Proteomic responses to elevated CO2 in wheat flag leaves are largely cultivar- and year-specific

Increasing atmospheric CO 2 concentrations are expected to alter carbon and nitrogen metabolism in C 3 crops such as wheat ( Triticum aestivum ), affecting not only grain yield and quality but also source tissues involved in assimilate supply. Among these, the flag leaf plays a central role during grain filling. Here, we investigated the effects of elevated CO 2 on protein content and protein composition in flag leaves of ten winter wheat cultivars grown under ambient (≈420 ppm) and elevated (≈600 ppm) CO 2 during the 2021 and 2022 growing seasons. To establish a robust proteomic workflow, four protein extraction methods were compared, identifying filter-aided sample preparation as the most suitable, followed by combined extraction, phenol extraction, and trichloroacetic acid/acetone precipitation. An SDS concentration of ≈0.5% was optimal, whereas higher concentrations markedly reduced protein identifications. Protein content ranged from 1.8 to 3.9% of fresh weight. Three-way ANOVA showed significant cultivar effects and a significant year × CO 2 interaction, but no overall CO 2 main effect, indicating strong environmental modulation of protein responses. Quantitative proteomics confirmed this pattern, with principal component analysis separating samples primarily by year rather than CO 2 treatment or cultivar. Only 22 proteins in 2021 and six proteins in 2022 were significantly altered in more than one cultivar, demonstrating limited common responses. Across all cultivars, 267 and 222 proteins were differentially abundant under elevated CO 2 in 2021 and 2022, respectively, but no pathways were consistently enriched across years. Overall, environmental variability exerted a stronger influence on wheat flag leaf protein metabolism than elevated CO 2 alone, highlighting pronounced genotype × environment interactions.