ZmbHLH81 Enhances Maize Drought Tolerance via Direct Transcriptional Activation of ABA Signaling and ROS Scavenging Genes

Drought severely limits maize production. Basic helix-loop-helix (bHLH) transcription factors act as key regulators of plant drought responses; however, the precise regulatory networks they coordinate in maize remain largely unclear. Here, we functionally characterized ZmbHLH81, a drought- and abscisic acid (ABA)-responsive bHLH transcription factor in maize. Subcellular localization confirmed that ZmbHLH81 is a nuclear protein. Overexpression of ZmbHLH81 in Arabidopsis enhanced drought tolerance, whereas CRISPR/Cas9-mediated targeted mutagenesis in maize significantly increased plant sensitivity to drought stress. Physiologically, these mutant lines exhibited accelerated water loss, delayed stomatal closure, compromised antioxidant enzyme activities and elevated malondialdehyde (MDA) accumulation under drought stress. DAP-seq analysis demonstrated that ZmbHLH81 specifically recognizes the conserved G-box motif (CACGTG). Furthermore, integrating DAP-seq and transcriptomic data successfully identified the key downstream targets governed by ZmbHLH81. Molecular assays confirmed that ZmbHLH81 directly targets and transactivates the core ABA signaling kinase gene ZmSnRK2.9 and stress-responsive transcription factor genes ZmNAC20 and ZmHDZ4. Taken together, ZmbHLH81 positively regulates maize drought tolerance by directly activating a specific regulatory module that orchestrates ABA-mediated stomatal closure and reactive oxygen species (ROS) scavenging, providing a promising genetic target for breeding climate-resilient crops.