Screening and functional research of methyltransferase genes involved in the biosynthesis of BIAs in Liriodendron tulipifera L.

Liriodendron tulipifera is a rich source of structurally diverse aporphine benzylisoquinoline alkaloids (BIAs), which exhibit various pharmacological activities including antioxidant, antitumor, and anti-rheumatic effects, indicating significant medicinal potential. Nevertheless, the clinical development of these compounds is limited by their low natural abundance in planta and poor synthetic biology yields, with enzyme resources and catalytic efficiency being the primary limiting factors for the latter. Methyltransferases (MTs) are critical for determining aporphine alkaloid diversity and bioactivity but remain largely uncharacterized in L. tulipifera . Here, we integrated genomic and evolutionary analyses to reveal a whole-genome duplication event in L. tulipifera , with expanded gene families significantly enriched in secondary metabolic pathways. Phylogenetic analysis of key BIA pathway genes revealed that they share a common ancestry between Magnoliids and Ranunculales, but evolved independently in Sapindales. Based on genomic data, a total of 65 methyltransferase genes (including 58 OMTs and 7 NMTs) were identified. Through phylogenetic analysis and expression pattern profiling, eight OMT and two NMT genes were selected as candidates. Enzyme activity assays demonstrated that LtOMT37 and LtOMT44 catalyze C7 O -methylation, while LtNMT4 and LtNMT6 catalyze N -methylation of multiple BIA pathway substrates. This study reports the first characterization of 7- O -methyltransferases in the Liriodendron , laying a foundation for deciphering the aporphine BIA pathway in magnoliids and providing new genetic resources for the sustainable production of BIAs.