Genome-Wide Identification of Binding Motifs and Drought-Responsive Target Genes by the Transcription Factor ZmNAC20 in Maize

Drought stress severely limits maize growth, and enhancing drought resistance remains a central objective of crop improvement. Plant-specific NAC (NAM/ATAF/CUC) transcription factors are critical regulators of abiotic stress responses. Previously, we identified ZmNAC20—a gene rapidly induced by both drought and abscisic acid (ABA)—as a positive regulator of stomatal closure and drought survival when over-expressed; however, its direct target genes and downstream regulatory network remained elusive. Here, we used DAP-seq to identify 3537 ZmNAC20 binding peaks genome-wide, revealing four enriched cis-motifs and 1439 target genes. GO and KEGG analyses showed pronounced enrichment in intracellular signal transduction. The intersection of RNA-seq of drought-stressed ZmNAC20-over-expressing and wild-type B104 seedlings with DAP-seq outputs defined 80 potential direct targets (48 up-, 32 down-regulated). Among the activated genes, ZmNAC20 binds and up-regulates genes of the ROS-producing enzyme ZmRBOH8, BURP domain-containing protein ZmBURP4, gibberellin (GA) catabolic enzyme ZmGA2OX6, and HD-Zip transcription factor ZmHB56. These results unveil a multi-layered drought-responsive network through which ZmNAC20 integrates hormone and ROS signaling, providing a molecular blueprint for breeding drought-resilient maize.