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 C₃ crop species. However, reducing g _s 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 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 C₄ 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. [ABSTRACT FROM AUTHOR]