The Study On The Accumulation And Mechanism Of Salvianolic Acids In Salvia Miltiorrhiza Hairy Roots Regulated By Plant-derived Smoke Water

Salvia miltiorrhiza Bunge belongs to the Lamiaceae plant,which is one of the traditional Chinese medicine in China.It was first recorded in Shennong’s Classic of Materia Medica.Salvianolic acids are the main active ingredients of S.miltiorrhiza.Based on the way of using water decoction in traditional Chinese medicine,phenolic acids compounds represented by salvianolic acid B（SAB）and rosmarinic acid（RA）are generally considered to be the main active substances of S.miltiorrhiza.In recent years,due to the increasing clinical demand of S.miltiorrhiza,it is very important to take effective methods to improve the synthesis and accumulation of active components of S.miltiorrhiza.Plant-derived smoke water（SW）is produced by combusting plant materials and bubbling smoke through water.Karrikinolide（KAR₁）,which is considered to be the main active ingredient in SW.Our research group has found that KAR₁could effectively promote the accumulation of salvianolic acids in S.miltiorrhiza,but the underlying mechanism is still unclear.In this study,S.miltiorrhiza hairy roots were used as experimental materials to investigate the effects of KAR₁on salvianolic acids accumulation and signal transduction mechanism by using exogenous signal molecule donors and specific inhibitors.The effects of KAR₁on differentially expressed genes,metabolites and proteins changes in salvianolic acids biosynthesis pathway were revealed by transcriptomics,metabonomics and proteomics.The main findings are as follows:（1）KAR₁treatment could significantly increase the contents of JA,H₂O₂and SAB in S.miltiorrhiza hairy roots,and SHAM and Pr Gall inhibited the generation of JA and H₂O₂and the accumulation of SAB induced by KAR₁.Application of exogenous H₂O₂could reverse the inhibition effect of KAR₁-induced SHAM and Pr Gall on SAB production.The results indicated that JA mediated the KAR₁-induced increase of SAB dependent on the generation of H₂O₂in S.miltiorrhiza hairy roots.In addition,the content of Ca²⁺–Ca M,BL,JA and SAB were significantly increased induced by KAR₁.The application of exogenous Ca Cl₂and BL could increase the level of JA,while SHAM/Pr Gall has no effect on the Ca²⁺and BL levels.The application of exogenous JAMe could reverse the total inhibitory effect of VERA,W₇,BRz and SHAM on the production of SAB induced by KAR₁,while exogenous Ca Cl₂and BL have no similar effect.All results indicated that dependent on the production of JA,Ca²⁺–Ca M and BL mediated the KAR₁-induced accumulation of SAB in S.miltiorrhiza hairy roots.（2）A total 199 differential metabolites were obtained induced by KAR₁in S.miltiorrhiza hairy roots,of which 113 metabolites were up-regulated and 86 metabolites were down-regulated.In these differential metabolites,it was found that most of the phenolic acids,organic acids,amino acids,flavonoids and carbohydrates were significantly up-regulated by treatment of KAR₁,while lipids,alkaloids and organic acids were down-regulated.The results showed that KAR₁treatment could significantly promote the phenolic acids biosynthesis in S.miltiorrhiza hairy roots.In addition,there are 59 unigenes annotated to the salvianolic acids pathway in S.miltiorrhiza hairy roots by treatment of KAR₁using transcriptomics technology,including C4H,RAS,TAT,HPPR,PAL and 4CL.We found that most of the genes related to the biosynthesis of phenolic acids were up-regulated by treatment with KAR₁,indicating that KAR₁could promote the expression of salvianolic acids biosynthesis-related genes.（3）A total 1678 differential proteins were obtained induced by KAR₁in S.miltiorrhiza hairy roots.Bioinformatics analysis indicated the differentially expressed proteins responding to KAR₁treatment mainly involved in macromolecule metabolic process,cell part,binding etc.,and most of the proteins were located at the cytoplasm and cell membrane,followed by nuclear.In addition,the proteins involved in salvianolic acids biosynthesis were up-regulated,including 4-coumarate-Co A ligase and shikimate O-Hydroxycinnamoyltransferase.The results indicated that proteins related to the biosynthesis of salvianolic acids were regulated by KAR₁in S.miltiorrhiza hairy roots.