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

## TL;DR

Nitric oxide boosts triterpenoid production in Inonotus obliquus by activating biosynthetic and signaling pathways.

## Contribution

NO's multilevel signaling regulation of triterpenoid biosynthesis in I. obliquus is newly revealed.

## Key findings

- NO increases betulin content by 70.59% in I. obliquus.
- NO activates downstream biosynthetic genes and antioxidant pathways.
- NO signaling interacts with Ca2+ and ROS pathways to enhance triterpenoid synthesis.

## Abstract

Triterpenoids are the bioactive components in Inonotus obliquus 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. obliquus 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, IoCAMP responded strongly to the NO signal, participating in the regulation of cytoplasmic Ca2+ concentration by altering the Ca2+ concentration of mitochondria together with IoCATP and IoCALM. 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 IoNOX after NO induction confirmed the activation of the ROS signaling pathway. It works in synergy with IoSOD and IoCAT 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. obliquus.