NtNAC50–NtEXLB1 module enhances cadmium tolerance by strengthening root antioxidant defence and maintaining root structural homeostasis

Cadmium (Cd) contamination inhibits plant growth and threatens the safe production of crops, but the root-mediated regulatory mechanisms that simultaneously enhance Cd tolerance and reduce tissue Cd accumulation remain incompletely understood. This study aimed to determine the function and upstream transcriptional regulation of the tobacco expansin-like gene NtEXLB1 under Cd stress. NtEXLB1 -overexpression and CRISPR/Cas9 knockout tobacco lines were analysed using yeast growth assays, physiological measurements, root histology, RNA-seq, yeast one-hybrid assays, and dual-luciferase reporter assays. NtEXLB1 was strongly induced by Cd stress, and its heterologous expression improved yeast growth under Cd treatment. In tobacco, NtEXLB1 overexpression alleviated Cd-induced inhibition of root growth and biomass and was associated with reduced Cd concentrations in roots, stems, and leaves, whereas knockout lines exhibited the opposite phenotypes. NtEXLB1 overexpression also maintained root tissue integrity, reduced ROS and malondialdehyde accumulation, and enhanced catalase and peroxidase activities. Transcriptomic analysis showed that NtEXLB1 -associated genes were enriched in oxidative-stress responses, ROS metabolism, cell-wall remodelling, and root development. NtNAC50 directly bound to the NtEXLB1 promoter and activated its transcription. Collectively, these results identify an NtNAC50 – NtEXLB1 regulatory module that contributes to Cd tolerance by strengthening root antioxidant defence and maintaining root structural integrity and is associated with altered Cd accumulation in tobacco tissues. NtEXLB1 therefore represents a potential target for improving Cd tolerance and reducing Cd accumulation in crops.