The aerial epidermis is a major site of quinolizidine alkaloid biosynthesis in narrow‐leafed lupin

Summary Lupins are promising protein crops that accumulate toxic quinolizidine alkaloids (QAs) in the seeds, complicating their end‐use. QAs are synthesized in green organs (leaves, stems, and pods) and a subset of them is transported to the seeds during fruit development. The exact sites of biosynthesis and accumulation remain unknown; however, mesophyll cells have been proposed as sources, and epidermal cells as sinks. We investigated the exact sites of QA biosynthesis and accumulation in biosynthetic organs of narrow‐leafed lupin (Lupinus angustifolius) using mass spectrometry‐based imaging (MSI), laser‐capture microdissection coupled to RNA‐Seq, and precursor feeding studies coupled to LC‐MS and MSI. We found that the QAs that accumulate in seeds (‘core’ QAs) were evenly distributed across tissues; however, their esterified versions accumulated primarily in the epidermis. Surprisingly, RNA‐Seq revealed strong biosynthetic gene expression in the epidermis, which was confirmed in leaves by quantitative real‐time polymerase chain reaction. Finally, feeding studies using a stably labeled precursor showed that the lower leaf epidermis is highly biosynthetic. Our results indicate that the epidermis is a major site of QA biosynthesis in narrow‐leafed lupin, challenging the current assumptions. Our work has direct implications for the elucidation of the QA biosynthesis pathway and the long‐distance transport network from source to seed.