Metabolic and Biochemical Responses of Heirloom and Hybrid Tomato (Solanum lycopersicum) Under Flooding, Specialist, and Generalist Insect Herbivory, and their Stress Combination

This study investigated how heirloom (Cherokee Purple) and hybrid (New Girl) tomato (Solanum lycopersicum L.) plant varieties respond metabolically and biochemically to flooding and specialist (Manduca sexta) and generalist (Spodoptera exigua) insect herbivory both individually and in combination. Using LC-MS/MS based untargeted metabolite profiling of foliar tissue and solid phase microextraction and GC-MS, we characterized and quantified metabolome and aboveground headspace volatile organic compounds (VOCs) changes in response to individual and combined stressors. We additionally measured aboveground and belowground biomass in each treatment. We hypothesized that (1) flooding would dominate metabolomic reprogramming and VOC emissions; (2) specialist and generalist herbivores would elicit distinct metabolic and VOC responses; (3) single and combined stresses would produce distinct metabolite and VOC profiles; and (4) VOC emissions would differ between heirloom and hybrid tomato varieties. Flooding was the primary driver of metabolic reprogramming, VOC emission, and reduced root biomass in tomato, with accumulated metabolites linked to oxidative stress mitigation. Differences between generalist and specialist herbivory were most evident in secondary sulfur metabolites. In addition, in the heirloom tomato variety, the generalist altered many more metabolites than the specialist, a trend that was reversed in the hybrid variety. Under combined stress, flooding-induced metabolic changes were largely preserved or additive, including the accumulation of indole metabolites associated with multifunctional roles including priming plant defenses against biotic stressors, whereas herbivore-driven metabolic changes were attenuated. Monoterpenes increased under flooding and combined stress, especially in the heirloom variety. Finally, we documented varietal differences in metabolic responses reflected in increased accumulation of several metabolites in the heirloom tomato variety compared with the hybrid. Together, our results reveal that flooding, herbivory by specialist and generalist caterpillar species and their interactions can dynamically change metabolites, volatile organic compounds, and plant growth patterns and suggest that breeding history shapes plants metabolic response and resillience under multi-stress combinations.