Functional Analysis Of SmMAPK3-SmARF1 Regulated Phenolic Acid Biosynthesis In Salvia Miltiorrhiza

The roots and rhizomes of Salvia miltiorrhiza Bunge(S.miltiorrhiza),a traditional Chinese herbal medicine,are evaluated for their herbal quality using water-soluble salvianolic acid B,lipid-soluble tanshinone I,cryptotanshinone,and tanshinone IIA.The mitogen-activated protein kinase(MAPK)cascade pathway is a classical signalling pathway that is highly conserved in eukaryotes,consisting of MAPK,MAPKK,MAPKKK,and MAPKKKK,which regulate the synthesis and accumulation of secondary metabolites in response to biotic and abiotic plant adversities.Members of the MAPK cascade in S.miltiorrhiza remain unknown,and their role in regulating secondary metabolism in S.miltiorrhiza has not been explored.In this study,we identified the members of the MAPK cascade in S.miltiorrhiza from the genome of S.miltiorrhiza and analysed the function of the SmMAPK3 gene in regulating the biosynthesis of secondary metabolites in S.miltiorrhiza using S.miltiorrhiza hairy roots as the material.We further explored the mechanism by which SmMAPK3 regulates the synthesis of phenolic acids through upstream SmMAPKK2/4/5/7 and downstream SmARF1.The main findings are as follows:1.Family members of the MAPK cascade pathway in S.miltiorrhiza were identified.Based on the genome of S.miltiorrhiza,six SmMAPKKKKs,83 SmMAPKKKs,nine SmMAPKKs,and 18 SmMAPKs genes were identified in this study.The gene expression pattern of each MAPK cascade member in S.miltiorrhiza was determined.Gene expression profiling showed that six SmMAPKKKKs,50 SmMAPKKKs,five SmMAPKKs,and 11 SmMAPKs genes were highly expressed in at least one S.miltiorrhiza tissue.Two SmMAPKKKKs,71 SmMAPKKKs,six SmMAPKKs,and 16 SmMAPKs genes were induced by at least one inducer.A few genes were also simultaneously induced by the three inducers.Co-expression analysis of SmMAPKs with genes encoding metabolic enzymes and transcription factors indicated that 18 SmMAPKs genes might participate in the biosynthesis of secondary metabolites in S.miltiorrhiza.Among these,SmMAPK3 is probably the most crucial in regulating the synthesis and accumulation of secondary metabolites in S.miltiorrhiza.2.The function of the SmMAPK3 gene in the biosynthesis and accumulation of tanshinones and phenolic acids was determined.We obtained transgenic hairy roots overexpressing SmMAPK3,SmMAPK3-CA,and RNAi SmMAPK3 via Agrobacterium ATCC15834-mediated genetic transformation.Compared to the wild-type S.miltiorrhiza hairy roots,the content of salvianolic acid B and rosmarinic acid in SmMAPK3-overexpressing lines were significantly increased.Overexpression of SmMAPK3-CA increased the tanshinone IIA content but decreased phenolic acid accumulation in hairy roots.The yield of phenolic acids in RNA interference of SmMAPK3 lines was significantly reduced;however,the tanshinone content was significantly increased.The expression of SmCYP98A14,SmHPPR1 and SmPAL1 were significantly increased in overexpressing SmMAPK3 lines,whereas the expression of SmPAL1 and SmRAS1 was significantly decreased in SmMAPK3 RNA interference lines.These results collectively suggest that SmMAPK3 may regulate the expression of metabolic enzymes via unknown transcription factors,which,in turn,regulate the synthesis and accumulation of phenolic acids.3.The proteins interacting with SmMAPK3 were also screened.The Y2 H and LCI results suggested that SmMAPK3 could interact with SmMAPKK2,SmMAPKK4,SmMAPKK5,and SmMAPKK7.The yeast two-hybrid(Y2H)assay results demonstrated that SmMAPK3 could interact upstream with SmPP2C14 and downstream with SmIAA9,SmIAA14,SmARF1,and SmJAZ2.Y2 H,subcellular localisation,and LCI results showed that SmARF1 interacted with SmMAPK3 and SmMAPK3-CA.All the above MAPK kinases,protein phosphatases,SmMAPK3,and substrates of SmMAPK3 form a regulatory network for secondary metabolism in S.miltiorrhiza.4.The function of the SmARF1 gene in the biosynthesis and accumulation of phenolic acids was also determined.We overexpressed SmARF1 and co-overexpressed SmMAPK3 and SmARF1 in transgenic hairy roots via Agrobacterium ATCC15834-mediated genetic transformation.Overexpression of SmARF1 reduced rosmarinic acid content in S.miltiorrhiza hairy roots,and co-overexpression of SmMAPK3 and SmARF1 enhanced the accumulation of phenolic acids in S.miltiorrhiza hairy roots.Overexpression of SmARF1 significantly reduced the expression of SmPAL1,SmRAS1,and SmRAS6.In contrast,overexpression of SmARF1+SmMAPK3 genes increased the expression of SmPAL1,SmRAS1,and SmRAS6.GUS and EMSA results showed that SmARF1 directly binds to the auxin responsive elements(Aux RE)in the promoter of the SmPAL1 gene and inhibits the expression of the SmPAL1 gene.In vitro phosphorylation showed that SmMAPK3-CA phosphorylated SmARF1.Dual-LUC results showed that SmMAPK3 and SmMAPK3-CA alleviate the transcriptional repressive activity of SmARF1 on the promoter of the SmPAL1 and also enhanced the expression of the SmPAL1 gene.These results suggested that SmMAPK3 positively regulates the biosynthesis of rosmarinic acid and salvianolic acid B via SmARF1.In summary,we combined phosphorylation modification and transcriptional regulation to elucidate the molecular mechanism by which SmMAPK3-SmARF1 regulates the synthesis of phenolic acids,providing a theoretical basis for selecting S.miltiorrhiza through genetic engineering techniques.