Ammonium and nitrate regulate NH4+ uptake activity of Arabidopsis ammonium transporter AtAMT1;3 via phosphorylation at multiple C-terminal sites

摘要

In plants, nutrient transporters require tight regulation to ensure optimal uptake in complex environments. The activities of many nutrient transporters are post-translationally regulated by reversible phosphorylation, allowing rapid adaptation to variable environmental conditions. Here, we show that the Arabidopsis root epidermis-expressed ammonium transporter AtAMT1;3 was dynamically (de-)phosphorylated at multiple sites in the cytosolic C-terminal region (CTR) responding to ammonium and nitrate signals. Under ammonium resupply rapid phosphorylation of a Thr residue (T464) in the conserved part of the CTR (CTR^C) effectively inhibited AtAMT1;3-dependent NH4⁺ uptake. Moreover, phosphorylation of Thr (T494), one of three phosphorylation sites in the non-conserved part of the CTR (CRT^NC), moderately decreased the NH4⁺ transport activity of AtAMT1;3, as deduced from functional analysis of phospho-mimic mutants in yeast, oocytes, and transgenic Arabidopsis. Double phospho-mutants indicated a role of T494 in fine-tuning the NH4⁺ transport activity when T464 was non-phosphorylated. Transient dephosphorylation of T494 with nitrate resupply closely paralleled a transient increase in ammonium uptake. These results suggest that T464 phosphorylation at the CTR^C acts as a prime switch to prevent excess ammonium influx, while T494 phosphorylation at the CTR^NC fine tunes ammonium uptake in response to nitrate. This provides a sophisticated regulatory mechanism for plant ammonium transporters to achieve optimal ammonium uptake in response to various nitrogen forms.