Two novel transporters NtNRAMP6a and NtNRAMP6b are involved in cadmium transport in tobacco (Nicotiana tabacum L.)

Plant natural resistance-associated macrophage protein (NRAMP) plays important roles in metal transport and tolerance. Tobacco is a typical cadmium (Cd) accumulator, while research on NRAMP in tobacco has been limited. In the current study, two novel NRAMP genes (NtNRAMP6a and NtNRAMP6b) were identified from the allotetraploid plant Nicotiana tabacum L. Real time‒PCR and GUS (β-glucuronidase) staining results showed that the two genes were expressed in roots, stems, leaves and flowers and induced by Cd stress. Subcellular localization revealed that they were located in the plasma membrane. Heterologously expressed NtNRAMP6a and NtNRAMP6b significantly increased the Cd sensitivity of the Δycf1 mutant, indicating that NtNRAMP6a and NtNRAMP6b had Cd transport functions in yeast. The difference in the manganese (Mn) transport activity of the two genes was demonstrated by point mutation, which was caused by the difference in the 18th amino acid. NRAMP6-N18K is a new key active site for manganese transport. After 50 μM Cd treatment for 7 days, the contents of Cd and Mn of the ntnramp6a/6b mutants was significantly lower than those of wild type in shoots, while the contents in roots were higher. Additionally, the mutant lines showed higher chorphyll contentration and lighter leaf damage. Knockout of NtNRAMP6a and NtNRAMP6b reduced Cd and Mn accumulation in tobacco shoots by influence root-to-shoot translocation. This provides new idea for cultivating tobacco varieties with low cadmium accumulation and high cadmium tolerance.