Effects of smoke-water and smoke-isolated karrikinolide on tanshinones production in Salvia miltiorrhiza hairy roots

Smoke–water (SW) and the biologically-active compound karrikinolide (KAR₁), isolated from plant-derived smoke, is known to show stimulating effects on the plant growth of agricultural and horticultural crops. However, little knowledge has been obtained on their effects on the secondary metabolism of medicinal plants. In this study, hairy root cultures of Salvia miltiorrhiza, one of the most renowned herbs in traditional Chinese medicine (commonly known as “danshen”), were treated with SW at dilutions of 1:500 (v/v), 1:1000 (v/v), 1:2000 (v/v) or KAR₁ solution at 10⁻⁹ M. The effects of SW and KAR₁ treatment on the content of tanshinones (tanshinone I, tanshinone IIA, cryptotanshinone and dihydrotanshinone) and the expressions pattern of genes involved in biosynthesis of tanshinones: Acetyl CoA acyltransferase (AACT), 3-hydroxy-3-methylglutaryl CoA synthase (HMGS), 3-hydroxy-3-methylglutaryl CoA redutase (HMGR), 1-deoxy-D-xylulose5-phosphate synthase (DXS), 1-deoxy-D-xylulose5-phosphate reductoisomerase (DXR), 4-(5-pyrophosphate cytidine) -2-C-methyl-Derythritol kinase (CMK), Cobaxyl pyrophosphate synthase (CPS), isopentenyl diphosphate isomerase (IPPI) and geranylgeranyl diphosphate synthase (GGPPS) in S.miltiorrhiza hairy root during elicitation in the S.miltiorrhiza hairy roots were evaluated. The results showed that both SW and KAR₁ treatments markedly increased the content of tanshinones in hairy roots of S.miltiorrhiza. The content of tanshinone I was significantly improved, and was evaluated as 3.1-, 3.3-, 1.3- and 1.2-fold of that in the control on 1, 3, 5 and 9 d after treatment with SW 1:1000, respectively. The content of tanshinone IIA was slightly increased by SW and KAR₁. The effects of SW treatments on cryptotanshinone content were concentration-dependent. Compared to the control, the accumulation of dihydrotanshinone increased significantly (p < 0.05) with SW and KAR₁(p < 0.05) treatments in the earlier stage (day 1–3post-treatment). Enzymes of the tanshinone pathways showed different responses to the treatments of SW and KAR₁ due to their multiple roles in the secondary metabolite pathways. The transcript of HMGS was more sensitive to the SW and KAR₁ elicitation, whilst the responses of DXS, DXR, CPS and GGPPS to SW and KAR₁ treatments were later than AACT and HMGS. Our results indicate that smoke–water and KAR₁ have the potential to be used as elicitors to increase the accumulation of secondary metabolites in S.miltiorrhiza hairy roots, as well as to provide insights into the mechanisms of biosynthesis of liposoluble tanshinones in S.miltiorrhiza.