Spatial Partitioning of Phenylpropanoid- and Auxin-Related Metabolites in Sparganium stoloniferum Tubers

Phenolic acids and flavonoids are major bioactive constituents of Sparganium stoloniferum tubers (SL), yet their spatial organization within this medicinal organ remains insufficiently characterized, limiting understanding of how anatomical structure relates to metabolite distribution. To address this, we integrated mass spectrometry imaging (MSI), non-targeted LC-MS and targeted LC-MS/MS metabolomics, and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis to characterize the spatial patterns of phenylpropanoid- and auxin-related metabolites and selected transcripts in SL. MSI and non-targeted LC-MS profiling showed that phenylpropanoid- and flavonoid-related metabolites were preferentially accumulated in the peripheral cortex rather than in the stele, although the stele constitutes the dominant internal tissue of the tuber. Targeted LC-MS/MS further confirmed that representative hydroxycinnamic acids and caffeoylquinic acid derivatives were enriched in the cortex and, for most validated metabolites, in the cell wall-enriched fraction. Notably, spatial metabolomic profiling also revealed a contrasting stele-biased distribution of indole-related metabolites, including indole-3-acetamide-related features detected by MSI and non-targeted LC-MS and indole-3-acetic acid (IAA) enrichment validated by targeted LC-MS/MS. RT-qPCR analysis showed that phenylpropanoid biosynthetic genes were generally more highly expressed in the cortex and cell wall-enriched fraction, whereas auxin-related genes showed higher expression in the stele. Together, these results show cortex-biased phenylpropanoid accumulation, preferential association of most validated phenylpropanoid-related metabolites with the cell wall-enriched fraction, and stele-associated IAA accumulation and auxin-related transcript expression. This study provides a spatial framework for understanding metabolite partitioning in medicinal aquatic storage organs and highlights the importance of integrating anatomical, metabolomic, and gene expression information in medicinal plant research.