SmJAZ4 interacts with SmMYB111 or SmMYC2 to inhibit the synthesis of phenolic acids in Salvia miltiorrhiza.

Jasmonic acid (JA), as an important plant hormone, can induce the synthesis of phenolic acids in Salvia miltiorrhiza Bunge, a model medicinal plant, but the specific mechanism remains to be further elucidated. JA-responsive SmMYB111 positively regulates the biosynthesis of salvianolic acid B (SalB), but the molecular mechanism is unclear. Here, we found that SmMYB111 directly binds to the promoters of SmTAT1 and SmCYP98A14 and activates their transcription. Yeast two hybrid and bimolecular fluorescent complementation assay indicated that SmMYB111 interacts with SmJAZ4. Furthermore, we systematically characterized the function of SmJAZ4, which was highly expressed in flowers and roots and located in the nucleus and cell membrane. The contents of phenolic acids in the SmJAZ4-overexpressed transgenic plantlets and SmJAZ4-overexpressed transgenic hairy roots decreased significantly. SmJAZ4 interacts with SmMYC2 or SmMYB111 to repress their transcriptional activation activity on target enzyme genes of the biosynthesis pathway of phenolic acids. Overall, the molecular mechanism of SmJAZ4-SmMYC2/SmMYB111 module participating in JA signaling regulation of SalB biosynthesis was elucidated, which give a clue for the molecular regulation of phenolic acids biosynthesis in S. miltiorrhiza.

R2R3-MYB Transcription Factor SmMYB52 Positively Regulates Biosynthesis of Salvianolic Acid B and Inhibits Root Growth in Salvia miltiorrhiza.

The dried root of Salvia miltiorrhiza is a renowned traditional Chinese medicine that was used for over 1000 years in China. Salvianolic acid B (SalB) is the main natural bioactive product of S. miltiorrhiza. Although many publications described the regulation mechanism of SalB biosynthesis, few reports simultaneously focused on S. miltiorrhiza root development. For this study, an R2R3-MYB transcription factor gene (SmMYB52) was overexpressed and silenced, respectively, in S. miltiorrhiza sterile seedlings. We found that SmMYB52 significantly inhibited root growth and indole-3-acetic acid (IAA) accumulation, whereas it activated phenolic acid biosynthesis and the jasmonate acid (JA) signaling pathway. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses revealed that SmMYB52 suppressed the transcription levels of key enzyme-encoding genes involved in the IAA biosynthetic pathway and activated key enzyme-encoding genes involved in the JA and phenolic acid biosynthesis pathways. In addition, yeast one-hybrid (Y1H) and dual-luciferase assay showed that SmMYB52 directly binds to and activates the promoters of several key enzyme genes for SalB biosynthesis, including SmTAT1, Sm4CL9, SmC4H1, and SmHPPR1, to promote the accumulation of SalB. This is the first report of a regulator that simultaneously affects root growth and the production of phenolic acids in S. miltiorrhiza.

MiR408-SmLAC3 Module Participates in Salvianolic Acid B Synthesis in Salvia miltiorrhiza.

MicroRNAs (miRNAs) are important regulators of gene expression involved in plant development and abiotic stress responses. Recently, miRNAs have also been reported to be engaged in the regulation of secondary plant metabolism. However, there are few functional studies of miRNAs in medicinal plants. For this study, we obtained Sm-miR408 interference lines to investigate the function of Sm-miR408 in a medicinal model plant (Salvia miltiorrhiza). It was found that inhibiting the expression of Sm-miR408 could increase the content of salvianolic acid B and rosmarinic acid in the roots. The SmLAC3 and Sm-miR408 expression patterns were analyzed by qRT-PCR. A 5' RLM-RACE assay confirmed that Sm-miR408 targets and negatively regulates SmLAC3. Moreover, the overexpression of SmLAC3 in S. miltiorrhiza promoted the accumulation of salvianolic acids in the roots. Furthermore, the lignin content of the roots in overexpressed SmLAC3 lines was decreased. Taken together, these findings indicated that Sm-miR408 modulates the accumulation of phenolic acids in S. miltiorrhiza by targeting SmLAC3 expression levels.