Genetic manipulation of stomatal patterning and function to optimise leaf gas exchange

Global food security depends on crops with enhanced productivity and resilience under increasingly variable environments to meet the growing demand. Photosynthetic carbon assimilation, a key determinant of yield, is often constrained by stomatal conductance, which governs CO₂ uptake and water loss. Under natural fluctuating light, slow stomatal responses cause lost carbon gain during increases and excess water loss during decreases, making stomatal behaviour a critical target for improving photosynthetic and water-use efficiency. This thesis investigates whether manipulating stomatal traits can alleviate diffusional limitations in strawberry (Fragaria × ananassa) and tobacco (Nicotiana tabacum). Transgenic strawberry lines were generated to overexpress STOMAGEN (increasing stomatal density), guard cell-targeted Hexokinase (reinforcing closure), and both constructs. Complementary tobacco studies were carried out in two parts: lines overexpressing STOMAGEN, ictB (enhancing carbon assimilation), and their combination; and lines with guard cell-specific Hexokinase, plasma membrane H⁺-ATPase (AHA2), and their combination to assess synergistic impacts on stomatal dynamics. STOMAGEN increased stomatal density and clustering but did not enhance conductance, with clustering impairing responses. Hexokinase had little effect in strawberry, except partially rescuing stomatal response times in clustered lines, but in tobacco accelerated stomatal responses and photosynthetic induction. ictB improved PSII efficiency, with some additional enhancement observed in combination with STOMAGEN. AHA2 reduced photosynthesis and biomass without altering conductance or kinetics. Most combinations showed no advantage over controls, with benefits being strongly environment-dependent and often failing to persist across generations. These findings reveal the trade-offs of manipulating stomatal traits across species. The thesis highlights both the opportunities and constraints of engineering stomatal development and guard cell function as a strategy to improve carbon assimilation and water-use efficiency, providing a framework for future crop optimisation.