Dissecting specialized metabolism in space: A MALDI-MSI atlas of Amaryllidaceae alkaloids in Hippeastrum papilio (Ravenna) Van Scheepen.

Amaryllidoideae produce specific specialized metabolites called Amaryllidaceae alkaloids (AAs), known for their pharmacological potential. The spatial distribution and biosynthesis within plant tissues, however, remain poorly understood. This study investigates organ- and tissue-specific localization in Hippeastrum papilio , from precursors to galanthamine and haemanthamine, using matrix-assisted-laser-desorption/ionization mass-spectrometry-imaging (MALDI-MIS). AAs consistently accumulated in epidermal and vascular tissues; leaves showed uniform distribution across ages and positions, bulbs had higher concentrations in outer-scales and basal-plates, while roots displayed compartmentalized patterns, with galanthamine uniquely abundant in vascular bundles. Haemanthamine and galanthamine were detected in leaf and bulb mucilage, while precursors were scarce. Multivariate analyses revealed that precursors clustered separately from end-products, enriched in middle-scales and apical leaves of bulbs. Nonetheless, biosynthetic intermediates occurred in all tissues, indicating widespread AA biosynthesis. Transcript profiling confirmed differential expression of biosynthetic genes across leaves, bulbs, and roots, consistent with the widespread and multi-organ biosynthesis of AAs revealed by MSI. These findings suggest a coordinated metabolic network in H . papilio , challenging existing hypotheses on organ-specific AA biosynthesis and hinting at the transport of end products. This study refines current models of alkaloid biosynthesis and underscores the value of H. papilio as a promising resource for sustainable production of therapeutic AAs. • MALDI-MSI mapped Amaryllidaceae alkaloids in Hippeastrum papilio . • Alkaloids accumulated mainly in epidermal and vascular tissues. • Bulbs showed scale-specific and basal-plate enrichment of alkaloids. • Roots showed very strong compartmentalization of Amaryllidaceae alkaloids. • Findings challenge organ-specific biosynthesis and imply metabolite transport.