Steroid alkaloids are the most important active substance in medicinal plant V. nigrum, which have many pharmacological effects such as anti-hypertension, anti-tumor and anti-insect. At present, cell suspension culture is an effective method to produce secondary metabolites, but the yield of secondary metabolites cultured in ordinary cell suspension is not high. Elicitation can overcome the limitation of low metabolite yield in plant cell culture platforms in vitro, and it is an effective method to improve productivity. The purpose of this study is to explore the best elicitor combination and apply it to cell suspension of V. nigrum to improve the yield of steroidal alkaloids. In this study, several abiotic elicitors (MeJA, SA, Co 2+, Cu 2+) and a biological elicitor yeast extract (YE) were used to treat V. nigrum callus. Among various treatments applied, MeJA (100 uM concentration) improved the highest in the accumulation of two main steroid alkaloids in V. nigrum veratramine and cyclopmine. The content of veratramine has increased to 0.6689 ± 0.089 mg/g DW as compared to the content of control 0.1495 ± 0.047 mg/g DW, and the content of cyclopmine has increased to 0.3869 ± 0.040 mg/g DW as compared to the content of control 0.3032 ± 0.031 mg/g DW. The addition of YE (200 mg/L) can effectively improve biomass accumulation (Fresh weight (FW): 1.8035 ± 0.079 g) as compared to the content of control (FW: 1.1565 ± 0.081 g). Subsequently, these two elicitors were applied to suspension cell culture, and the effects of MeJA concentration, YE concentration, inoculation amount and MeJA induction time on the total contents of veratramine and cyclopmine produced by cell suspension were investigated. The experimental results show that the total yield of veratramine and cyclopmine can be increased from the initial 0.0338 ± 0.002 mg to 0.0638 ± 0.004 mg with the addition of elicitors. Through the optimization of response surface experiment and combined with the actual experimental conditions, the final optimal suspension cell culture conditions were determined as follows: MeJA concentration: 120 um/L; YE concentration: 450 mg/L; Inoculation amount: 0.45 g; MeJA induction time: the 18th day. Finally, the total yield of veratramine and cyclopmine reached 0.0827 ± 0.003 mg under this culture condition. It is possible to scale the current strategy to a bioreactor for higher productivity of steroid alkaloids of interest for various pharmaceutical industries.

Tartary buckwheat ( Fagopyrum tataricum) is rich in flavonoids, which not only play an important role in plant-environment interaction, but are also beneficial to human health. Rutin is a therapeutic flavonol which is massively accumulated in Tartary buckwheat. It has been demonstrated that transcription factors (TFs) control rutin biosynthesis. However, the transcriptional regulatory network of rutin is not fully clear. In this study, through transcriptome and target metabolomics profiling analysis together with homolog prediction, we identified and validated the role of FtMYB102 and FtbHLH4 TFs at the different developmental stages of Tartary buckwheat. The elevated accumulation of rutin in the sprout appears to be closely associated with the expression of FtMYB102 and FtHLH4. Yeast two-hybrid, transient luciferase activity and co-immunoprecipitation analysis demonstrated that FtMYB102 and FtbHLH4 can interact and form a transcriptional complex. Moreover, yeast one-hybrid showed that both FtMYB102 and FtbHLH4 directly bind to the promoter of chalcone isomerase ( CHI), and they can coordinately induce CHI expression as shown by transient luciferase activity assay. Finally, we transferred the FtMYB102 and FtbHLH4 into the hairy roots of Tartary buckwheat and found that they both can promote the accumulation of rutin. Our results indicate that FtMYB102 and FtbHLH4 can form a transcriptional complex by inducing CHI expression to coordinately promote the accumulation of rutin.