Unexpectedly, we noted that overexpression of SmbHLH53 did not significantly influence the concentrations of rosmarinic acid and salvianolic acid B in transgenic plants. Results from yeast one-hybrid assays showed that SmbHLH53 binds to the promoters of SmTAT1, SmPAL1, and Sm4CL9, the key genes for enzymes in the pathway for phenolic acid synthesis. Assays of transient transcriptional activity demonstrated that SmbHLH53 represses the promoter of SmTAT1 while activating the promoter of Sm4CL9. Thus, the present work revealed that SmbHLH53 may play dual roles in regulating the genes for enzymes in the pathway for Sal B biosynthesis.