Chitosan elicitation of saponin accumulation in Psammosilene tunicoides hairy roots by modulating antioxidant activity, nitric oxide production and differential gene expression.

Elicitation is one of the most effective strategies for enhancing plant bioactive compounds, such as triterpenoid saponins. Chitosan gained worldwide attention for biostimulant activity, but little is known about its roles in the elicitation of triterpenoid saponin in medicinal plants. Here, we explored the regulatory network of chitosan on saponin accumulation in hairy root cultures of Psammosilene tunicoides, a valuable medicinal herb known for its pain-relieving properties endemic to China. Compared with control, the highest total saponin accumulation exhibited a 4.55-fold enhancement in hairy roots elicited by 200 mg L-1 chitosan for nine days. High-performance liquid chromatography (HPLC) revealed the yields of quillaic acid, gypsogenin and gypsogenin-3-O-β-D-glucuronopyranoside were significantly increased after chitosan treatments. Moreover, exogenous chitosan application dramatically triggered the reactive oxygen species (ROS) scavenging enzyme activities and nitric oxide (NO) content in hairy roots. Comparative transcriptome analysis from chitosan-treated (1 and 9 d) or control groups revealed that differentially expressed genes (DEGs) were greatly enriched in plant-pathogen interaction and metabolic processes. The transcriptions of candidate DEGs involved in chitosan-elicited saponin metabolism were increased, especially genes encoding antioxidant enzymes (SOD, POD and GR), stress-responsive transcription factors (WRKYs and NACs) and terpenoid biosynthetic enzymes (DXS, GPPS and SE). Taken together, these results indicate that chitosan elicitor promotes triterpenoid saponin biosynthesis by enhancing antioxidant activities, NO production and differential gene expression in P. tunicoides hairy roots.