High-throughput stomatal phenotyping provides selection targets for stress-resilient wheat

Phenotyping stomatal traits and their developmental plasticity is time-consuming but holds potential to improve water use efficiency and photosynthesis for designing stress-tolerant crops under climate change. Here, we develop a robust, high-throughput pipeline for phenotyping 14 stomatal traits in winter wheat related to size, variation, maximum conductance, and spatial patterning. We (1) analyze over 25,000 images from 60 wheat cultivars grown in growth chamber, greenhouse, and field conditions; (2) investigate the impact of light, temperature, and reduced water and nitrogen supply on stomatal traits and their developmental plasticity across adaxial and abaxial surfaces; and (3) evaluate genetic diversity and breeding progress of stomatal traits. Stomatal traits were highly broad-sense heritable, were largely plastic in response to environmental conditions, and showed genotype-specific responses. Stomatal traits of third leaves under controlled environments with stable light and temperature conditions reliably captured the genetic variance of flag leaves under field conditions. Our data suggests that the upper leaf surface contributed more to transpiration and cooling through consistently higher stomatal density, area, and maximum conductance, while the lower surface facilitated CO₂ diffusion via systematic proper patterning and spacing. Breeding maintains the genetic diversity of stomatal traits, and our pipeline facilitates breeders to target them to enhance water use efficiency in high-yielding modern cultivars.