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Rho of Plants (ROPs) are signaling small G proteins implicated in diverse signaling cascades at the interface of development and stress responses. Here, ROP signaling in stomata, was studied with a guard cell ROP activation marker and the effect of ROP inactivation was studied with null mutants in SlROP9, the single tomato homolog of Arabidopsis AtROP10/11. Analysis of guard cell ROP activation status indicated that abscisic acid (ABA) represses ROP signaling. The Slrop9 mutants display reduced stomata aperture, conductance and transpiration rates with remarkably negligible effects on photosynthesis, fruit quality and quantity, translating into higher water use efficiency. A continuous whole plant transpiration analysis revealed that the transpiration of Slrop9 mutants is specifically reduced during mid-day when water pressure deficit (VPD) values are high but not in the early morning and late afternoon hours when the VPD is lower. Intriguingly, the Slrop9 mutants are not ABA hypersensitive. However, activated SlROP9 interacts with Respiratory Burst Oxidase Homologs (RBOHs) NADPH oxidases, the slrop9 guard cells have high reactive oxygen species (ROS) levels and inhibition of RBOH activity reduced ROS levels in the Slrop9 mutants and induced stomata opening. Collectively, results in this work indicate that ABA suppression of ROPs leads to increased RBOH-dependent ROS formation and stomata closure and that SlROP9 and its homologs may be targeted for increasing crop water use efficiency.