Editing cis-regulatory elements towards generating rice stomatal morphological variation for adaptation to broad and dynamic environments

Cis-regulatory element editing can generate quantitative trait variation while mitigating against extreme phenotypes and harmful pleiotropy associated with coding sequence mutations. Here, we applied a multiplexed guide RNA design approach, informed by bioinformatic datasets, to generate genotypic variation in the promoter of OsSTOMAGEN, a positive regulator of stomatal density in rice. Engineered genotypic variation corresponded to broad and continuous variation in stomatal density, ranging from 70% to 120% of wild-type stomatal density. This near-isogenic panel of stomatal variants was leveraged in physiological assays to establish discrete relationships between stomatal morphological variation and stomatal conductance, carbon assimilation, and intrinsic water use efficiency in steady-state and fluctuating light conditions. Additionally, promoter alleles were subjected to vegetative drought regimes to assay the effects of the edited alleles on developmental response to drought. Notably, the capacity for drought-responsive stomatal density reprogramming in stomagen and two cis-regulatory edited alleles was reduced. Collectively our data demonstrate that cis-regulatory element editing can generate near-isogenic trait variation that can be leveraged for establishing relationships between anatomy, physiology, and crop improvement along diverse environmental clines.