NO Activates the Triterpenoid Biosynthetic Pathway in Inonotus obliquus Through Multilevel Signaling Regulation to Enhance Its Production

Triterpenoids are the bioactive components in <i>Inonotus obliquus</i> with extensive medicinal prospects, but their low content in fermentation production is the main limiting factor for their application. This study focuses on nitric oxide (NO), an important signaling molecule within organisms, aiming to explore its inducing effect on the synthesis of triterpenes in <i>I. obliquus</i> and the potential signaling transduction mechanisms involved. Compared with the control group, the content of representative triterpenoid betulin increased by 70.59% after adding the NO donor sodium nitroprusside. Gene expression level detection revealed that NO mainly promotes its biosynthesis by activating the transcription of key enzyme genes in the downstream pathway of betulin biosynthesis, thereby increasing its abundance. Tracing upstream, the NO signal was found to induce the upregulation of genes related to cellular antioxidant and calcium ion signaling pathways. Notably, <i>IoCAMP</i> responded strongly to the NO signal, participating in the regulation of cytoplasmic Ca²⁺ concentration by altering the Ca²⁺ concentration of mitochondria together with <i>IoCATP</i> and <i>IoCALM.</i> Additionally, the signaling of changes in Ca²⁺ concentrations is likely to crosstalk with the reactive oxygen species (ROS) signaling pathway. The increase in enzyme activity of <i>IoNOX</i> after NO induction confirmed the activation of the ROS signaling pathway. It works in synergy with <i>IoSOD</i> and <i>IoCAT</i> to reduce oxidative damage and promote downstream triterpenoid biosynthesis. This study not only contributes to clarify the signaling pathways regulating NO-mediated triterpenoid biosynthesis but also provides a theoretical basis for the efficient production of triterpenoid active components in <i>I. obliquus</i>.