Ion mobility mass spectrometry imaging for in situ characterisation of phytohormone changes within Pseudomonas syringae-infected Arabidopsis thaliana

Abstract Background Phytohormones are low-abundance biomolecules essential for regulating plant growth and responses to various environmental factors. Direct identification and quantification of these compounds in heterogeneous tissues, especially after wounding or pathogen infection, remain challenging due to the presence of naturally occurring isomers and structurally similar molecules. Conventional liquid chromatography-mass spectrometry (LC–MS)-based metabolomics profiling enables the detection of major stress-related hormones but does not provide spatial or temporal resolution within infected tissues. Results To enhance in situ separation and localisation of low-abundance metabolites, we applied ion mobility-assisted desorption electrospray ionisation mass spectrometry imaging (DESI‑IM‑MSI). This method was used to characterise key stress‑related phytohormones in Arabidopsis thaliana leaves inoculated with Pseudomonas syringae . DESI‑IM‑MSI revealed distinct drift ion distribution patterns reflecting the in situ abundance of targeted phytohormones and their metabolites (salicylic acid, jasmonic acid, auxin, abscisic acid and cytokinins), while minimising interference from background isomers. Importantly, changes in salicylic acid and its metabolites were consistent with parallel LC‑MS analysis, demonstrating the accuracy of the spatially resolved measurements. Conclusions DESI‑IM‑MSI provides precise spatial and temporal insights into selected biomolecules, including structurally related isomers. The method represents a promising approach for generating comprehensive hormonomic and metabolomic maps during plant responses to pathogen attack, offering an alternative to conventional LC‑MS profiling.