Plant-derived Smoke Water Regulates The Accumulation And Mechanism Of Tanshinones In Hairy Roots Of Salvia Miltiorrhiza

Danshen is derived from the dried roots and rhizomes of Salvia miltiorrhiza Bunge,which was originally published in the Shennong Materia Medica.S.miltiorrhiza has the effect of promoting blood circulation,removing blood stasis,regulating menstruation and relieving pain,and is one of the most valuable Chinese medicinal materials commonly used in China.Its main active ingredients were divided into two categories,water-soluble salvianolic acids and fat-soluble tanshinones,of which tanshinones include dihydrotanshinone,cryptotanshinone,tanshinone I and tanshinone IIA.The quantity of wild S.miltiorrhiza has decreased year after year and the cultivar has been severely degraded,which led to a decline in the quality of S.miltiorrhiza.Therefore,how to improve the secondary metabolites of S.miltiorrhiza by using new technology and means has become an important content.Plant-derived smoke water（SW）refers to an aqueous solution obtained by the smoke from the plant simmering into the water,KAR₁ was considered to be the main active component in the smoke water.Research suggests that smoke water had significant effects on seed germination,seedling survival rate and crop yield,there were few studies on traditional Chinese medicine,however.Our research team found that smoke water can effectively promote the accumulation of secondary metabolites of S.miltiorrhiza,however,its mechanism is not clear.In this study,the S.miltiorrhiza hairy roots were considered as a research platform,HPLC and Metabolomics were used to study the effect of SW and KAR1 on the accumulation of tanshinones of S.miltiorrhiza,signal molecule donors,quenchers and inhibitors was used to study the effect of KAR1 on the signal molecules involved in tanshinones biosynthesis,transcriptomics was used to study the effect of SW and KAR1 on the signal transduction and molecular mechanism of the accumulation of tanshinone.It is hopeful to provide data support for revealing the influence of smoke water on the secondary metabolism of S.miltiorrhiza.The main results are as follow:（1）SW and KAR₁ treatment caused a significant improves the content of tanshinones in hairy roots.The content of tanshinone I was significantly improved（P<0.05）,and was evaluated as 1.4-,2.6-,1.2-and 1.2-fold of that in the control after treatment of SW 1:500,and 3.1-,3.3-,1.3-and 1.2-fold of that in the control after treatment of SW 1:1000 at 1,3,5and 9 d.Compared with the control,the accumulation of tanshinone I was significantly increased（P<0.05）by KAR₁ treatments at 1,3 d.The content of tanshinone IIA in the hairy roots was increased slight more by different concentration of SW and KAR₁.The content of tanshinone IIA was significantly induced by SW 1:1000（P<0.05）,reaching143.36 mg/kg on 1d post-treatment.One day after KAR1 treatment,the content of tanshinone IIA was significantly induced（P<0.05）,reaching was 117.89 mg/kg.The effect of SW on the content of cryptotanshinone increased in a concentration-dependent manner.The content of cryptotanshinone increased significantly（P<0.05）on the 5 d after KAR₁ treatment.Compared with the control,the content of dihydrotanshinone in the hairy roots increased significantly（P<0.05）on the 1 and 3 d after treatment with SW and KAR1.The results indicated that SW and KAR1 have an important effect on the accumulation of tanshinones in hairy roots,and the promotion effect of tanshinone I was particularly significant.（2）Untargeted metabolomics results show SW and KAR₁ treatment significantly changed the hairy root metabolism level.Compared with the control,SW treatment caused 93metabolites were up-regulated,of which 43 metabolites in positive ion mode and 50metabolites in negative ion mode;SW treatment caused 85 metabolites were down-regulation,of which 60 metabolites in positive ion mode and 25 metabolites in negative ion mode.Compared with the control,KAR₁ treatment caused 112 metabolites were up-regulated,of which 59 metabolites in positive ion mode and 52 metabolites in negative ion mode;KAR1treatment caused 86 metabolites were down-regulation,of which 54 metabolites in positive ion mode and 32 metabolites in negative ion mode.Further analysis found that these metabolites mostly involved in a series of metabolic pathways,such as secondary metabolite synthesis,amino acid metabolism,sugar metabolism,fatty acid metabolism,etc.The results showed that SW and KAR1 had a great effect on the metabolites of hairy root of S.miltiorrhiza.（3）KAR₁-induced the synthesis of tanshinone I in hairy roots depends on the accumulation of endogenous signaling molecules NO and JA.The contents of NO and JA in S.miltiorrhiza after KAR1 treatment changed significantly（P<0.05）.The content of NO in hairy roots increased by 30.69%and 34.03%respectively after 12 and 24 h of KAR1 treatment,and the content of JA in the hairy root increased in a time-dependent manner after KAR₁ treatment.The content of JA was evaluated as 1.41-fold of that in the control after treatment of KAR1 at12 h.PBITU and cPITO（NO specific scavenger）significantly inhibited the KAR₁-induced accumulation of JA content（P<0.05）,howere SHAM and PrGall（JA synthesis inhibitor）had no significant effect on NO content（P>0.05）.SHAM and PrGall significantly inhibited the promoting effect of SNP on tanshinone I（P<0.05）,the promoting effect of JAMe on tanshinone I in hairy roots was not inhibited by PBITU or cPITO（P>0.05）.The results showed that the synthesis of tanshinone I in hairy root of S.miltiorrhiza promoted by KAR₁depended on the accumulation of NO and JA,and NO regulated the synthesis of tanshinone I induced by KAR₁ through JA signal pathway.（4）Transcriptomics analysis showed that the hairy root transcript level changed significantly after treatment with SW and KAR1.A total of 654,959,272 raw reads were obtained by sequencing,643,853,930 clean reads were obtained after filtering,158,555Transcripts and 138,993 Unigenes were obtained,the main life activities of hairy roots were almost covered.In-depth analysis of these Unigenes revealed that SW and KAR₁ treatments caused a variety of strong reactions in the hairy roots,and annotated the expression levels of glycolytic metabolism genes,glycosyltransferase genes,terpene secondary metabolic process genes,etc.,which led to changes in the overall metabolic profile of hairy roots.The expressions of AACT,HMGs,HMGR,DXS,DXR and CMK in tanshinone synthesis pathway after SW and KAR₁ treatment was verified by qRT-PCR,which was basically consistent with the transcriptome data.The results showed that the transcription level of hairy roots changed greatly after SW and KAR1 treatment.