A telomere-to-telomere genome of Dactylicapnos scandens reveals O -methyltransferase-mediated diversification of benzylisoquinoline alkaloids

Abstract Aporphine alkaloids, a bioactive subclass of benzylisoquinoline alkaloids (BIAs) in Papaveraceae, include isocorydine and corydine. However, the genomic basis and enzymatic mechanisms underlying their structural diversification remain poorly understood, mainly due to limited high-quality reference genomes for aporphine-producing species. Here, we report a telomere-to-telomere (T2T) genome assembly of Dactylicapnos scandens, a medicinal plant rich in aporphine-type BIAs. The 300.86 Mb genome was anchored to eight pseudochromosomes and shows high continuity and completeness, with a scaffold N50 of 36.91 Mb and BUSCO completeness of 99.0%. Comparative genomic analysis revealed an ancestral whole-genome duplication signal shared across Ranunculales and identified 132 candidate genes involved in BIA biosynthesis, including expanded NCS and OMT families with 29 and 23 members, respectively. Duplication analysis showed that tandem duplication is the predominant mode contributing to pathway expansion, accounting for 40.91% of total duplication events and 56.52% of OMT family expansion. Functional characterization of 23 DsOMTs revealed that several enzymes exhibit broad catalytic promiscuity across benzylisoquinoline, protoberberine, and aporphine-type substrates. DsOMT catalytic activity is influenced by SAM-binding sites, substrate positioning regions, and regulatory residues. D216 and D236 are essential for catalytic competence, whereas the 254–255 region contributes to substrate positioning and regioselectivity, and D304, N308, and Q111 modulate reaction efficiency and substrate preference. Notably, DsOMT18 and DsOMT19 methylated corytuberine to yield isocorydine and corydine. The higher C1-methylation activity of DsOMT19 may be associated with hydrogen-bond formation between the conserved H254 residue and the C1-hydroxyl group. Our findings provide valuable genomic resources for elucidating aporphine alkaloid biosynthesis and reveal evolutionary mechanisms underlying BIA structural diversification in Papaveraceae.