GhNAC029 transcription factor negatively regulates cold tolerance in cotton through an ICE1-NAC-COR regulatory module

Transcription factors play key roles in plant responses to cold stress; however, the underlying regulatory mechanisms in cotton remain unclear. Transcriptome analysis revealed that GhNAC029 expression was rapidly upregulated within 1 h of cold treatment. Functional analyses showed that, compared with overexpression (OE) and wild-type (WT) plants, GhNAC029 knockout (KO) lines exhibited enhanced cold tolerance, higher survival and increased reactive oxygen species (ROS) scavenging capacity. GhNAC029, a member of the NAM, ATAF and CUC (NAC) transcription factor family, directly regulated 549 cold-responsive ( COR ) genes (52.84% of its targets), and bound to the G-box motif (5′-CACGTG-3′). A zinc-finger transcription factor (GhZAT10) and an ethylene-response factor (GhERF4) were identified as direct downstream targets. Their expression was elevated in KO plants, indicating that GhNAC029 functions as a transcriptional repressor of positive regulators of cold tolerance. The upstream regulator inducer of CBF expression 1 ( GhICE1 ) binds to the GhNAC029 promoter and negatively regulates its expression. Silencing GhICE1 caused severe leaf wilting and plant death under cold stress, confirming its positive role in cold tolerance. Collectively, these findings establish an ICE1–GhNAC029–COR regulatory network that elucidates the molecular basis of GhNAC029 -mediated cold responses and provides insights for breeding cold-tolerant cotton varieties.