Temporal transcriptional regulatory network and key factors controlling nitrate deprivation and resupply responses in maize roots

To establish a foundation for improving maize (Zea mays L.) nitrogen efficiency, we conducted RNA-sequencing analysis of maize roots under nitrogen deprivation and resupply at eight time points. Weighted Gene Co-expression Network Analysis (WGCNA) identified 21 nitrogen response modules. KEGG and GO analyses further revealed the biological functions of these modules. Under nitrogen deprivation, various nitrogen response modules implicated in maintaining redox homeostasis, reshaping root architecture, and remodeling cell walls were temporally initiated under the signal transduction of multiple hormones. During nitrogen resupply, modules associated with carbon/nitrogen metabolism and amino acid metabolism exhibited rapid recovery. An inferred temporally organized maize nitrogen response regulatory network, constructed through Enrichr queries, suggested coordinated actions of diverse transcription factors (TFs) from multiple TF families. It also highlighted the predicted bZIP-NAC-ARF and NLP-GLK regulatory modules, identifying members of the bZIP (ZmbZIP54, ZmbZIP96, ZmbZIP113), NAC (ZmNAC32), and ARF (ZmARF10, ZmARF13) families as candidate regulators in response to nitrogen deprivation, as well as members of the NLP (ZmNLP6, ZmNLP3), GLK (ZmGLK16, ZmGLK52), and ZmEREB102 during nitrogen resupply. Notably, in vitro and transient in vivo assays, including DAP-seq and dual-luciferase reporter systems, further supported the role of ZmNLP3 as a core transcriptional activator during the rapid metabolic reset. These findings offer novel insights and candidate targets that may facilitate genetic improvement of nitrogen use efficiency in maize.