Mutation of NtNRAMP3 improves cadmium tolerance and its accumulation in tobacco leaves by regulating the subcellular distribution of cadmium.

Cadmium (Cd) is a harmful element that affects plant growth and development. Genetic improvements could be applied for enhancing Cd tolerance and accumulation in plants. Here, a novel Cd stress-induced gene, NtNRAMP3, was identified in tobacco. We constructed two NtNRAMP3-knockout (KO) tobacco lines using the CRISPR/Cas9 system, which enhanced Cd tolerance and Cd accumulation in tobacco leaves compared with those in the wildtype (WT). Subcellular localization analysis suggested that NtNRAMP3 is a tonoplast protein and GUS (β-glucuronidase) histochemical analysis showed that NtNRAMP3 is highly expressed in the conductive tissue of leaves. NtNRAMP3-KO tobacco showed reduced Cd translation from vacuole to cytosol in leaves compared with the WT, and its vacuolar Cd concentration was significantly higher (20.78-22.81%) than that in the WT; in contrast, Cd concentration in the cytosol was reduced by 13.72-20.15%, preventing chlorophyll degradation and reducing reactive oxygen species accumulation in the leaves. Our findings demonstrate that NtNRAMP3 is involved in regulating Cd subcellular distribution (controlling Cd transport from vacuoles to the cytosol) and affects Cd tolerance and its accumulation in tobacco. This provides a key candidate gene to improve the phytoremediation efficiency of plants via genetic engineering.