The EIN3/EIL-ERF9-HAK5 transcriptional cascade positively regulates high-affinity K⁺ uptake in Gossypium hirsutum

High-affinity K⁺ (HAK) transporters play essential roles in facilitating root K⁺ uptake in higher plants. Our previous studies revealed that GhHAK5a, a member of the HAK family, is crucial for K⁺ uptake in upland cotton. Nevertheless, the precise regulatory mechanism governing the expression of GhHAK5a remains unclear. The yeast one-hybrid screening was performed to identify the transcription factors responsible for regulating GhHAK5a, and ethylene response factor 9 (GhERF9) was identified as a potential candidate. Subsequent dual-luciferase and electrophoretic mobility shift assays confirmed that GhERF9 binds directly to the GhHAK5a promoter, thereby activating its expression. Silencing of GhERF9 decreased the expression of GhHAK5a and exacerbated K⁺ deficiency symptoms in leaves, also decreased K⁺ uptake rate and K⁺ content in roots. Additionally, it was observed that the application of ethephon (an ethylene-releasing reagent) resulted in a significant upregulation of GhERF9 and GhHAK5a, accompanied by an increased rate of K⁺ uptake. Expectedly, GhEIN3b and GhEIL3c, the two key components involved in ethylene signaling, bind directly to the GhERF9 promoter. These findings provide valuable insights into the molecular mechanisms underlying the expression of GhHAK5a and ethylene-mediated K⁺ uptake and suggest a potential strategy to genetically enhance cotton K⁺ uptake by exploiting the EIN3/EILs-ERF9-HAK5 module.