Integrative network analysis and GWAS identify TaERF-4A and TaFd-5B as key regulators of low-nitrogen tolerance in wheat

Nitrogen (N) limitation constrains wheat productivity and motivates the development of cultivars with improved nitrogen use efficiency (NUE), yet the causal genes and regulatory logic underlying low nitrogen (LN) tolerance remain insufficiently resolved in wheat. Here, we integrated controlled hydroponic seedling phenotyping, genome-wide association mapping, regulatory-network prioritization and functional genetics to dissect LN adaptation in a panel of 284 wheat (Triticum aestivum L.) accessions. Low N significantly reduced shoot biomass and plant N accumulation while increasing root biomass and root-to-shoot ratio, revealing extensive natural variation in biomass allocation and N-related plasticity. Using six relative response indices to decouple stress responsiveness from baseline vigor, GWAS identified 70 significant marker–trait associations, including pleiotropic regions affecting multiple indices. Integration of GWAS intervals with an integrative wheat gene regulatory network (wGRN) and rice N-related orthology prioritized TaERF-4A, TaHD-ZIP-5A and TaFd-5B as high-confidence candidates. Expression analyses in extreme tolerant and sensitive genotypes indicated genotype-dependent regulation, with stress-induced TaERF-4A showing stronger induction in sensitive lines, attenuated repression of TaHD-ZIP-5A in tolerant lines, and differential repression of TaFd-5B between groups. EMS stop-gained mutants validated TaERF-4A and TaFd-5B as required for maintaining biomass and N homeostasis under N deficiency. We further identified a functional T/C promoter polymorphism in TaFd-5B and developed a diagnostic dCAPS marker, with the favorable ‘C’ allele associated with improved LN performance. Together, these results define key components of the wheat LN response network and provide actionable phenotypic indices, elite germplasm, validated gene targets and a breeding-ready marker to accelerate the development of N-efficient wheat.