AbstractThe atmospheric pollutant ozone (O₃) is a strong oxidant that causes extracellular reactive oxygen species (ROS) formation, has significant ecological relevance, and is used here as a non‐invasive ROS inducer to study plant signalling. Previous genetic screens identified several mutants exhibiting enhanced O₃ sensitivity, but few with enhanced tolerance. We found that loss‐of‐function mutants in Arabidopsis MLO2, a gene implicated in susceptibility to powdery mildew disease, exhibit enhanced dose‐dependent tolerance to O₃ and extracellular ROS, but a normal response to intracellular ROS. This phenotype is increased in a mlo2 mlo6 mlo12 triple mutant, reminiscent of the genetic redundancy of MLO genes in powdery mildew resistance. Stomatal assays revealed that enhanced O₃ tolerance in mlo2 mutants is not caused by altered stomatal conductance. We explored modulation of the mlo2‐associated O₃ tolerance, powdery mildew resistance, and early senescence phenotypes by genetic epistasis analysis, involving mutants with known effects on ROS sensitivity or antifungal defence. Mining of publicly accessible microarray data suggests that these MLO proteins regulate accumulation of abiotic stress response transcripts, and transcript accumulation of MLO2 itself is O₃ responsive. In summary, our data reveal MLO2 as a novel negative regulator in plant ROS responses, which links biotic and abiotic stress response pathways.