Reducing stomatal density by expression of a synthetic epidermal patterning factor increases leaf intrinsic water use efficiency and reduces plant water use in a C4 crop

Enhancing crop water use efficiency (WUE) is a key target trait for climatic resilience and expanding cultivation on marginal lands. Engineering lower stomatal density to reduce stomatal conductance (g(s)) has improved WUE in multiple C-3 crop species. However, reducing g(s) in C-3 species often reduces photosynthetic carbon gain. A different response is expected in C-4 plants because they possess specialized anatomy and biochemistry which concentrates CO2 at the site of fixation. This modifies the relationship of photosynthesis (A(N)) with intracellular CO2 concentration (c(i)), such that photosynthesis is CO2 saturated and reductions in g(s) are unlikely to limit A(N). To test this hypothesis, genetic strategies were investigated to reduce stomatal density in the C-4 crop sorghum. Constitutive expression of a synthetic epidermal patterning factor (EPF) transgenic allele in sorghum led to reduced stomatal densities, reduced g(s), reduced plant water use, and avoidance of stress during a period of water deprivation. In addition, moderate reduction in stomatal density did not increase stomatal limitation to A(N). However, these positive outcomes were associated with negative pleiotropic effects on reproductive development and photosynthetic capacity. Avoiding pleiotropy by targeting expression of the transgene to specific tissues could provide a pathway to improved agronomic outcomes.