Phosphorylation-mediated cadmium and manganese selectivity uptake of SaNRAMP5 in nightshade

Identifying the selectivity of cadmium (Cd) and manganese (Mn) in transporters has long been a challenging scientific issue. Here, we identified the gene SaNRAMP5 from Solanum americanum, an orthologue of OsNRAMP5. SaNRAMP5 is predominantly expressed in root and localizes to the plasma membrane (PM). Knockout of SaNRAMP5 significantly reduced Cd accumulation in nightshade, while its overexpression in Arabidopsis increased Cd uptake in roots. Given the close relationship between nightshade and vegetables like potatoes, tomatoes, eggplants, and peppers, we compared the Cd absorption capabilities of NRAMP5 homologs in these species. Our results indicated that SaNRAMP5 exhibited a greater Cd uptake capacity than its homologs within the Solanaceae family. Interestingly, the Mn uptake capacities of these NRAMP5s varied independently of their Cd uptake capacities. Amino acid sequence analysis revealed that the N-terminal STNP residues, which mediate phosphorylation in SaNRAMP5, are crucial for the selective uptake of Cd and Mn. Mutating these STNP residues to a non-phosphorylatable form, SaNRAMP5(AANP), resulted in reduced Cd uptake without affecting Mn uptake. Conversely, StNRAMP5 and SlNRAMP5, which naturally lack STNP residues, demonstrated enhanced Cd uptake upon the introduction of STNP but not AANP. Notably, neither the introduction of STNP nor AANP affected their Mn uptake capacities. The reduced Cd uptake of SaNRAMP5(AANP) without compromising Mn uptake was attributed to alterations in PM localization due to continuous Cd exposure, rather than Mn exposure. Our findings provide novel insights into phosphorylation-mediated selective uptake of Cd and Mn, paving the way for engineering low-Cd crops without compromising yield.