The PopGRF3-PopKNAT3 module orchestrates salt tolerance through transcriptional control of stress-responsive genes in...

Plants are often exposed to various stressors during their growth and development. Sustaining intracellular ion equilibrium, especially the balance between Na⁺ and K⁺, plays a vital role in plant adaptation to salt stress. In this study, we identified growth-regulating factor 3 (PopGRF3), a gene encoding a transcription factor (TF) that acts as a negative regulator of salt stress responses. PopGRF3-overexpressing (OE) and PopGRF3-RNA interference (RNAi) lines exhibited increased sensitivity and enhanced tolerance to salt stress, respectively. Notably, when exposed to salt stress, PopGRF3 interacts with the promoter region of the sodium/hydrogen exchanger 2 (PopNHX2), leading to the suppression of its transcriptional activity. It is demonstrated that knotted-like homeobox 3 (PopKNAT3), a gene encoding a TF, physically interacts with PopGRF3 and mitigates the latter's inhibitory effect on PopNHX2. Although PopKNAT3 does not directly regulate PopNHX2 expression, the PopKNAT3-PopGRF3 protein complex prevents PopGRF3 from binding to the PopNHX2 promoter. Consequently, transgenic plants overexpressing PopKNAT3 exhibited improved salt tolerance, while those with RNAi-mediated silencing of PopKNAT3 showed increased salt sensitivity. The dual role of the PopGRF3-PopKNAT3 module in regulating salt stress reveals a novel mechanism in Populus, thereby offering new insights into the complex networks that control these stress responses.