The ADF4-RCAR12 module regulates ABA-mediated drought tolerance in Arabidopsis

Introduction Drought is one of the most severe abiotic stresses limiting crop productivity, and abscisic acid (ABA)-induced stomatal closure is a key mechanism underlying plant drought resistance. Although ABA signaling has been extensively characterized in Arabidopsis thaliana , the role and regulatory mechanisms of microfilaments (F-actin) in this process remain poorly understood. Previously, we identified the actin-binding protein CAP1 as an interactor of the ABA receptor RCAR12 and a phosphorylation target of OPEN STOMATA 1 (OST1), suggesting a potential link between actin dynamics and ABA signaling. Methods To investigate the role of Actin Depolymerizing Factor 4 (ADF4) in ABA signaling, we examined its interaction with RCAR12 using bimolecular fluorescence complementation (BiFC), GST pull-down, and co-immunoprecipitation (Co-IP) assays. Genetic interactions between ADF4 and RCAR12 were further analyzed using ADF4 -overexpressing lines in the ABA receptor quadruple mutant 1124 ( pyr1 pyl1 pyl2 pyl4 ) and the adf4 × OE- RCAR12 genotype. ABA responses and drought tolerance were evaluated through seed germination, cotyledon greening, stomatal closure, and drought assays. Results ADF4 physically interacted with RCAR12 in vivo and in vitro . Overexpression of ADF4 only weakly rescued the ABA-insensitive phenotype of the 1124 quadruple mutant during seed germination and seedling establishment, but completely restored its drought sensitivity. In addition, the adf4 × OE-RCAR12 genotype partially rescued the defects of the adf4 mutant in ABA-mediated seed germination, cotyledon greening, ABA-induced stomatal closure, and drought tolerance. Discussion These findings demonstrate that ADF4 functionally interacts with the ABA receptor RCAR12 and that the two proteins act synergistically in regulating ABA responses. Our results establish a link between actin dynamics and ABA receptor-mediated signaling and suggest that ADF4–RCAR12 coordination contributes to ABA-induced stomatal regulation and drought tolerance in Arabidopsis.