Differential responses of stomatal and leaf anatomical traits explain wheat cultivar sensitivity to ozone

Abstract Tropospheric ozone (O3) is a major air pollutant that threatens wheat production. However, the mechanisms underlying cultivar-specific responses remain unclear, particularly how stomatal traits and leaf structural characteristics contribute to differences in O3 sensitivity among cultivars. A field experiment was conducted using 13 winter wheat (Triticum aestivum L.) cultivars exposed to ambient (A-O3) or elevated (E-O3; 1.5 times A-O3) O3 treatment in a Free-Air O3–Concentration Enrichment system (O3–FACE). Elevated O3 significantly increased stomatal density on both adaxial and abaxial leaf surfaces, with a stronger response on the adaxial side, indicating independent regulation between surfaces. At the anthesis stage, stomatal and pore width significantly increased under E-O3, whereas their length was not affected. Elevated O3 significantly reduced adaxial epidermal thickness, abaxial epidermal thickness, mesophyll tissue thickness, and total leaf thickness during grain filling, with significant O3 × cultivar interactions for all traits except abaxial epidermal thickness. Cultivars were classified into sensitive, intermediate, and tolerant groups. Sensitive cultivars exhibited significant reductions in leaf structure, whereas tolerant cultivars largely maintained or even enhanced structural integrity. Overall, cultivar sensitivity to O3 was not directly associated with stomatal traits but rather with the ability to preserve leaf anatomy. These findings suggest that maintaining flag leaf structure is critical for improving wheat resilience to rising O3 levels.