Members of the clade A protein phosphatase 2 C (PP2C) family serve as critical negative regulators of abscisic acid (ABA) signaling pathways and mediate plant adaptation to abiotic stresses. However, systematic genome-wide characterization of clade A PP2C members in Populus trichocarpa remains limited. In this study, we identified 16 clade A PtrPP2C genes through a comprehensive genome-wide analysis. Additionally, their evolutionary relationships, cis-acting elements, and expression patterns were investigated. Phylogenetic reconstruction revealed significant evolutionary conservation of the poplar clade A PtrPP2Cs with Arabidopsis homologs. Moreover, promoter analysis identified abundant ABA-responsive elements (ABREs) and stress-related elements, suggesting conserved regulatory mechanisms in stress adaptation. Transcriptomic profiling demonstrated that most of clade A PtrPP2Cs exhibited significant upregulation under both drought stress and ABA treatment, with PtrPP2C-9 as a representative example. Additionally, functional validation through osmotic stress assays using mannitol revealed that PtrPP2C-9-overexpressing (PtrPP2C-9-OE) transgenic plants displayed enhanced sensitivity to osmotic stress compared to wild-type controls, as evidenced by reduced root elongation and compromised stress tolerance. Furthermore, PtrPP2C-9 may influence poplar tolerance to drought stress by mediating a transcriptional regulatory network centered on ABF3 and GBF3. This study provides the first systematic investigation of clade A PP2Cs in poplar, establishing their critical roles in osmotic stress responses and offering potential molecular targets for improving stress resilience in woody plants.
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