The transcription factor ZmMYB3R targets CYCB1;2 to modulate maize drought tolerance.
The R1R2R3-MYB (3R-MYB) transcription factor subfamily is associated with stress tolerance; however, the underlying mechanisms in crops remain poorly understood. This study investigates the function of maize MYB3R in regulating seedling drought tolerance. We characterised MYB3R overexpression lines and CRISPR-Cas9 loss-of-function mutants in maize and rice using physiological assays and transcriptome profiling. DNA affinity purification sequencing (DAP-seq) and molecular interaction assays were employed to identify direct downstream targets. MYB3R overexpression enhanced drought tolerance by promoting root development, stomatal closure and antioxidant defence, whereas mutants displayed hypersensitivity. MYB3R binds the mitosis-specific activator (MSA) motif to directly transactivate the B-type cyclin gene CYCB1;2, and cycb1;2 mutants phenocopied the myb3r drought defects. These findings establish that the MYB3R-CYCB1;2 module positively regulates maize drought tolerance by coordinating developmental and physiological adaptations. This pathway provides a valuable molecular target for breeding drought-resilient crops.