Wheat Tae-MIR1118 Constitutes a Functional Module With Calmodulin TaCaM2-1 and MYB Member TaMYB44 to Modulate Plant Low-N Stress Response

Abstract

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.

Keywords: N deprivation‐associated processes; Tae‐MIR1118 and target expression; low‐N stress; protein interaction; transgene analysis; wheat (Triticum aestivum L.).