Multiplexed LC-MS analysis reveals novel insights into grapevine defense mechanisms by expanding metabolome coverage

Comprehensive metabolome characterization in complex plant matrices remains a major analytical challenge, particularly when studying biochemical responses to disease. Here, we present a multiplexed strategy integrating biphasic extraction with three orthogonal LC-MS methods—reverse-phase (RP), hydrophilic interaction chromatography (HILIC), and lipidomics-focused RP—to maximize the coverage of the grapevine (Vitis vinifera L.) wood metabolome. This approach enabled the annotation of 1,425 unique features and unveiled a comprehensive landscape of grapevine chemical responses. Using a multi-block statistical integration framework (DIABLO), we delineated the contributions of each analytical modality and revealed distinct metabolomic and lipidomic signatures associated with fungal trunk pathogens (Phaeomoniella chlamydospora, Phaeoacremonium minimum) and a biocontrol agent (Trichoderma atroviride, strain Vintec®). Notably, lipidomic analysis uncovered a critical role for oxidized fatty acids—specifically hydroxy-eicosatetraenoic acids (13-HETE, 16(R)-HETE, and 11(R)-HETE)—as potential signaling molecules mediating plant defense. Compound classification using NPClassifier highlighted diverse biosynthetic classes, including phenylpropanoids, terpenoids, and sphingolipids, emphasizing the chemical heterogeneity of the grapevine response. This work establishes a versatile analytical pipeline for untargeted metabolomics and lipidomics in plant systems and underscores the necessity of method integration to elucidate signaling molecules in plant–pathogen interactions.