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 <i>ZmRBOH8</i>, BURP domain-containing protein <i>ZmBURP4</i>, gibberellin (GA) catabolic enzyme <i>ZmGA2OX6</i>, and HD-Zip transcription factor <i>ZmHB56</i>. 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.