Abstract

Drought stress significantly constrains agricultural productivity by impairing plant growth and development. Although WRKY transcription factors have been widely studied in the context of plant stress responses, their role in orchestrating comprehensive signaling cascades under drought conditions remains incompletely understood. Here, we characterize WRKY49 as a pivotal regulator of abscisic acid (ABA)‐mediated drought adaptation in Arabidopsis. Genetic analysis revealed that the wrky49‐1  T‐DNA insertion mutant and two independent WRKY49 CRISPR‐Cas9 knockout lines exhibited reduced ABA sensitivity and enhanced drought susceptibility, whereas WRKY49 overexpression (WRKY49‐OX) transgenic lines displayed opposite responses. Complementation assays confirmed that the ABA‐insensitive and drought‐sensitive phenotypes of wrky49‐1 are attributable to the loss of WRKY49 function. Physiological assays further established WRKY49 as a positive regulator of osmotic stress tolerance. Transcriptomic profiling combined with qRT‐PCR validation demonstrated that WRKY49 directly activates the transcription of ERF55 and ABA biosynthetic genes ( ABA2 , NCED3 ) during drought stress. Protein interaction studies revealed that MPK6 physically interacts with and phosphorylates WRKY49 at Ser130 and Thr231. Notably, drought stress‐induced expression of both WRKY49 and ERF55 was significantly attenuated in mpk6‐2 mutants. Furthermore, phospho‐mimicking WRKY49 2D , but not the phospho‐dead WRKY49 2A , exhibited enhanced transcriptional activation of ERF55 and stronger binding affinity to its promoter. Our findings delineate a novel MPK6‐WRKY49‐ERF55‐ABA2 signaling module that enhances drought resilience through coordinated ABA biosynthesis, providing mechanistic insight into WRKY‐mediated stress adaptation. Identifies WRKY49 as a positive regulator of ABA signaling and drought tolerance in Arabidopsis. Reveals the MPK6‐WRKY49‐ERF55‐ABA2 signaling module, where MPK6 phosphorylates