Functional characterization of SmNRAMP1 allelic variants conferring reduced cadmium uptake in Salvia miltiorrhiza

Cadmium (Cd) pollution in soil poses a significant threat to the production of traditional Chinese medicine. Reducing Cd accumulation in medicinal plants can mitigate potential risks to human health. Salvia miltiorrhiza, a prominent traditional Chinese medicinal plant, is known for promoting blood circulation and eliminating blood stasis through its roots and rhizomes. Identifying genetic targets and understanding the molecular basis of low Cd accumulation in S. miltiorrhiza are crucial to expedite gene-editing efforts aimed at cultivating low-Cd varieties. Although SmNRAMP1 is widely recognized as a Cd transporter in plants, its precise regulatory mechanism remains elusive. SmNRAMP1 alleles were isolated from high- and low-Cd-accumulating S. miltiorrhiza ecotypes (SD and SC, respectively). Comparative analysis of in vitro and in vivo Cd transport capacity demonstrated that the SmNRAMP1 allelic protein variant from the SC ecotype exhibited significantly lower transport capacity than that from the SD ecotype, with a 32.95% reduction. Analysis of the two SmNRAMP1 allelic protein variants from naturally high- (SD) and low- (SC) Cd-accumulating ecotypes through comparative sequence alignment, centrality analysis, and secondary structure estimation revealed a novel leucine-to-histidine substitution at position 301 (L301H) in the SC variant. Site-directed mutagenesis of the cloned gene followed by expression in a transgenic hairy root system demonstrated that the L301H substitution reduced Cd uptake by 15.34%. These results demonstrate that although the L301H mutation significantly impairs the protein’s Cd uptake capacity, it fails to reduce Cd accumulation to the low level observed in the SC ecotype. Further molecular dynamics simulation analysis indicated that other residues may also contribute to reduced Cd transport by SmNRAMP1. And the L301H mutation alters key stability-governing residues, disrupting SmNRAMP1’s conformational cycle during metal transport and ultimately reducing root Cd uptake.