Multi-omics insights into quinolizidine alkaloid biosynthetic architecture in narrow-leafed lupin genotypes with contrasting alkaloid regulation

Quinolizidine alkaloids restrict the use of lupin seeds as food and feed. In narrow-leafed lupin, low-alkaloid content in most cultivars has been traced to the recessive iucundus locus, with the RAP2-7 transcription factor as a candidate regulator, yet the molecular basis of alternative low-alkaloid sources remains unclear. Here we provide a comparative view of alkaloid pathway and its regulation across two genetic backgrounds, the iucundus and Iucundus contrast and the Bryansk low-alkaloid background carrying the Iucundus-type RAP2-7 allele. This multi-omic framework integrates alkaloid profiling and transcriptomics alongside RAP2-7 DNA binding characterization, and sequence-level motif and variant analyses. Alkaloid profiles revealed genotype-specific differences, with Bryansk lines showing a distinct, sparteine-enriched and lupanine-depleted chemotype relative to iucundus and Iucundus. Using Iucundus line as a reference, transcriptome analyses highlighted candidate genes associated with low-alkaloid iucundus and Bryansk backgrounds, spanning enzymes, transporters and putative regulators. Consistent with a key role of RAP2-7, DAP-seq summits in Iucundus and Bryansk contained a clear AP2-like motif, whereas the iucundus background showed both strong depletion of high-confidence peaks and no defined motif. In silico modelling of RAP2-7 bound to its DNA motif, combined with DAP-seq and expression data, supported reduced binding of the iucundus variant relative to Iucundus, as well as a crucial mutation within the promoter of key acyltransferase (LaAT). Collectively, these data extend lupin transcriptomic resources and refine models of alkaloid biosynthesis beyond classical iucundus sources, and within this comparative framework, provide the first comprehensive molecular characterization of Bryansk low-alkaloid lines.