The Study On Molecular Mechanism Of SmJAZ9-SmMYB76 And SmbHLH60 Regulating The Biosynthesis Of Medicinal Ingredients In Salvia Miltiorrhiza

Salvia miltiorrhiza is a traditional Chinese medicine used to treat cardiovascular and cerebrovascular diseases.The dried roots and rhizomes of S.miltiorrhiza are used as medicine,and the main medicinal components include lipid-soluble tanshinone and water-soluble phenolic acid.With the progress of research,the genome of S.miltiorrhiza has been published and the genetic transformation system has become more mature,which also make S.miltiorrhiza a medicinal model plant.Due to the increasing pressure of modern work and life,the incidence of cardiovascular and cerebrovascular diseases is also increasing gradually.With the increasing market demand,the shortage of wild S.miltiorrhiza resources,and the low content of medicinal components in commonly cultivated S.miltiorrhiza,it is particularly important to use biotechnology to improve the quality of S.miltiorrhiza,which has become a research hotspot.Treating with various plant hormones is an effective means to promote the accumulation of tanshinone and phenolic acid in S.miltiorrhiza.In particular,the effect of Jasmonic acid(JA)and its derivatives has attracted attention.MeJA has been confirmed to promote phenolic acid biosynthesis in Salvia miltiorrhiza,but the specific regulation mechanism remains unclear.The JASMONATE ZIM DOMAIN(JAZ)proteins function as negative regulators in JA signaling pathways.Little is known about the role of SmJAZ9 in the biological regulation of phenolic acid even though SmJAZ9 has been previously reported to negatively affect tanshinone biosynthesis.Here,we firstly reported that SmJAZ9 negatively affects the biosynthesis of phenolic acid.We found that the content of phenolic acids in SmJAZ9 OE hairy roots was significantly reduced,and the transcription levels of key enzyme genes involved in phenolic acid biosynthesis were decreased to varying degrees.In SmJAZ9-CRSPR hairy roots,the content of phenolic acid in hairy roots increased,and the gene expression of key enzymes was activated.Furthermore,we identified a novel R2R3-type MYB transcription factor that interacted with SmJAZ9 through Y2 H screening,named SmMYB76.Meanwhile,the interaction was verified by different assays such as Y2 H,Bi FC and pull down.SmMYB76 belonged to the S4 subfamily,and the expression level of SmMYB76 gene decreased under MeJA treatment.Overexpression of SmMYB76 significantly reduced the accumulation of phenolic acids.Conversely,the content of phenolic acids was increased in SmMYB76 CRISPR/Cas9 hairy roots.SmMYB76 was able to bound to and to repressed the promoters of SmPAL1,Sm4CL2 and SmRAS1.SmJAZ9 promoted SmMYB76 transcription level and enhanced the transcriptional repression of SmMYB76 on downstream genes.Taken together,our findings reveal a novel phenolic acid biosynthetic pathway regulatory network,providing new insights into how the JA signaling pathway regulates metabolism in plants.The JA signaling pathway plays important roles in the induction of secondary metabolites production,mediated by methyl jasmonate(MeJA)and the basic helixloop-helix transcription factor MYC2 as a major transcriptional facilitator.However,whether other molecular players are involved in the regulation of secondary metabolism biosynthesis in Salvia miltiorrhiza are under continues investigations.In this study,we identified that SmbHLH60 was one of the most significantly negatively regulated bHLH genes by MeJA.The mechanism of secondary metabolism regulation of Salvia miltiorrhiza by transcription factors negatively regulated by MeJA is rarely reported.CRISPR/Cas9 system was used for SmbHLH60 knock-out resulting in an increase of phenolic acids and total anthocyanins in Salvia miltiorrhiza hairy roots,in accordance with the upregulation of phenolic acid and anthocyanin biosynthesis genes.Conversely,the content of phenolic acids and total anthocyanins in SmbHLH60 overexpression lines were decreased.We identified that SmbHLH60 protein directly interacted with SmMYC2 by Y2 H assays.The results of Dual-LUC,Y1 H and EMSA assays indicated that SmbHLH60 repressed SmTAT1 and SmDFR expression by binding to the G-box in the promoter region impairing the transcriptional activation through competition with SmMYC2.Our results suggest that SmbHLH60 and SmMYC2 can form a heterodimer and play an antagonistic role in the biosynthesis of phenolic acids and anthocyanins.Our study provides new insights into the molecular mechanism of MeJA regulating the secondary metabolism in S.miltiorrhiza.