# Integrated metabolite profiling and transcriptome analysis identify candidate genes involved in diterpenoid alkaloid biosynthesis in Aconitum pendulum

## TL;DR

This study combines metabolite and gene analysis in Aconitum pendulum to identify genes involved in producing valuable diterpenoid alkaloids, which are important for Tibetan medicine.

## Contribution

The study identifies candidate genes and regulatory networks for diterpenoid alkaloid biosynthesis in Aconitum pendulum using integrated metabolomic and transcriptomic analysis.

## Key findings

- 198 alkaloids were identified, with 61 being diterpenoid alkaloids, showing tissue-specific abundance.
- 34 differentially expressed genes were identified as potential regulators of diterpenoid alkaloid synthesis.
- Co-expression analysis revealed 116 significant correlations between 30 DAs and 58 enzyme genes.

## Abstract

Aconitum pendulum is a well-known Tibetan medicine that possesses abundant diterpenoid alkaloids (DAs) with high medicinal value. However, due to the complicated structures of DAs and the associated challenges in vitro synthesis presents, plants like Aconitum pendulum remain the primary source for DAs.

Given the underutilization of the A. pendulum, a thorough metabolomic and transcriptomic analysis was conducted on its flowers, leaves, and stems to elucidate the regulatory network underlying DA biosynthesis.

Metabolomic profiling (utilizing UPLC-QQQ-MS/MS) identified 198 alkaloids, of which 61 were DAs and the relative abundance of DAs was different among different tissues. Without a reference genome, we performed de novo assembly of the transcriptome of A. pendulum. We generated 181,422 unigenes, among which 411 candidate enzyme genes related to the DA synthesis pathway were identified, including 34 differentially expressed genes (DEGs). Through joint analysis of transcriptome and metabolome data, we found a correlation between the detected metabolite levels in various tissues and the expression of related genes. Specifically, it was found that ApCYP1, ApCYP72, and ApCYP256 may be related to turupellin accumulation, while ApBAHD9, ApBAHD10, ApBAHD12 positively associated with the accumulation of aconitine. Furthermore, our study also revealed that genes involved in the diterpene skeleton synthesis pathway tend to be highly expressed in flowers, whereas genes related to DA skeleton synthesis and their subsequent modifications are more likely to be highly expressed in leaf and stem tissues. Functional analysis of gene families identified 77 BAHD acyltransferases, 12 O-methyltransferases, and 270 CYP450 enzyme genes potentially involved in the biosynthesis of DAs. The co-expression network between metabolites and related genes revealed 116 significant correlations involving 30 DAs and 58 enzyme genes.

This study provides valuable resources for in-depth research on the secondary metabolism of A. pendulum, not only deepening our understanding of the regulatory mechanisms of DA biosynthesis but also providing valuable genetic resources for subsequent genetic improvement and metabolic engineering strategies.