Two-component high-affinity nitrate transport system in barley: Membrane localization, protein expression in roots and a direct protein-protein interaction

A high affinity transport system (HATS) for nitrate in plants is operated by a two-component NRT2/NAR2 transport system. However, the regulation and localization of NRT2 and NAR2 at protein level are largely unknown and especially so in crop plant species. In this study with barley (Hordeum vulgare), membrane localization, protein expression in the roots, and a direct protein-protein interaction of HvNRT2 and HvNAR2 proteins were investigated. Immunochemical analysis showed that both HvNRT2 and HvNAR2 proteins were co-localized in the plasma membrane of the roots. Expression of HvNRT2 and HvNAR2 proteins was more strongly induced by treatment with higher concentrations of external nitrate, while HATS activity and transcripts for HvNRT2 and HvNAR2 were markedly repressed. An affinity Column binding analysis using recombinant proteins suggests that the C-terminus of HvNRT2.1 is possibly involved in its binding to the HvNAR2.3 central region and that the Ser463 present in the HvNRT2.1 C-terminus plays a role in the binding ability.