| The roots and rhizomes of Salvia miltiorrhiza Bunge(S.miltiorrhiza)are traditional Chinese herbal medicines.Salvianolic acids are the important medicinal components of S.miltiorrhiza,which have remarkable efficacy in treating cardiovascular and cerebrovascular diseases.Our previous studies have found that exogenous application of salicylic acid(SA)can induce the expression of genes involved in salvianolic acid biosynthesis and thereby promote its accumulation.However,the molecular mechanism by which SA regulates phenolic acid biosynthesis remains unclear.Nonexpressor of pathogenesis-related gene 1(NPR1)and its family members serve as key components in the SA signaling pathway,responsible for sensing and transmitting SA signals to promote plant defense responses.Whether NPR proteins are involved in the SA signaling pathway to regulate salvianolic acid biosynthesis is still unclear.In this study,we analyzed the roles of SmNPR1 and SmNPR4 in the salvianolic acid biosynthesis using S.miltiorrhiza hairy roots as the material.Furthermore,we explored the functions of SmNPR-SmTGA modules in the salvianolic acid biosynthesis,aiming to elucidate the molecular mechanism by which SA regulates the biosynthesis of salvianolic acid.The main results are as follows:1.Genome-wide identification and analysis of the SmNPR gene family in S.miltiorrhiza.Five SmNPR genes,namely SmNPR1,SmNPR3,SmNPR4,SmNPR5,and SmNPR6,were identified from the S.miltiorrhiza genome.Evolutionary tree analysis showed that SmNPRs were classified into three subclades,of which SmNPR1 belonged to subclade I,SmNPR3/4belonged to subclade II,and SmNPR5/6 belonged to subclade III.Amino acid sequence alignment showed that all SmNPRs contained the BTB/POZ domain and Ankyrin repeats domain,while SmNPR1/3/4 also contained the NPR1＿like＿C domain.Tissue-specific expression analysis revealed that SmNPR1/3/4 had the highest expression in roots,SmNPR5 in roots and flowers,and SmNPR6 in flowers.After SA treatment,the expression of SmNPR1 and SmNPR4 peaked at 12 h(4.87-fold and 7.24-fold of the control group,respectively),while SmNPR3/6 reached their highest expression at 1 h and 3 h,respectively.In contrast,the expression of SmNPR5 was inhibited.The expression of SmNPR1/4 showed a strong correlation with that of Sm TAT1/Sm PAL1,the key enzyme genes involved in phenolic acid biosynthesis,indicating that SmNPR1 and SmNPR4 may play significant roles in SA-induced phenolic acid biosynthesis.2.SmNPR1 and SmNPR4 were found to regulate the biosynthesis of salvianolic acid.Transgenic hairy roots with SmNPR1,SmNPR4 overexpression and RNA interference were generated using genetic transformation mediated by Agrobacterium rhizogenes ATCC15834.Compared to the control group,the contents of rosmarinic acid,salvianolic acid B,and total phenolic acids,as well as the expression of genes related to phenolic acid biosynthesis,were elevated in SmNPR1-overexpressing hairy roots and decreased in SmNPR1 RNA-interfering hairy roots,suggesting that SmNPR1 positively regulate phenolic acid biosynthesis.On the contrary,overexpression of SmNPR4 in hairy roots reduced the contents of phenolic acids and expression of the enzyme genes.However,the contents of phenolic acids and enzyme genes expression in the SmNPR4 RNAi hairy roots were significantly upregulated after SA treatment,indicating that SmNPR4 functions as a negative regulator of SA-mediated phenolic acid biosynthesis.Extensive targeted metabolomics combined with transcriptomics analysis showed that SmNPR1 and SmNPR4 function in both primary and secondary metabolism of S.miltiorrhiza.3.The transcription factors that interacted with SmNPR1 and SmNPR4 were screened.The Y2 H analysis revealed that SmNPR1 interacted with SmTGA2,while SmNPR4 interacted with SmTGA1,SmTGA2,SmTGA5,Sm PAP1,Sm MYB111,Sm TTG1,Smb HLH37,Sm MYB36,and Sm MYB39,respectively.The interaction between SmNPR1 and SmTGA2,SmNPR4 and SmTGA5 were further confirmed by LCI and GST pull-down assays.4.SmTGA2 and SmTGA5 were identified as positive regulators of salvianolic acid biosynthesis.Transgenic hairy roots with SmTGA2,SmTGA5 overexpression and antisense expression were generated using ATCC15834-mediated genetic transformation method.Compared with control group,the contents of phenolic acids and the expression of enzyme genes were increased in the SmTGA2 and SmTGA5 overexpressing hairy roots,while decreased in the antisense expressing hairy roots.Dual-LUC,Y1 H,and EMSA results demonstrated that SmTGA2 and SmTGA5 could bind to the as-1 element on the promoter regions of the Sm CYP98A14 and Sm TAT1 genes,respectively,thereby activating their expression.These results indicate that SmTGA2 and SmTGA5 are positive regulators of salvianolic acid biosynthesis.5.The mechanism by which the SmNPR4-SmTGA5 module regulates SA-induced salvianolic acid biosynthesis was revealed.Transgenic hairy roots with double overexpression of SmNPR4 and SmTGA5 were generated using genetic transformation mediated by ATCC15834.Compared to the control group,the contents of salvianolic acid B and total phenolic acids were significantly reduced in the transgenic hairy roots with double overexpression.However,the expression of genes involved in phenolic acid biosynthesis varied among the three lines.Dual-LUC and EMSA results demonstrated that SmNPR4 could attenuate the DNA binding ability of SmTGA5 to the promoter of the Sm TAT1 gene,thereby inhibiting the transcriptional activity of SmTGA5 on the Sm TAT1 gene promoter.Moreover,exogenous SA treatment could alleviate the inhibitory effect of SmNPR4 on the transcriptional activity of SmTGA5.6.The mechanism by which the SmNPR4-SmNPR1-SmTGA2 module regulates SAinduced salvianolic acid biosynthesis was elucidated.Transgenic hairy roots were generated through ATCC15834-mediated genetic transformation with SmNPR1 overexpression and SmTGA2 antisense expression.Compared to the control group,the contents of phenolic acids were reduced in the transgenic hairy roots with SmNPR1 overexpression and SmTGA2 antisense expression,indicating that the role of SmNPR1 in promoting phenolic acid biosynthesis is dependent on SmTGA2.Dual-LUC and EMSA results demonstrated that SmNPR1 could enhance the DNA binding ability of SmTGA2 to the promoter of the Sm CYP98A14 gene,thereby increasing the transcriptional activation activity of SmTGA2.Additionally,SmNPR4 could interact with SmNPR1 and inhibit the enhancement of SmNPR1 on the transcriptional activation activity of SmTGA2,which could be relieved by exogenous SA treatment.Taken together,this study has identified the critical roles of SmNPR-SmTGA modules in SA-mediated salvianolic acid biosynthesis,elucidated the molecular mechanism by which SA signaling regulates salvianolic acid biosynthesis,expanded the biological functions of NPR proteins in plant science,and provided new insights into the SA signaling pathway in regulating plant secondary metabolism. |
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