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

Lupins are promising legume crops that accumulate toxic alkaloids in the seeds, complicating their use as high-protein crops. The alkaloids 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 have been suggested as sinks. We examined the spatial localization of the alkaloids in biosynthetic organs of narrow-leafed lupin using mass spectrometry-based imaging (MSI). The alkaloids that accumulate in seeds ("core" alkaloids) were evenly distributed across tissues, however their esterified versions accumulated primarily in the epidermis. In addition, we generated a tissue-specific RNAseq dataset of biosynthetic organs using laser-capture microdissection. The dataset revealed that alkaloid biosynthetic genes are strongly expressed in the epidermis. To confirm the biosynthetic capacity of the leaf epidermis, we combined precursor feeding studies with mass spectrometry imaging, which showed that the lower epidermis is highly biosynthetic. Our work challenges the current assumptions on the precise sites of lupin alkaloid biosynthesis, with direct implications for the elucidation of the alkaloid biosynthesis pathway and the long-distance transport network from source to seed. ### Competing Interest Statement The authors have declared no competing interest.