A type 2C protein phosphatase activates high-affinity nitrate uptake by directly dephosphorylating NRT2.1 in Arabidopsis

Nitrate transporter NRT2.1, which plays a central role in high-affinity nitrate uptake in roots, is activated at the post-translational level in response to nitrogen (N) starvation. However, critical enzymes required for post-translational activation of NRT2.1 remain to be identified. Here, we show that a type 2C protein phosphatase, designated CEPD-induced phosphatase (CEPH), activates high-affinity nitrate uptake by directly dephosphorylating S501 of NRT2.1, a residue that functions as a negative phospho-switch. CEPH is predominantly expressed in epidermal and cortex cells in roots and up-regulated by N starvation via a CEPDL2/CEPD1/2-mediated long-distance signaling from shoots. Loss of CEPH leads to a marked decrease in high-affinity nitrate uptake, tissue nitrate content, and plant biomass. Collectively, our results identify CEPH as a crucial enzyme in N starvation-dependent activation of NRT2.1, providing molecular and mechanistic insights into how plants regulate high-affinity nitrate uptake at the post-translational level in response to the N environment. We prepared total RNA from roots of wild type, CEPD1ox, CEPD2ox and CEPDL2ox plants grown on N-replete vertical plates for 14 d, and used a microarray analysis to identify genes specifically induced by CEPD family peptides. Total RNA isolated from the roots were subjected to microarray analysis. Labeled complementary RNA was hybridized with an Affymetrix Genechip Arabidopsis Gene 1.0 ST array.