Plants deploy chemical defense strategies to respond to abiotic and biotic stresses, including the glucosinolate-myrosinase (GM) system ("mustard oil bomb") characteristic of Brassicales. Glucosinolates (GLSs) are specialized metabolites that, upon hydrolysis by myrosinases, generate bioactive products that deter herbivores and pathogens. While the biochemical functions of GLS breakdown products are well studied, the cell-type-specific organization of the GM system and its relevance to stomatal immunity remain incompletely defined. Here, we compare cell-type-enriched GLS profiles and myrosinase activities in guard cells (GCs) and mesophyll cells (MCs) of Arabidopsis thaliana. Using untargeted metabolomics, we identified 20 GLSs across seeds, leaves, GCs, and MCs with high mass accuracy, followed by targeted quantitative analysis of GLSs based on a selected reaction monitoring approach. Comparative analyses revealed pronounced differences in GLS composition between GCs and MCs, accompanied by distinct myrosinase activity profiles. Together, these findings establish a cell-type-specific model for GLS metabolism in A. thaliana and demonstrate differential organization of the GM system between GCs and MCs. This work provides foundational evidence linking the spatial organization of GLS metabolism to stomatal immune function and will springboard future mechanistic studies of the GM system in plant defense signaling.

Plants are multicellular systems composed of highly specialized cell types, whose distinct transcriptomes, proteomes, and metabolomes collectively shape growth, development, defense, and environmental adaptation. While single-cell transcriptomics has transformed our understanding of plant cellular heterogeneity, metabolites provide a direct and integrative readout of cellular biochemical activity and physiological state and can offer valuable insights into phenotype. They provide essential information that gene expression alone does not capture. Recent advances in mass spectrometry-based single-cell metabolomics (scMet) have enabled direct measurement of metabolic variation across individual cells and subcellular domains. In parallel, mass spectrometry imaging (MSI) has emerged as a powerful approach for mapping the spatial organization of metabolites within intact plant organs, offering new opportunities to connect chemical diversity with anatomy, cell identity, and biological functions. This review summarizes current technologies for plant single-cell sampling, scMet, and MSI for spatial metabolomics. It further highlights emerging efforts to illuminate the plant’s cellular and spatial dark metabolome through spectral libraries, computational metabolomics, and AI-assisted annotation. A long-term objective is to build quantitative, spatially resolved, and biologically interpretable plant signaling and metabolic networks at single-cell resolution.