Effects of Combined Elicitors on Tanshinone Metabolic Profiling and SmCPS Expression in Salvia miltiorrhiza Hairy Root Cultures

Tanshinones are abietane-type norditerpenoid quinone natural products found in a well-known traditional Chinese medicinal herb, Salvia miltiorrhiza Bunge. The copalyl diphosphate synthase of S. miltiorrhiza (SmCPS) is the key enzyme in the first step for transformation of geranylgeranyl diphosphate (GGPP) into miltiradiene, which has recently been identified as the precursor of tanshinones. Based on previous gene-to-metabolite network, this study examined the influences of various combined elicitors on the expression of SmCPS and production of tanshinones in S. miltiorrhiza hairy root cultures. Combined elicitors were composed of three classes of elicitors, a heavy metal ion (Ag(+)), a polysaccharide (yeast extract, YE), and a plant response-signalling compound (methyl jasmonate, MJ). YE + Ag(+), Ag(+) + MJ, YE + MJ, and YE + Ag(+) + MJ were the combinations we tested. The effect of elicitors on the SmCPS expression level was detected by quantitative real-time PCR (qRT-PCR), and the tanshinones accumulation responses to elicitation were analysed by Ultra Performance Liquid Chromatography (UPLC) metabolite profiling. Of these combined elicitors, the expression of SmCPS was significantly enhanced by elicitation, especially at 24 h and 36 h. Of four tanshinones detected, the contents of cryptotanshinone and dihydrotanshinone I were enhanced by treatment with YE + Ag(+), Ag(+) + MJ, and YE + Ag(+) + MJ. Our results indicate that appropriate combined elicitors can enhance tanshinones production in hairy root cultures.