A Universal Untargeted Modification Metabolomics Pipeline for Profiling Plant Metabolite Modifications: Application to Wheat-Fusarium Head Blight Interactions

Modification of metabolites after synthesis, such as glycosylation, acylation, and glutathionylation, plays critical roles in regulating their stability, activity, and signaling during plant stress responses. However, systematic profiling of these modified metabolites remains technically challenging due to the lack of high-throughput analytical pipelines. Here, we established a novel untargeted modification metabolomics workflow integrating UPLC-QE-MS with neutral loss and diagnostic ion scanning, enabling simultaneous profiling of over 20 modification types from complex plant extracts without prior suspect lists. The pipeline was validated through rigorous quality control (QC sample correlations R > 0.99) and applied to a time-course study of Fusarium head blight (FHB)-resistant (Wangshuibai) and susceptible (Fielder) wheat lines. Approximately 19% of detected metabolites were identified as modified forms, with glycosylation, acylation, and glutathionylation being the most predominant. Comparative analysis revealed distinct temporal modification dynamics between genotypes, and identified indole-3-acetyl-beta-1-D-glucoside (IAA-Glc) as a resistance-associated modified metabolite. This workflow provides a robust, broadly applicable tool for exploring the “modificome” in plant–pathogen interactions and offers new targets for resistance breeding through modification-oriented metabolic engineering.