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-ß-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.

Full-Length Transcriptome Analyses of Genes Involved in Triterpenoid Saponin Biosynthesis of Psammosilene tunicoides Hairy Root Cultures With Exogenous Salicylic Acid.

Triterpenoid saponins constitute a diverse class of bioactive compounds in medicinal plants. Salicylic acid (SA) is an efficient elicitor for secondary metabolite production, but a transcriptome-wide regulatory network of SA-promoted triterpenoid saponin biosynthesis remains little understood. In the current study, we described the establishment of the hairy root culture system for Psammosilene tunicoides, a triterpenoid saponin-producing medicinal herb in China, using genetic transformation by Agrobacterium rhizogenes. Compared to controls, we found that total saponin content was dramatically increased (up to 2.49-fold) by the addition of 5 mg/L SA in hairy roots for 1 day. A combination of single-molecule real-time (SMRT) and next-generation sequencing (Illumina RNA-seq) was generated to analyze the full-length transcriptome data for P. tunicoides, as well as the transcript profiles in treated (8 and 24 h) and non-treated (0 h) groups with 5 mg/L SA in hairy roots. A total of 430,117 circular consensus sequence (CCS) reads, 16,375 unigenes and 4,678 long non-coding RNAs (lncRNAs) were obtained. The average length of unigenes (2,776 bp) was much higher in full-length transcriptome than that derived from single RNA-seq (1,457 bp). The differentially expressed genes (DEGs) were mainly enriched in the metabolic process. SA up-regulated the unigenes encoding SA-binding proteins and antioxidant enzymes in comparison with controls. Additionally, we identified 89 full-length transcripts encoding enzymes putatively involved in saponin biosynthesis. The candidate transcription factors (WRKY, NAC) and structural genes (AACT, DXS, SE, CYP72A) might be the key regulators in SA-elicited saponin accumulation. Their expression was further validated by quantitative real-time PCR (qRT-PCR). These findings preliminarily elucidate the regulatory mechanisms of SA on triterpenoid saponin biosynthesis in the transcriptomic level, laying a foundation for SA-elicited saponin augmentation in P. tunicoides.

Rorippa indica Regeneration via Somatic Embryogenesis Involving Frog Egg-like Bodies Efficiently Induced by the Synergy of Salt and Drought Stresses.

Frog egg-like bodies (FELBs), novel somatic embryogenesis (SE) structures first observed in Solanum nigrum, were induced in Rorippa indica. NaCl-mediated salt and mannitol-mimicked drought stresses induced FELBs in R. indica, which is very different from the induction by plant growth regulators (PGRs) under low light condition that was used in S. nigrum FELB induction. It demonstrated that NaCl or mannitol supplements alone could induce FELBs in R. indica, but with low induction rates, while the synergy of NaCl and mannitol significantly increased the FELB induction rates. For the combination of 5.0 g/L mannitol and 10.0 g/L NaCl the highest FELB induction rate (100%) was achieved. It suggests that the synergy of drought and salt stresses can replace PGRs to induce FELBs in R. indica. On medium supplemented with 1.0 mg/L gibberellic acid all the inoculated in vitro FELBs developed into multiple plantlets. Morphological and histological analyses confirmed the identity of FELBs induced in R. indica and revealed that FELBs originate from root cortex cells.