Reduced mesophyll conductance under chronic O₃ exposure in poplar reflects thicker cell walls and increased subcellular diffusion pathway lengths according to the anatomical model

Ozone (O₃) is one of the most harmful and widespread air pollutants, affecting crop yield and plant health worldwide. There is evidence that O₃ reduces the major limiting factor of photosynthesis, namely CO₂ mesophyll conductance (g_m), but there is little quantitative information of O₃-caused changes in key leaf anatomical traits and their impact on g_m. We exposed two O₃-responsive clones of the economically important tree species Populus × canadensis Moench to 120 ppb O₃ for 21 days. An anatomical diffusion model within the leaf was used to analyse the entire CO₂ diffusion pathway from substomatal cavities to carboxylation sites and determine the importance of each structural and subcellular component as a limiting factor. g_m decreased substantially under O₃ and was found to be the most important limitation of photosynthesis. This decrease was mostly driven by an increased cell wall thickness and length of subcellular diffusion pathway caused by altered interchloroplast spacing and chloroplast positioning. By contrast, the prominent leaf integrative trait leaf dry mass per area was neither affected nor related to g_m under O₃. The observed relationship between g_m and anatomy, however, was clone-dependent, suggesting that mechanisms regulating g_m may differ considerably between closely related plant lines. Our results confirm the need for further studies on factors constraining g_m under stress conditions.