Spatial metabolomics based on desorption electrospray ionization-ion mobility-mass spectrometry imaging reveals distinct accumulation patterns and biosynthetic sites of diosgenin-type and pennogenin-type saponins in Paris polyphylla var. yunnanensis.

Although the spatial distribution of plant metabolites is critical for understanding their biosynthesis, transformation, transport, and accumulation, the tissue-specific organization of saponins in Paris polyphylla Smith var. yunnanensis (PPY) remains poorly understood. In this study, saponins, flavonoids, and ecdysteroids in the rhizomes, fibrous roots, stems, leaves, and calyces of PPY were analyzed using UPLC-IM-MS. Based on these results, desorption electrospray ionization-ion mobility-mass spectrometry imaging (DESI-IM-MSI) was employed to map their spatial distributions, with collision cross section (CCS) information enabling the in situ discrimination of isomeric saponins. Integration with transcriptomic analysis of key cytochrome P450 hydroxylase genes further provided insights into the biosynthesis and transformation of tissue-specific saponins. Diosgenin-type saponins were predominantly enriched in the rhizome cortex and pith and exhibited pronounced spatial redistribution during rhizome development, whereas pennogenin-type saponins were consistently localized in the epidermis and fibrous root scar regions. Transcriptomic evidence further suggested that the rhizome served as a major site of saponin biosynthesis, while their occurrence in aerial tissues likely reflected subsequent transport and local metabolic modification. Overall, this study establishes a DESI-IM-MSI-based spatial metabolomics workflow and reveals a spatial metabolic trajectory of saponins involving rhizome biosynthesis, peripheral conversion of diosgenin-type to pennogenin-type saponins, and subsequent transport and modification in other tissues.