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.

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