Hybrid genome and enzyme characterization uncover novel glycosyltransferases associated with benzylisoquinoline alkaloid chemodiversity in lotus

INTRODUCTION: Hybridization is a key evolutionary force that enriches genetic diversity and is associated with metabolic innovation. Lotus (Nelumbo), comprising Asian lotus (N. nucifera) and American lotus (N. lutea), is both a culturally important ornamental and medicinal plant rich in benzylisoquinoline alkaloids (BIAs). However, the genomic and biochemical basis of BIA glycoside biosynthesis remains poorly understood. OBJECTIVES: A high-quality hybrid lotus genome was presented to dissect the formation of benzylisoquinoline alkaloid glycosides and the mechanism underlying their synthesis. METHODS: The chromosomal-level hybrid genome was constructed through PacBio HiFi, ONT Ultra-long, and Hi-C sequencing data combined assembly strategies. The metabolite and enzyme function were assayed by LC-MS. Multi-omics and molecular dynamics simulations (MD) revealed the catalytic mechanism of seven tandemly duplicated UGT75C genes. RESULTS: Here, we present a high-quality haplotype-resolved genome of an Asian-American hybrid lotus (Nn × Nl CB), complemented by new assemblies of one N. lutea and four N. nucifera accessions. Comparative genomics revealed divergence time between the two species and extensive interspecific recombination in hybrids. Metabolite profiling uncovered armepavine-4'-O-glucoside, a novel BIA glycoside preferentially accumulated in cotyledons and hybrid cultivars. Weighted gene co-expression network analysis (WGCNA) and Gene Ontology (GO) analysis identified a cotyledon-specific module enriched in UDP-glycosyltransferases (UGTs). Seven tandemly duplicated UGT75C genes, derived from both parental genomes, were cloned and biochemically characterized. Functional assays demonstrated strict substrate and donor specificity, with NnUGT75C2 exhibiting the highest catalytic efficiency toward armepavine and norarmepavine. MD and MM/PBSA analyses identified key catalytic residues, and site-directed mutagenesis validated E321D as an activity-enhancing substitution. CONCLUSION: Together, these results uncover the genetic and biochemical basis of BIA glycoside biosynthesis in lotus, highlight tandem duplication and hybridization as promoters of chemodiversity, and provide molecular targets for breeding and metabolic engineering of high-value medicinal cultivars.