Distinct target genes are modulated by microRNA members and affect various biological processes associated with abiotic stress responses in plants. In this study, we characterized a functional module comprising miRNA/target and a downstream MYB transcription factor partner, Tae‐MIR1118/TaCaM2/TaMYB44, in Triticum aestivum to mediate the plant low‐nitrogen (N) stress response. Dual luciferase (LUC) assay and expression analysis indicated that TaCaM2 is regulated by Tae‐MIR1118 through a posttranscriptional cleavage mechanism. Reporter LUC activity in N. benthamiana leaves co‐transformed with effector CaMV35S::Tae‐MIR1118 and reporter TaCaM2::LUC was significantly reduced, and the transcripts of Tae‐MIR1118 and TaCaM2 in tissues exhibited converse expression patterns under varying N levels. Specifically, the transcripts of Tae‐MIR1118 decreased, whereas those of TaCaM2 increased under low‐N stress in a temporal‐dependent manner. Yeast two‐hybrid, bimolecular fluorescence complementation (BiFC) and co‐immunoprecipitation (Co‐IP) assays indicated that TaCaM2 interacted with the MYB transcription factor TaMYB44. Transgene analysis revealed the negative roles of Tae‐MIR1118 and the positive functions of TaCaM2 and TaMYB44 in regulating plants for low‐N stress adaptation by modulating glutamine synthetase activity, N uptake capacity, and root morphology. Yeast one‐hybrid, transcriptional activation, and chromatin immunoprecipitation‐quantitative polymerase chain reaction (ChIP‐PCR) assays indicated that TaMYB44 could bind to the promoters of genes TaGS2.2, TaNRT2.1, and TaPIN4 and induce transcription of these stress‐defensive genes. Knockdown of these three genes reduced GS activity, N accumulation, and root growth traits in plants subjected to N starvation. The yield in the wheat variety panel was highly correlated with the transcripts of Tae‐MIR1118, TaCaM2, and TaMYB44 in plants cultured under N‐deprived field conditions. A major haplotype of Tae‐MIR1118, TaMIR1118‐Hap1, enhanced the low‐N stress tolerance of plants. Our findings indicate that the Tae‐MIR1118/TaCaM2/TaMYB44 pathway primarily affects the low‐N response of plants by modulating associated physiological processes.
