Difference In Tanshinone And Phenolic Acid Metabolism Between Salvia Miltiorrhiza And Salvia Castanea Diels F.tomentosa

Salvia miltiorrhiza Bunge(S.miltiorrhiza)is a medicinal model plant that has been widely used to treat cardiovascular diseases.Salvia castanea Diels f.tomentosa Stib(S.castanea)is used as a substitute product because of its similar medicinal activities to S.miltiorrhiza.The two species accumulate different levels of natural products,such as tanshinones and phenolic acids.However,the mechanism of diverse specialized metabolism remains unknown.We investigated and compared specialized metabolism between S.miltiorrhiza and S.castanea hairy roots under normal culture and different induction conditions,which revealed diverse accumulation of specialized metabolites and their responses to biotic stress,abiotic stress and signal molecule.This provides a foundation for studying diverse specialized metabolism between S.miltiorrhiza and S.castanea,along with beneficial reference for plant tissue engineering.Moreover,we compared the transcript levels of key genes involved in different pathways and screened some candidate genes involved in specialized metabolism,which provides foundations for functional characterization of these genes.1.HPLC analysis of specialized metabolites in S.miltiorrhiza and S.castanea hairy roots under normal culture condition showed that S.castanea produced higher levels of cryptotanshinone(CT),tanshinone IIA(T-IIA),caffeic acid(CA),and rosmarinic acid(RA),but lower dihydrotanshinone I(DT-I)and salvianolic acid B(SAB)levels than S.miltiorrhiza.2.Lactalbumin hydrolysate(LH)was applied to induce S.miltiorrhiza and S.castanea hairy root cultures.We optimized the concentration of LH,and then performed a time gradient experiment.Concentration screening of LH showed that 1 g/L could be regarded as an optimum concentration of LH,which benefitted both growth status and tanshinone accumulation in hairy roots of both species.The time gradient experiment showed that 1 g/L LH positively influenced tanshinone accumulation for both species,but had less effect on phenolic acid accumulation.LH with 1 g/L contributed more to tanshinone biosynthesis in S.castanea than that in S.miltiorrhiza.Moreover,the DT-I,CT,tanshinone I(T-I),and T-IIA levels in S.castanea peaked at day 9,and they were 2.00-,3.55-,3.91-,and 1.24-fold of the control levels,respectively.LH induced more persistent upregulation of HMGR,DXR,GGPPS,and CYP76AH1 in S.castanea than those in S.miltiorrhiza,and potentially contributed to more sensitive metabolic responses of tanshinones in S.castanea than those in S.miltiorrhiza.3.To elucidate diverse responses to biotic and abiotic elicitors between S.miltiorrhiza and S.castanea,yeast extract(YE),Ag+,and methyl jasmonate(MJ)were used to induce hairy root cultures of both species.The metabolic profiles indicated that YE stimulated the production of DT-I,CT,CA,and RA in S.miltiorrhiza,while siginificantly enhanced accumulations of DT-I,CT,T-I,T-IIA,and CA in S.castanea.Ag+ increased the CT,T-I,T-IIA,CA,and RA levels in S.miltiorrhiza at specific time,while enhanced the CT,T-IIA,RA,and SAB levels in S.castanea.MJ dramatically enhanced accumulations of DT-I,CT,T-IIA,CA,RA,and SAB in S.castanea,while only changed the production of DT-I,CT,CA,and SAB in S.miltiorrhiza.Gene expression analysis revealed that gene responses to elicitors potentially led to different metabolic responses of tanshinones and phenolic acids between the two species.4.The transcript levels of key genes in S.miltiorrhiza and S.castanea hairy roots were investigated to screen candidate genes involved in specialized metabolism.Ten genes involved in terpenoid biosynthesis were highly exressed in S.castanea,while 31 genes were highly expressed in S.miltiorrhiza.Six genes involved in phenolic acid biosynthesis were highly expressed in S.castanea,while 11 genes were highly expressed in S.miltiorrhiza.Twelve genes encoding cytochromes P450 were highly expressed in S.castanea,while another 12 genes were highly expressed in S.miltiorrhiza.Three genes encoding transcription factors were highly expressed in S.castanea,while one transcription factor gene was highly expressed in S.miltiorrhiza.5.The tissue-specific expression analysis indicated that 7 genes were highly expressed in roots,including CYP71AT90,CYP94C55,CYP82V2,CYP749A39,CYP721A38,CYP76AK3,and ORA1.However,CYP736A123 and CYP716A89 were highly expressed in leaves.Moreover,plant expression vectors of Sm CYP749A39,SmCYP71AT90 and SmWRKY61 were constructed by Gateway technology.