Wheat miRNA TaMIR5062-5A Targets Calmodulin TaCML31 That Cooperates With MYB Member TaMYB77 to Modulate Drought and...

MicroRNAs (miRNAs) are key regulators of plant growth, development, and abiotic stress tolerance, acting via posttranscriptional control of target genes. In this study, we identified and characterized TaMIR5062-5A, a miRNA member in Triticum aestivum, focusing on its role in mediating drought and salt stress responses. TaMIR5062-5A showed downregulated expression upon exposure to drought and salt stress conditions, whereas its target gene TaCML31, a calmodulin-encoding gene verified by dual luciferase (LUC) assay, displayed the opposite expression trend under the above stress conditions. These findings suggested its involvement in osmotic stress responses through the Ca²⁺-dependent signaling pathway. Protein interaction assays (i.e., yeast two-hybrid, bimolecular fluorescence complementation, and co-immunoprecipitation) revealed that TaCML31 could interact with the MYB transcription factor TaMYB77, indicating that TaMIR5062-5A, its target gene TaCML31, and TaMYB77 constitute a regulatory module in plant osmotic stress response. Transgene analysis confirmed that TaMIR5062-5A negatively while TaCML31 and TaMYB77 positively regulated drought and salt stress tolerance by regulating osmolyte accumulation, stomata closure, root formation, and reactive oxygen species (ROS) homeostasis. Yeast one-hybrid, transcriptional activation, and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-PCR) assays indicated that TaMYB77 could bind to the promoters of osmotic stress-defense genes, namely TaP5CS2, TaNCED1, and TaDREB3, and regulate their transcription. The transgenic lines with knockdown expression of these stress response-associated genes exhibited impaired plant growth, reduced proline accumulation, dysregulated photosynthetic function, and compromised ROS homeostasis under drought and salt stress conditions, suggesting their roles in regulating plant osmotic stress responses underlying the miRNA module regulation. Strong correlations were observed between yield and the transcript levels of TaMIR5062-5A, TaCML31, and TaMYB77 in a wheat variety panel cultured under field drought conditions. Moreover, haplotype characterization of TaMIR5062-5A indicated that TaMIR5062-5A-Hap1 conferred enhanced drought tolerance in wheat plants. Overall, our findings establish a miRNA regulatory module, namely TaMIR5062-5A-TaCML31-TaMYB77, that plays an essential role in plant osmotic stress response in T. aestivum.