Pumpkin CmoNAC1 enhances salt tolerance in grafted cucumbers. However, the potential interactions with other proteins that may co-regulate salt tolerance alongside CmoNAC1 have yet to be explored. In this study, we identified pumpkin CmoDREB2A as a pivotal transcription factor that interacts synergistically with CmoNAC1 in the co-regulation of salt tolerance. Both transcription factors were observed to bind to each other’s promoters, forming a positive regulatory loop of their transcription. Knockout of CmoDREB2A in the root resulted in reduced salt tolerance in grafted cucumbers, whereas overexpression demonstrated the opposite effect. Multiple assays in our study provided evidence of the protein interaction between CmoDREB2A and CmoNAC1. Exploiting this interaction, CmoDREB2A facilitated the binding of CmoNAC1 to the promoters of CmoRBOHD1, CmoNCED6, CmoAKT1;2, and CmoHKT1;1, inducing H₂O₂ and ABA synthesis and increasing the K+/Na+ ratio in grafted cucumbers under salt stress. Additionally, CmoNAC1 also promoted the binding of CmoDREB2A to CmoHAK5;1/CmoHAK5;2 promoters, further contributing to the K+/Na+ homeostasis. In summary, these findings reveal a crucial mechanism of CmoNAC1 and CmoDREB2A forming a complex enhancing salt tolerance in grafted cucumbers.

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