CmoNAC1 in pumpkin rootstocks improves salt tolerance of grafted cucumbers by binding to the promoters of CmoRBOHD1, CmoNCED6, CmoAKT1;2 and CmoHKT1;1 to regulate H2O2, ABA signaling and K+/Na+ homeostasis.

The NAC transcription factor is a type of plant-specific transcription factor that can regulate plant salt tolerance, but the underlying mechanism is unclear in grafted vegetables. H₂O₂ and ABA in pumpkin rootstocks can be transported to cucumber scion leaves, promoting stomatal closure to improve salt tolerance of grafted cucumbers. Despite these observations, the regulatory mechanism is unknown. Here, our research revealed that CmoNAC1 is a key transcription factor that regulates H₂O₂ and ABA signaling in pumpkin roots under salt stress. The function of CmoNAC1 was analyzed using root transformation and RNA-seq, and we found that pumpkin CmoNAC1 promoted the production of H₂O₂ and ABA via CmoRBOHD1 and CmoNCED6, respectively, and regulated K⁺/Na⁺ homeostasis via CmoAKT1;2, CmoHKT1;1, and CmoSOS1 to improve salt tolerance of grafted cucumbers. Root knockout of CmoNAC1 resulted in a significant decrease in H₂O₂ (52.9% and 32.1%) and ABA (21.8% and 42.7%) content and K⁺/Na⁺ ratio (81.5% and 56.3%) in leaf and roots of grafted cucumber, respectively, while overexpression showed the opposite effect. The root transformation experiment showed that CmoNCED6 could improve salt tolerance of grafted cucumbers by regulating ABA production and K⁺/Na⁺ homeostasis under salt stress. Finally, we found that CmoNAC1 bound to the promoters of CmoRBOHD1, CmoNCED6, CmoAKT1;2, and CmoHKT1;1 using yeast one-hybrid, luciferase, and electrophoretic mobility shift assays. In conclusion, pumpkin CmoNAC1 not only binds to the promoters of CmoRBOHD1 and CmoNCED6 to regulate the production of H₂O₂ and ABA signals in roots, but also binds to the promoters of CmoAKT1;2 and CmoHKT1;1 to increase the K⁺/Na⁺ ratio, thus improving salt tolerance of grafted cucumbers.