Cell-type–specific compartmentalization and function of the glucosinolate-myrosinase system in Arabidopsis thaliana

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.