CpWRKY51 enhances drought and salt resistance in Cucurbita pepo via activating CpPYL2-mediated PYL-PP2C-SnRK2 pathway

This study characterized the function of CpWRKY51, a WRKY transcription factor from Cucurbita pepo, and explored its role in responding to salt and drought stresses. The function of the CpWRKY51 gene was verified using a virus-mediated transient overexpression system. After salt and drought stress treatments, C. pepo plants with transient overexpression of CpWRKY51 showed significantly milder wilting symptoms than the control group; similarly, Arabidopsis thaliana with CpWRKY51 overexpression also exhibited stronger tolerance to salt and drought stresses. Physiological detection revealed that under stress conditions, the accumulation of reactive oxygen species (ROS) such as malondialdehyde (MDA), superoxide anion (O₂^-), and hydrogen peroxide (H₂O₂) in the leaves of CpWRKY51-overexpressing lines was reduced, while the activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were significantly higher than those in the control group. At the molecular mechanism level, DNA Affinity Purification Sequencing (DAP-seq) identified the canonical W-box motif ("TTGAC") as the core binding sequence of CpWRKY51. Electrophoretic mobility shift assay (EMSA) and yeast one-hybrid (Y1H) assay confirmed that CpWRKY51 could specifically bind to the promoter of Pyrabactin Resistance 1-Like 2 (PYL2). Yeast two-hybrid (Y2H) assay and bimolecular fluorescence complementation (BiFC) assay showed that PYL2 could interact with Protein Phosphatase 2C (PP2C1 and PP2C4) in the presence of 10 μM ABA, and CpSnRK2.6 from C. pepo could specifically interact with PP2C1 and PP2C4. These results indicate that CpWRKY51 positively regulates the tolerance of Cucurbita pepo to salt and drought stresses by coordinately modulating the antioxidant defense system and the PYL-PP2C-SnRK2 pathway. This study provides a theoretical reference for elucidating the molecular mechanisms underlying WRKY transcription factor-mediated stress adaptation in horticultural crops, and clarifies that CpWRKY51 can serve as a potential target for the genetic improvement of crop stress resistance.