Allelic variation of NtNramp5 associated with cultivar variation in cadmium accumulation in Tobacco

Abstract

Tobacco (Nicotiana tabacum) is a cadmium (Cd) accumulator, and smoking is a major source of Cd exposure. In the present study, we identified two tobacco cultivars with contrasting phenotypes of Cd and manganese (Mn) accumulation in both hydroponic and soil pot experiments. Physiological experiments showed that the two cultivars differed in Cd uptake, but not in Cd translocation from roots to shoots. A homolog of OsNramp5 (natural resistance-associated macrophage protein 5), NtNramp5, was isolated from both cultivars. There was no significant difference in the expression level of NtNramp5 in the roots between the two cultivars. Sequence analysis revealed that the low Cd/Mn-accumulating cultivar possesses an NtNramp5 allele with a predicted mutation for early translation termination, resulting in a truncated protein missing 104 amino acids in the C-terminus of the full-length NtNramp5 found in the high Cd/Mn-accumulating cultivar. Both proteins were found to be localized to the plasma membrane. Heterologous expression of the two alleles of NtNramp5 in yeast showed that the full-length protein had transport activities for both Mn and Cd, whereas the truncated protein had no transport activity for Mn and a weak transport activity for Cd. These results suggest that NtNramp5 is a transporter for Mn and Cd, and the allelic variation in the coding region of NtNramp5 probaby explains the cultivar difference in Cd and Mn accumulation.