Drought is a major limiting factor for growth, development, and yield in wheat. TaPP2C62-2A, a clade A type phosphatase (PP2C) member in T. aestivum, was identified as a mediator of drought response. TaPP2C62-2A expression was downregulated in tissues in response to drought and abscisic acid signaling, which is linked to a transcriptional silencer in the promoter region. Yeast two-hybrid, bimolecular fluorescence complementation, and co-immunoprecipitation assays revealed interaction between TaPP2C62-2A and SnRK2 kinase TaSnRK2.5-2D and that TaSnRK2.5-2D also interacted with bZIP transcription factor TaABI5-3B with both interactions occurring via distinct conserved domains to establish a regulatory module of TaPP2C62-2A with the above partners. Transgene analyses revealed that this module is essential in modulating drought responses. TaPP2C62-2A negatively regulates drought tolerance, whereas TaSnRK2.5-2D and TaABI5-3B positively regulate plant growth and agronomic traits under drought conditions. The modified drought response mediated by TaPP2C62-2A and its associated components was ascribed to their functions in regulating osmotic stress-related physiological processes, including stomatal movement, osmolyte biosynthesis, leaf water retention, root morphology, and homeostasis of reactive oxygen species. TaABI5-3B binds with the promoters of stress-responsive genes, including S-type channel gene TaSLAC1-3, P5CS gene TaP5CS1, PIP gene TaPIP2;1, PIN-FORMED gene TaPIN4, and catalase gene TaCAT6, leading to their transcriptional activation. Transgene analysis confirmed the positive roles of these stress-responsive genes in regulating drought tolerance. Significant correlations were observed between yield and TaPP2C62-2A transcription and its module genes in a wheat variety panel subjected to drought conditions, with haplotype TaPP2C62-2A_h1 enhancing drought adaptation capacity. Our study presents novel insights into plant drought response associated with PP2C regulatory module and provides substantial genetic resources for breeding drought-tolerant wheat cultivars.

SNF1-related protein kinase 2 (SnRK2) family members are essential components of the plant abscisic acid (ABA) signaling pathway initiated by osmotic stress and triggering a drought stress response. This study characterized the molecular properties of TaSnRK2.4 and its function in mediating adaptation to drought in Triticum aestivum. Transcripts of TaSnRK2.4 were upregulated upon drought and ABA signaling and associated with drought- and ABA-responsive cis-elements ABRE and DRE, and MYB and MYC binding sites in the promoter as indicated by reporter GUS protein staining and activity driven by truncations of the promoter. Yeast two-hybrid, BiFC, and Co-IP assays indicated that TaSnRK2.4 protein interacts with TaPP2C01 and an ABF transcription factor (TF) TaABF2. The results suggested that TaSnRK2.4 forms a functional TaPP2C01-TaSnRK2.4-TaABF2 module with its upstream and downstream partners. Transgene analysis revealed that TaSnRK2.4 and TaABF2 positively regulate drought tolerance whereas TaPP2C01 acts negatively by modulating stomatal movement, osmotic adjustment, reactive oxygen species (ROS) homeostasis, and root morphology. Expression analysis, yeast one-hybrid, and transcriptional activation assays indicated that several osmotic stress-responsive genes, including TaSLAC1-4, TaP5CS3, TaSOD5, TaCAT1, and TaPIN4, are regulated by TaABF2. Transgene analysis verified their functions in positively regulating stomatal movement (TaSLAC1-4), proline accumulation (TaP5CS3), SOD activity (TaSOD5), CAT activity (TaCAT1), and root morphology (TaPIN4). There were high correlations between plant biomass and yield with module transcripts in a wheat variety panel cultivated under drought conditions in the field. Our findings provide insights into understanding plant drought response underlying the SnRK2 signaling pathway in common wheat.