Abstract: Drought stress orchestrates a phosphorylation-dependent signaling cascade that reprograms transcriptional networks to enhance crop resilience. Through a large-scale transgenic screening, we identified ZmCRK5A, a Ca²⁺-independent calcium-dependent protein kinase (CDPK)-related kinase, as a master regulator of drought tolerance in maize. Mechanistically, ZmCRK5A directly phosphorylates the MYB transcriptional repressor ZmSMH4 (Single MYB Histone 4) at three conserved serine residues (Ser42/43/59) within its SANT domain, as demonstrated by in vitro kinase assays and site-directed mutagenesis. This post-translational modification abolishes ZmSMH4's DNA-binding capacity to ACC cis-elements, thereby de-repressing the potassium influx channel gene ZmKCH1 (K⁺ Channel 1). Functional validation revealed that ZmKCH1 overexpression confers drought resilience through optimized stomatal dynamics and water retention, whereas clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9(Cas9)-generated zmkch1 mutants display hypersensitivity to water deficit. Crucially, field evaluations demonstrated preserved grain yield alongside enhanced drought tolerance in plants with activated ZmCRK5A–ZmSMH4–ZmKCH1 signaling. Our findings delineate a kinase-transcription factor-ion channel axis that dynamically fine-tunes drought responses while maintaining productivity, providing a strategic framework for engineering stress-adapted crops without yield penalties.

Key words: drought response, maize, phosphorylation, transcription factor, ZmCRK5A, ZmSMH4