Abstract
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.
Highlights
Triterpenoid saponins constitute a diverse class of natural products, and their biosynthesis is responsible for the pharmacological properties of numerous traditional medicinal plants (Moses et al, 2013)
To investigate the elicitation effects of Salicylic acid (SA) on triterpenoid saponin accumulation, 35-day-old P. tunicoides hairy roots were treated with a series of concentrations (5, 10, 20 mg/L) of SA, cultivated continuously for 9 days
SA acted as an effective elicitor to positively modulate triterpenoid saponin production in P. tunicoides hairy root culture (PTHRC), and 5 mg/L SA might be the optimal concentration for further analysis
Summary
Triterpenoid saponins constitute a diverse class of natural products, and their biosynthesis is responsible for the pharmacological properties of numerous traditional medicinal plants (Moses et al, 2013). Due to the high market demand and overexploitation, the natural resources of P. tunicoides have dwindled and become endangered It has been listed in the National Key Protected Plants in China. As an alternative to wild exploitation, hairy root cultures using genetic transformation by Agrobacterium rhizogenes have been developed for sustainable production of the bioactive components (Gutierrez-Valdes et al, 2020) These culture systems were effectively used in elucidating the biosynthesis pathway of bioactive molecules, such as in Isatis indigotica (Chen et al, 2015) and Salvia miltiorrhiza (Zhou et al, 2017). This approach provides the foundation for in vitro triterpenoid saponin production by synthetic biology strategies in P. tunicoides
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