Stomata regulate the plant’s gas exchange and water balance, and their density may be a crucial factor in the response to abiotic stresses. The aim of this study was to investigate the response of leaf gas exchange of three spring wheat genotypes with different stomatal density to progressive drought and combined heat and drought stress. The stomatal conductance (gs) was the most sensitive parameter that declined with increasing drought stress. This negatively affected transpiration and leaf cooling, and limited photosynthesis (A) when gs decreased to < 550 mmol m−2 s−1. The treatments affected all three genotypes similarly over time irrespective of stomatal density. However, when related to the fraction of transpirable soil water (FTSW) in the pot, gs and A of the low and high stomatal density cultivars responded differently when heat was added to the drought stress. The high stomatal density cultivar showed no difference in maximum gs at FTSW > 0.3, and a similar decline of gs and A at FTSW < 0.3 in drought alone and combined drought and heat. The low stomatal density cultivar showed a higher maximum gs and the most severe decline of gs under combined heat and drought stress and a significantly slower decline of gs under drought alone, which was also reflected in a significantly slower reduction in A under drought compared to the combined stress. Overall, the drought response of stomatal closure dominated the physiological response under simultaneous heat and drought irrespective of stomatal density, and it was only in the combined stress that the maximum photochemical efficiency Fv/Fm was negatively affected. In conclusion, to elucidate the effect of drought and combined drought and heat on the leaf gas exchange in wheat cultivars with varied stomatal density, it is crucial to relate the parameters to the available soil water, not the duration of the drought.
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