Fritillaria unibracteata var. wabuensis is an important resource plant for the famous traditional Chinese medicine Fritillariae cirrhosae bulbus (Chuanbeimu in Chinese). F. cirrhosae bulbus is the dried bulbs of several species from Fritillaria genus, with isosteroidal alkaloids components assumed as the bioactive ingredients. However, the biosynthesis pathway of isosteroidal alkaloids remains elusive. Here, we adopted F. unibracteata var. wabuensis as a material to identify genes involved in the biosynthesis of isosteroidal alkaloids. We first constructed the multi-tissue metabolome and transcriptome dataset of F. unibracteata var. wabuensis. Interestingly, imperialine-3-β-d-glucoside, an isosteroidal glycoalkaloid, was found to be the major tissue-specific accumulated alkaloid. Through phylogenetic and co-expression analysis, we identified two UDP-glucosyltransferases from UGT73 family catalyzing 3-O-glucosylation of isosteroidal and steroidal alkaloids: imperialine 3-O-glucosyltransferase (FuwI3GT) can use both isosteroidal alkaloid imperialine and steroidal alkaloid solanidine as substrates, while solanidine 3-O-glucosyltransferase (FuwS3GT) can only use steroidal alkaloid solanidine as a substrate. We further approved that the W201 residue of FuwI3GT determined its substrate preference of isosteroidal alkaloids. Overall, our results identified enzymes involved in 3-O-glucosylation of isosteroidal and steroidal alkaloids in F. unibracteata var. wabuensis and paved the way to fully elucidate the isosteroidal alkaloid biosynthesis pathway in Fritillaria species. © 2025 Society for Experimental Biology and John Wiley & Sons Ltd.
