Single-nucleus RNA-seq and ATAC-seq analyses provide molecular insights into cadmium-stress response in alfalfa roots

Soil cadmium (Cd) pollution threatens global food security. Elucidating plant cellular responses to Cd stress is critical for the breeding of low-Cd-accumulating crops. Here, we investigated Cd-responsive regulatory mechanisms in alfalfa at single-cell resolution and identified key Cd-associated genes. Eight major root cell types were annotated, with significant remodeling under Cd stress. Under Cd stress, root endodermal and phloem cells adopt distinct adaptive strategies: endodermal cells shift toward a Cd sequestration and detoxification state, whereas phloem cells exhibit a response gradient ranging from basic defense to systemic regulation. Integrated multiomics analyses revealed cell-type-specific genome-wide changes in chromatin accessibility, which positively correlated with gene expression-particularly in promoter regions. Key genes including MsGSH1, MsMT2A, MsHMP47, and MsABCC3 were shown to increase Cd tolerance in yeast. Coexpression network analysis revealed 10 cell-type-specific modules, with the calmodulin-like gene MsCML acting as a highly interconnected hub gene, whose overexpression significantly improved Cd tolerance. These findings provide valuable genetic resources and a theoretical basis for the precise breeding of low-Cd-accumulating forage, with implications for understanding Cd-responsive epigenetic and transcriptional regulation in plants.