Biosynthesis And Salt Resistance Regulation Of Salvianolic Acids Based On Multi-omics Analysis

Salvia miltiorrhiza Bunge is a common medicinal plant of Salvia in Labiatae.Its dried roots or rhizomes are used as traditional chinese medicine.Water-soluble phenolic acids are one of major groups of active ingredients which responsible for the pharmacological effects of S.miltiorrhiza.Phenolic acids have many pharmacological activities such as antioxidant,antibacterial,anti-inflammatory,anti-tumor,and so on.Although the biosynthetic pathway of salvianolic acids has attracted much attention,it has not been fully elucidated,and the regulatory mechanism is still unclear.It is reported that wild S.miltiorrhiza has higher phenolic acids and stronger stress resistance than cultivated S.miltiorrhiza.Here,based on the integration of genome,comparative metabolomics and transcriptomics of wild and cultivated S.miltiorrhiza,we focused on the screen of transcription factors related to phenolic acids biosynthesis and genes related stress resistance,and used genetic manipulation technology to study the function.Our study laid a solid theoretical foundation for deeply understanding the biosynthesis and regulatory mechanism of phenolic acids,and revealed the influence of environmental factors on the formation of secondary metabolites.The main conclusions are as follows.1.Broad targeted metabolomics of S.miltiorrhiza based on UPLC-MS/MS.The metabolomics of wild and cultivated S.miltiorrhiza were determined and analyzed.A total of 658 metabolites were detected and 286 metabolites with significant difference were obtained.The results of comparative metabolomics analysis showed that there were 159 metabolites with significant difference in root comparative group.Among the differential phenolic acids,the change of salvianolic acid C was the most significant.KEGG enrichment analysis implied that the differential metabolites in the biosynthetic pathway of phenolic acids,flavonoids and phenylpropanoids were the chemical basis of the differences between wild and cultivated S.miltiorrhiza.The metabolites correlation analysis results demonstrated that the content of salvianolic acid B and C exhibited significantly positive correlated with each other,suggesting that they may share the same metabolic regulation mechanism,and salvianolic acid C may participate in the biosynthesis of phenolic acids as a precursor of salvianolic acid B.2.Transcriptome analysis of S.miltiorrhiza based on Illumina sequencingTranscriptome sequencing of wild and cultivated S.miltiorrhiza was carried out.A total of 88.64 GB clean data were obtained and 30,286 Unigenes were annotated.Comparative transcriptome analysis indicated that the function of differentially expressed genes in wild and cultivated S.miltiorrhiza was mainly related to plant metabolism,response to stimulus,plant stress resistance,and antioxidant activity,which may be the genetic basis for the differences in stress resistance,secondary metabolite biosynthesis and nutrient absorption capacity of wild and cultivated S.miltiorrhiza.The expression levels of key enzyme genes in phenolic acid biosynthetic pathway of cultivated roots were generally higher than those in wild roots,while the expression levels in leaves were lower than those in wild leaves.Expression of flavonoid biosynthetic pathway enzyme genes showed the same trend.Among the genes related to antioxidant and stress resistance,the expression of SODs showed significant difference in wild and cultivated S.miltiorrhiza,indicating the critical role in the response of stress.3.Integration analysis of transcriptomics and metabolomics of S.miltiorrhizaThrough the correlation analysis of metabolites and gene expression,21 cytochrome P450 family genes and 11 laccase coding genes related to salvianolic acid C biosynthesis were screened,including 16 MYBs,23 bHLHs,13 WRKYs,23 ERFs,10 NACs,4 TIFYs,4 heat stress transcription factors,4 trihelix,and 2 bZIP.The key enzyme genes in lignin metabolism and flavonoid biosynthesis were negatively correlated with the content of salvianolic acid C.4.Function of SmMYB13 and SmMYB70 in the regulation of salvianolic acid and flavonoid biosynthesisSmMYB13,a transcription factor related to salvianolic acid C biosynthesis,and SmMYB70,a transcription factor specifically expressed in roots,were cloned.Both SmMYB13 and SmMYB70 response to wound,dehydration,CuSO4,chilling,NaCl,PEG8000,GA,and MeJA treatment.Overexpression of SmMYB13 and SmMYB70 increased the contents of total phenolic acids and total flavonoids in S.miltiorrhiza.In the SmMYB13 overexpressed transgenic plants,the expression of SmPAL2,Sm4CL1,SmC4H,SmHPPR,SmRAS,and SmCYP98A14 in phenolic acids biosynthetic pathway were significantly up-regulated,and the expression of SmCHS1,SmCHS2,SmFLS1,and SmFLS2 in flavonoids biosynthetic pathway were significantly increased.In the SmMYB70 overexpressed transgenic plants,the expression of SmPAL2,Sm4CL1,SmC4H,SmHPPR,and SmCYP98A14 in phenolic acids biosynthetic pathway were significantly up-regulated,and SmPAL1,Sm4CL2,and SmTAT were significantly down-regulated,while SmRAS was not significantly changed.The upstream genes in flavonoids biosynthetic pathway were up-regulated and downstream genes were down regulated.These results suggested that SmMYB13 and SmMYB70 play an important role in the regulation of phenolic acids and flavonoids.5.Molecular mechanism of SmMYB13 and SmMYB70 overexpressed S.miltiorrhiza under stressStudies have shown that MYB transcription factor can affect plant response to various biotic and abiotic stresses by regulating the expression of SOD.The SmSOD gene family related to stress tolerance was systematically identified and analyzed in the genome wide.Overexpression of SmMYB13 and SmMYB70 respectively promoted the expression of most SmSODs,and the total SOD activity was elevated.Under salt stress,the MDA content in cells of transgenic lines was lower than that in non-transgenic lines.The overexpression of SmMYB13 and SmMYB70 enhanced the tolerance of S.miltiorrhiza to salt stress.In conclusion,the differential metabolites and differentially expressed genes between wild and cultivated S.miltiorrhiza were analyzed based on the integrated analysis of multiple omics.The function of two screened MYB transcription factors were studied,and the molecular mechanism of S.miltiorrhiza in response to salt stress was preliminarily clarified.This research is of great significance for comprehensively elucidating the phenolic acids biosynthetic pathway and its regulatory mechanism,and for breeding the S.miltiorrhiza with high content and strong stress resistance.