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Changes in stomatal conductance and density allow plants to acclimate to changing environmental conditions. In the present paper, the influence of atmospheric CO 2  concentration and light intensity on stomata were investigated for two barley genotypes—Barke and Bojos, differing in their sensitivity to oxidative stress and phenolic acid profiles. A novel approach for stomatal density analysis was used—a pair of convolution neural networks were developed to automatically identify and count stomata on epidermal micrographs. Stomatal density in barley was influenced by genotype, as well as by light and CO 2  conditions. Low CO 2  conditions resulted in increased stomatal density, although differences between ambient and elevated CO 2  were not significant. High light intensity increased stomatal density compared to low light intensity in both barley varieties and all CO 2  treatments. Changes in stomatal conductance were also measured alongside the accumulation of pentoses, hexoses, disaccharides, and abscisic acid detected by liquid chromatography coupled with mass spectrometry. High light increased the accumulation of all sugars and reduced abscisic acid levels. Abscisic acid was influenced by all factors—light, CO 2 , and genotype—in combination. Differences were discovered between the two barley varieties: oxidative stress sensitive Barke demonstrated higher stomatal density, but lower conductance and better water use efficiency (WUE) than oxidative stress resistant Bojos at saturating light intensity. Barke also showed greater variability between treatments in measurements of stomatal density, sugar accumulation, and abscisic levels, implying that it may be more responsive to environmental drivers influencing water relations in the plant.

Barley Genotypes Vary in Stomatal Responsiveness to Light and CO2 Conditions