A phospho-switch in the N-terminus of NRT2.1 affects nitrate uptake by controlling the interaction of NRT2.1 with NAR2.1

Abstract

NRT2.1 can be phosphorylated at five different sites within N- and C-terminus. Here, we provide a systematic functional characterization of phosphorylation at S21 and S28 within the N-terminus of NRT2.1. We used existing phosphoproteomic data sets of nitrate starvation and nitrate resupply to construct a site-specific correlation network identifying kinase candidates to phosphorylate NRT2.1. By this approach, we identified NITRATE UPTAKE REGULATORY KINASE 1 (AT5G49770) which itself was regulated by phosphorylation at S839 and S870 within its kinase domain. In the active state, when S839 was dephosphorylated and S870 was phosphorylated, NURK1 was found to interact with NRT2.1 at dephosphorylated S28. Upon that interaction, NURK1 can phosphorylate NRT2.1 at S21. Phosphorylation of NRT2.1 at S21 resulted in low interaction of NRT2.1 with its activator protein NAR2.1. By contrast, phosphorylation of NRT2.1 at S28 by a yet unknown kinase enhanced the interaction with NAR2.1, but inhibited the interaction with NURK1. We propose that serines S21 and S28 are involved in a phospho-switch mechanism and by which the interaction of NRT2.1 with its activator NAR2.1, and thus NRT2.1 activity, is modulated. NURK1 here was identified as the kinase affecting this phospho-switch through phosphorylation of NRT2.1 at S21 leading to inactivation of NRT2.1.

Plasma membrane calcineurin B‐like calcium‐ion sensor proteins function in regulating primary root growth and nitrate uptake by affecting global phosphorylation patterns and microdomain protein distribution

“…Moreover, we discovered differential phosphorylation for the C-terminal phosphorylation site T521. Upon LN supply, an increase of S28 phosphorylation, which activates NRT2.1 (Li et al, 2020), occurred in WT but was absent in cbl5x (Fig. 5a).…”

“…Remarkably, loss of CBL function inversely affected degree of phosphorylation of inhibitory and activating P‐sites within NRT2.1 under N starvation, providing an example for coordinated regulation within an individual protein. In this way, reduced phosphorylation of the inhibitory residue S11 and enhanced phosphorylation of the activating residue S28 in LN conditions allows for effective activation of this high‐affinity nitrate transporter (Li et al ., 2020). Notably, the nitrate transceptor NRT1.1 is also subject to CBL–CIPK phosphorylation, which switches this transporter into the high affinity state (Ho et al ., 2009).…”

“…7). (1) LN exposure leads to NRT2.1 activation by phosphorylation at activating S28 and by dephosphorylation at inhibitory S11 (Li et al ., 2020) in a CBL‐dependent manner to facilitate HATS. Accordingly, in cbl5x , we detected significantly lower S11 phosphorylation, especially after N starvation and at LN resupply for 15 min.…”

Genome-wide identification of high-affinity nitrate transporter 2 (NRT2) gene family in alfalfa (Medicago sativa)

“…For most dry-land plants, the main form of nitrogen absorbed and utilized is nitrate nitrogen. Nitrate is the most abundant inorganic nitrogen in soil and its uptake and translocation within plants have a significant influence on their nitrogen use efficiency [8][9] . Nitrate is not only the most important source of nitrogen for most plants, but also an important signaling molecule during plant growth and development 10 .…”

Regulation of the nitrate uptake process in grass species

“…HATS transporters, responsible for the larger proportion of nitrate uptake, are regulated in different levels by N-plant status and nitrate availability (Laugier et , Nacry et al 2013, Jacquot et al 2019. Chromatin modifications (Bellegarde et and phospho-proteome analysis (Engelsberger and Schulze 2012, Menz et al 2016, Jacquot et al 2019, Li et al 2020) recently seem to drag much attention among researchers. There is the huge potential to manipulate TFs targeting to improve NUpE in crops since those regulators can control a cascade of genes involved in nitrate uptake, rather than modulating a single or handful of NRTs genes simultaneously (Cao et al.…”