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.
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.
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