Machine learning-enabled non-targeted metabolomics reveals nutritional and metabolic responses of Brachypodium distachyon to drought and elevated CO2.

Rising atmospheric CO2 and intensified drought are reshaping nutrient dynamics in C3 plants, with implications for ecosystem function and food security. To investigate how these stressors jointly affect nutrient homeostasis, we examined Brachypodium distachyon, a model for C3 cereal grasses, grown under ambient (400 ppm) or elevated (800 ppm) CO2, factorially combined with well-watered or drought treatments. Integrative analyses of physiology, ionomics, transcriptomics, and non-targeted metabolomics revealed that plant elemental composition and metabolomic responses to elevated CO2 strongly depend on water availability. The CO2 fertilization effect on biomass was abolished under drought, coinciding with reduced nitrogen content, altered carbon-to-nitrogen ratios, and nutrient-specific translocation changes. These shifts were partly linked to reduced stomatal conductance and transpiration but also reflected active regulation. Nitrogen status declined, accompanied by greater repression of root nitrate transporter genes than ammonium transporters and increased accumulation of the polyamine spermidine. Under combined stress, foliar iron increased alongside elevated expression of chelator synthesis genes and accumulation of S-adenosylmethionine, suggesting enhanced support for Fe homeostasis. Lipid metabolism was reprogrammed, notably via root sphingolipid accumulation, potentially contributing to ionome stabilization. Together, these findings highlight coordinated molecular and metabolic strategies governing nutrient regulation under interacting climate-related stressors.