Key message <p>Ozone stress reconfigures wheat metabolism by downregulating glycolysis and the TCA cycle while channelizing the pentose phosphate pathway and amino acid biosynthesis to enhance secondary metabolite synthesis and oxidative stress resilience.</p> Abstract <p>This study offers a comprehensive analysis of metabolic pathway reconfigurations in the ozone-sensitive wheat cultivar PBW-550 during the milking stage under elevated ozone (O₃) stress. Utilizing UHPLC-HRAMS, we observed a significant shift in primary metabolic pathways, with glycolysis and the tricarboxylic acid (TCA) cycle downregulated, while alternative pathways such as the pentose phosphate pathway (PPP) and amino acid biosynthesis were upregulated. This metabolic shift facilitated enhanced production of secondary metabolites, particularly through the phenylpropanoid pathway, which plays a crucial role in oxidative stress defense. Key enzymes, including phenylalanine ammonia-lyase (PAL), were significantly upregulated, driving the synthesis of phenolic compounds and flavonoids that strengthen stress resilience. In addition, resource reallocation led to increased levels of amino acids, purines, and unsaturated fatty acids, further diverting the carbon pool toward secondary metabolite production. This adaptive strategy highlights the plant’s prioritization of repair and defense mechanisms under O₃ stress. Our findings underscore wheat’s metabolic plasticity in response to ozone, providing valuable insights for developing strategies to enhance crop resilience in ozone-affected environments.</p> Graphical abstract <p></p>