Manipulating stomatal density in the C4 crop Zea mays; in a background of low photorespiration

As climate change intensifies, there is a growing demand for crops that require less water and can withstand the increasing frequency and intensity of droughts. One possible solution involves engineering the number of stomata that develop on the leaf epidermis to limit plant water loss. By manipulating the expression of the Epidermal Patterning Factor (EPF) gene family, which regulate stomatal development, water-use-efficiency and drought resistance has been improved in several C3 species. However, this is often accompanied by a reduction in photosynthetic carbon assimilation since stomata also facilitate the uptake of CO2. If we are to achieve global food security, it is paramount that improvements in crop resilience do not come at a cost to grain yield. Using several Arabidopsis thaliana and Oryzae sativa L. (rice) EPF mutants with a range of stomatal densities (SD), this work revealed that a significant negative relationship exists between stomatal conductance and photorespiration. When assessed under low photorespiratory conditions, mutants with fewer stomata no longer displayed significantly reduced carbon assimilation rates. This suggests that the photosynthetic penalty incurred by reducing stomatal density in C3 species can partly be explaining by their enhanced rates of Rubisco oxygenation. Furthermore, this work found that low-SD mutants had increased photoprotection under normal conditions, and the responses of these mechanisms to drought stress were explored. The remainder of this thesis focussed on manipulating SD in Zea mays L. (maize); a crop species that utilises C4 photosynthesis and thus circumvents photorespiration. Three ZmEPFL9 paralogous genes were first identified, with one found to be the primary positive regulator of maize stomatal development. Using CRISPR/Cas9, gene-edited maize plants were generated with around 60% fewer stomata, by editing ZmEPFL9-1a, which led to a significant reduction in stomatal conductance and water consumption under well-watered conditions. These plants were able to conserve more soil water when water was restricted, allowing at least one additional day of gas exchange. Unlike the C3 EPF mutants analysed, most low-SD maize lines had similar photosynthetic rates to control plants under saturating light and showed no increase in photorespiration or deleterious impact on yield. Together, this work suggests that stomatal engineering may be a viable option for improving drought resistance in maize, without compromising on carbon fixation.