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. The molecular basis of alternative low-alkaloid sources remains unclear. The aim of this study was to elucidate the molecular determinants underlying low-alkaloid content through a comparative analysis of the classical iucundus and alternative Bryansk backgrounds, with particular emphasis on the molecular characterization of RAP2–7. 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 lines, 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 possible role for a mutation within the promoter of key acyltransferase (LaAT). Collectively, these data extend lupin transcriptomic resources and refine models of alkaloid biosynthesis beyond the classical iucundus background, while also providing the first comprehensive molecular characterization of Bryansk low-alkaloid lines.