Integrating Metabolomics and Transcriptomics to Unveil Atisine Biosynthesis in Aconitum gymnandrum Maxim

Abstract

Diterpene alkaloids (DAs) are characteristic compounds in Aconitum, which are classified into four skeletal types: C18, C19, C20, and bisditerpenoid alkaloids. C20-DAs are thought to be the precursor of the other types. Their biosynthetic pathway, however, is largely unclear. Herein, we combine metabolomics and transcriptomics to unveil the methyl jasmonate (MJ) inducible biosynthesis of DAs in the sterile seedling of A. gymnandrum, the only species in the Subgenus Gymnaconitum (Stapf) Rapaics. Target metabolomics based on root and aerial portions identified 51 C19-DAs and 15 C20-DAs, with 40 inducible compounds. The highest content of C20-DA atisine was selected for further network analysis. PacBio Isoform sequencing integrated with RNA sequencing not only provided the full-length transcriptome but also their response to induction, revealing 1994 genes that exhibited up-regulated expression. Further, 38 genes involved in terpenoid biosynthesis were identified, including 7 diterpene synthases. In addition to the expected function of the four diterpene synthases, AgCPS5 was identified to be a new ent-8,13-CPP synthase in Aconitum and could also combine with AgKSL1 to form the C20-DAs precursor ent-atiserene. Combined with multiple network analyses, six CYP450 and seven 2-ODD genes predicted to be involved in the biosynthesis of atisine were also identified. This study not only sheds light on diterpene synthase evolution in Aconitum but also provides a rich dataset of full-length transcriptomes, systemic metabolomes, and gene expression profiles, setting the groundwork for further investigation of the C20-DAs biosynthesis pathway.

Overview of the chemistry and biological activities of natural atisine-type diterpenoid alkaloids

“…15,16 Briey, the formation of geranylgeranyl pyrophosphate (GGPP) from three IPP units is catalyzed by geranylgeranyl pyrophosphate synthase (GGPPS), and GGPP is further catalyzed to form ent-copalyl diphosphate (ent-CPP). Ent-CPP is the only precursor in the biosynthesis of all types of DAs, 17,18 and it is further cyclized to form ent-kaurane and ent-atisane, which are aminated to obtain the corresponding DAs. The pathways for the formation of DAs from the amination of other diterpenoids lack the support of intermediates found in nature, but there are more reports of ent-atisane-type diterpenoids.…”

“…3). The rst class of compounds includes six compounds (15)(16)(17)(18)(19)(20) that feature an N-C-20-O-C-7 group. 32,33 Among these compounds, compound 19 possesses a rare OMeBu substituent and compound 20 is the only atisine-type DA possessing an OBz substituent.…”