Functional Study Of Salvia Miltiorrhiza Transcription Factors SmbHLH59, SmMYB97 And SmWRKY14 Mediated By Jasmonin Signaling

Salvia miltiorrhiza Bunge is a perennial herb plant in the labiatae family,which contains two active components,salvianolic acid and tanshinone.It is effective in the treatment of cardiovascular and cerebrovascular diseases.Uncover the biosynthesis pathway and regulatory mechanism could contribute to understanding the quality formation of S.miltiorrhiza.There are some ways to increase the secondary metabolites in S.miltiorrhiza:controlling the expression of enzyme-encoding genes to change the accumulation of secondary metabolites,application of biological or abiotic elicitors to stimulate the production of secondary metabolites,and regulating secondary metabolites biosynthesis by transcription factors.There have been a lot of reports on the functional identification of enzyme-encoding genes,but the research on the mechanism of elicitors and transcription factors is still to be improved.It’s reported that jasmonate（JA）could regulate secondary metabolism in S.miltiorrhiza.Treat with methyl jasmonate（MeJA）affects the expression of related genes of JA signaling pathway,activates the enzyme-encoding genes of related biosynthesis pathway,and promotes the accumulation of secondary metabolites.However,the potential mechanism of JA signaling pathway has not been clarified.By Referencing the JA signaling in other species and combining with previous study,we found that JAZ protein and bHLH,MYB,and WRKY transcription factors play important roles in JA signaling,which is the molecular basis of JA function.Therefore,identifying the transcription factors that interact with JAZ protein and analyzing the effect of JAZ protein on the transcription factors could contribute to clarifying the molecular mechanism of JA signal in secondary metabolism of S.miltiorrhiza.The main results and conclusions are as follows:1.Based on the previous study,we screened three transcription factors,SmbHLH59,SmMYB97,and SmWRKY14,which respond to MeJA treatment and may involved in the secondary metabolism of S.miltiorrhiza.Yeast two-hybrid and bimolecular fluorescent complementation assays were used to identify the interaction between SmJAZ proteins and transcription factors.The results showed that SmbHLH59 interacts with SmJAZ1 and SmJAZ8,SmMYB97 interacts with SmJAZ8,and SmWRKY14 interacts with SmJAZ 1,SmJAZ3 and SmJAZ8.It’s suggested that transduction units JAZs-TFs may mediate the regulation of JA signaling in S.miltiorrhiza.2.A bHLH transcription factor,SmbHLH59,was identified from S.miltiorrhiza.Bioinformatics analysis showed that SmbHLH59 belongs to the Ⅲe subfamily,and members of this family play a positive regulatory role in the JA signaling pathway.Expression pattern analysis revealed that SmbHLH59 was highly expressed in leaves and responded to MeJA treatment,which may be involved in JA signal.Then,Gateway and CRISPR/Cas9 technology were employed to construct overexpression and knockout vector of SmbHLH59 gene,and overexpression and knockout transgenic lines were obtained by Agrobacterium-mediated transformation of S.miltiorrhiza.The contents of secondary metabolites and expression of enzyme-encoding genes in the transgenic lines were detected.It found that overexpression of SmbHLH59 significantly increased the contents of salvianolic acid and tanshinone,and activated enzyme-encoding genes in the biosynthetic pathway,especially SmPAL1,SmC4H1,SmHPPR1,SmCPS1 and SmKSL1 genes were induced.Knockout of SmbHLH59 decreased the accumulation of salvianolic acid and tanshinone,and down-regulated the expression of most key genes.Subsequently,yeast one-hybrid and transient transcriptional activity assays showed that SmbHLH59 activates SmPAL1,SmC4H1,SmHPPR1,SmCPSl and SmKSL1 promoters by binding to E/G-box elements.Therefore,SmbHLH59,as a positive regulator,promotes the accumulation of secondary metabolites via binding and activating the target gene promoter in S.miltiorrhiza.3.We identified and characterized the JA-responsive gene SmMYB97.Multiple sequence alignment and phylogenetic tree analyses showed that SmMYB97 was clustered with AtMYB11,AtMYB12,and ZmP1 in the subgroup S7 regulating flavonol biosynthesis.SmMYB97 was highly expressed in S.miltiorrhiza leaves and induced by MeJA.SmMYB97 overexpression increased salvianolic acid and tanshinone biosynthesis and upregulated the genes implicated in these processes.Yeast-one hybrid and transient transcriptional activity assays disclosed that SmMYB97 binds the SmPAL1,SmTAT1,SmCPS1,and SmKSL1 promoter regions,inducing the accumulation of secondary metabolites of S.miltiorrhiza.4.We compared the different expression genes under MeJA treatment in iCOI1 lines and WT,a novel COI1-dependent WRKY TF,SmWRKY14 was identified in S.miltiorrhiza.Bioinformatics and expression pattern results show that SmWRKY14 belongs to the Ⅱc subfamily,with the highest expression in roots,and induced by MeJA.Compared with wild-type line,the contents of salvianolic acid and tanshinone were significantly higher in overexpression lines,and the key genes of the biosynthetic pathway were also activated.Among them,the SmPAL1,SmC4H1,SmTAT1 and SmRAS1 genes in the salvianolic acid biosynthesis pathway and the tanshinone biosynthesis gene SmCPS1 were strongly induced,which suggest that SmWRKY14 targets these genes and regulates the biosynthesis of salvianolic acid and tanshinone.Further,we found that SmWRKY14 could bind and activate the promoters of SmPAL1,SmC4H1,SmTAT1,SmRAS1,and SmCPS1 via W-box elements,then regulate the target genes and increase the accumulation of salvianolic acid and tanshinone.5.Transient transcriptional activity assays were used to investigate whether SmJAZ8 interferes the DNA binding between transcription factors and promoters of target gene.Compared with the control,SmbHLH59 and SmMYB97 activate the promoters of target genes.SmJAZs did not affect the activity of the promoters,but SmJAZ8 and SmbHLH59 or SmMYB97 co-transformation significantly reduced the activity of the promoters.It’s indicated that SmJAZs protein blocks transcription factors and inhibits target gene promoters.We speculate that SmJAZs protein interacts with SmbHLH59 or SmMYB97 transcription factors,and suppresses the activation activity of target gene promoters,thereby down-regulating the expression of target genes,leading to decrease the contents of salvianolic acid and tanshinone.In short,the functions and regulatory mechanisms of three JA-induced transcription factors SmbHLH59,SmMYB97 and SmWRKY14 involved in secondary metabolism were analyzed in S.miltiorrhiza.We preliminarily explained that JAZs-TFs as the JA signal transduction unit to mediate the secondary metabolism,which provides information about improving the contents of secondary metabolites in S.miltiorrhiza.