Promoter editing of OsGTL1 improves instantaneous water use efficiency via targeted stomatal reduction in rice

KEY MESSAGE: OsGTL1 promoter modification decreases stomatal density (SD) and is inherited as a recessive trait compared to the wild type. Lower SD increases water use efficiency but lowers yield components. GT2-LIKE1 (GTL1) is a predicted drought-responsive gene in rice involved in stomatal development, yet its regulatory relationship with putative targets like Stomatal Density and Distribution 1 remains complex. Here, we employed CRISPR/Cas-based editing to alter GTL1 expression by designing a tRNA-based CRISPR to mutagenize its promoter, targeting ~ 1,500 bp 5' of the transcription start site with 7 gRNAs. Two promoter-edited lines with decreased stomatal density were selected and tested for inheritance by crossing with wild type and demonstrated to be recessive alleles, which regulated the lower expression of OsGTL1 in the developing leaves, leading to a tendency of OsSDD1 higher expression. Lower OsGTL1 expression did not significantly affect meristemoid formation, but it prevented some guard mother cells from completing symmetrical division to form mature guard cells. Lower stomatal density did not decrease the photosynthesis rate, but increased water use efficiency in the tillering stage under normal conditions and in the reproductive stage under drought stress. However, yield components in the edited lines were lower than in WT under normal conditions, but drought stress at the reproductive stage reduced yield components in the edited lines less than in WT. These findings indicate that altered OsGTL1 expression can affect stomatal development and water-use efficiency, while also revealing a trade-off between water conservation and yield components under different water regimes.