Reduced stomatal density in wheat overexpressing EPIDERMAL PATTERNING FACTOR1 differentially affects red and blue light responses

Stomatal pores govern the trade-off between CO2 assimilation and water loss, and optimizing their performance is critical for crop resilience, particularly under dynamic field environments. Here, we show that overexpression of Triticum aestivum EPIDERMAL PATTERNING FACTOR1 (TaEPF1) in bread wheat (T. aestivum) reduces leaf stomatal density (SD) in a leaf surface-specific manner, with a greater decline on the abaxial surface than on the adaxial surface. TaEPF1 overexpressors exhibited substantially lower stomatal conductance than wild-type (WT) control plants, which resulted in diffusional constraints that limited photosynthesis when measured under monochromatic red light. However, upon partial substitution of red light with blue light, EPIDERMAL PATTERNING FACTOR1 overexpressors displayed an amplified and rapid stomatal opening response, particularly on the abaxial surface, where relative conductance increased by up to 90% versus 49% observed in the WT. Despite anatomical limitations in maximum conductance rate, this blue light sensitivity effectively compensated for the lower baseline gas exchange. The enhanced sensitivity to blue light was also concomitant with lower intercellular CO2 levels under red light. When gsw responses were normalized relative to SD, stomatal sensitivity to red light was reduced at the pore level, while blue light sensitivity increased, which was particularly evident during abaxial surface illumination. Finally, the transgenic lines maintained a 15% to 20% higher water use efficiency across light regimes. These findings show a compensatory mechanism in which genetically induced reduction in SD is offset by heightened blue light sensitivity.