NtGCN2 is associated with multilayer cadmium tolerance in tobacco via coordinated transcriptional changes in growth, antioxidant, and detoxification pathways.

UNLABELLED: Cadmium (Cd) severely inhibits plant growth. While our previous physiological study established that the protein kinase gene NtGCN2 enhances Cd tolerance in tobacco, the underlying molecular mechanisms remained unknown. Here, we conducted comparative transcriptomic profiling of NtGCN2-overexpressing, NtGCN2-silenced, and K326 under Cd stress to bridge this molecular gap. Critically, our analysis moves beyond confirming known physiological phenotypes (e.g., enhanced antioxidant activity) to reveal the unexpected and multi-layered transcriptional logic orchestrated by NtGCN2. We discovered that NtGCN2 expression correlates with a coordinated transcriptional program that simultaneously (1) preserves glutathione pools by downregulating the glutathione-degrading enzyme Chac2-a previously unrecognized mechanism supporting both ROS scavenging and phytochelatin synthesis; (2) restructures cell wall architecture via coordinated upregulation of cellulose (CSLG2) and pectin-modifying (PME45) genes alongside suppression of a lignin inhibitor (SCL14); (3) sustains nitrogen assimilation by upregulating nitrate transporters (NPF8.1, NRT2.5) and reductase (NIA); and (4) orchestrates multilayered Cd detoxification including uptake suppression (bHLH37↓), chelation (PCS1↑), and vacuolar sequestration (HIPPs, CAX6↑). WGCNA further identified co‑expression modules correlated with Cd tolerance traits, and hub genes independently supported the DEG‑based findings. These findings suggest that NtGCN2 is associated with a putative transcriptional hub that may integrate growth, metabolism, and detoxification pathways, providing candidate genes and a testable framework for future functional validation. We provide a mechanistic model and a rich set of candidate genes for engineering heavy metal tolerance in crops. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at https://doi.org/10.1007/s12298-026-01776-6.