Reducing stomatal density by expression of a synthetic EPF 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. Reducing stomatal conductance (gs) through manipulating stomatal density has been observed to translate to improved WUE in multiple C3 crop species. However, reducing gs in C3 species often reduces photosynthetic carbon gain. A different response is expected in C4 plants because they possess specialized anatomy and biochemistry which concentrates CO2 at the site of fixation. This modifies the photosynthesis (AN) relationship with intracellular CO2 concentration (ci) so that photosynthesis is CO2-saturated and reductions in gs are unlikely to impair AN. To test this hypothesis, genetic strategies were investigated to reduce stomatal density in the C4 crop sorghum. Constitutive expression of a synthetic epidermal patterning factor (EPF) transgenic allele in sorghum, lead to reduced stomatal densities. A moderate reduction in stomatal density did not strengthen stomatal limitation to AN, improved WUE, reduced water use, and avoided loss of carbon fixation during a period of water deprivation. 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 provides a potential pathway to optimal agronomic outcomes.