Phosphoregulation in the N-terminus of NRT2.1 affects nitrate uptake by controlling the interaction of NRT2.1 with NAR2.1 and kinase HPCAL1 in Arabidopsis.

NRT2.1, the major high affinity nitrate transporter in roots, can be phosphorylated at five different sites within the N- and the C-terminus. Here, we characterized the functional relationship of two N-terminal phosphorylation sites, S21 and S28, in Arabidopsis. Based on a site-specific correlation network, we identified a receptor kinase (HPCAL1, AT5G49770), phosphorylating NRT2.1 at S21 and resulting in active nitrate uptake. HPCAL1 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, HPCAL1 was found to interact with the N-terminus of NRT2.1, mainly when S28 was dephosphorylated. Phosphorylation of NRT2.1 at S21 resulted in a reduced interaction of NRT2.1 with its activator NAR2.1, but nitrate transport activity remained. By contrast, phosphorylated NRT2.1 at S28 enhanced the interaction with NAR2.1, but reduced the interaction with HPCAL1. Here we identified HPCAL1 as the kinase affecting this phospho-switch through phosphorylation of NRT2.1 at S21.

Characterization of the signalling pathways involved in the repression of root nitrate uptake by nitrate in Arabidopsis thaliana.

In Arabidopsis thaliana, root high-affinity nitrate (NO3-) uptake depends mainly on NRT2.1, 2.4, and 2.5, which are repressed by high NO3- supply at the transcript level. For NRT2.1, this regulation is due to the action of (i) feedback down-regulation by N metabolites and (ii) repression by NO3- itself mediated by the transceptor NRT1.1(NPF6.3). However, for NRT2.4 and NRT2.5, the signalling pathway(s) remain unknown as do the molecular elements involved. Here we show that unlike NRT2.1, NRT2.4 and NRT2.5 are not induced in an NO3- reductase mutant but are up-regulated following replacement of NO3- by ammonium (NH4+) as the N source. Moreover, increasing the NO3- concentration in a mixed nutrient solution with constant NH4+ concentration results in a gradual repression of NRT2.4 and NRT2.5, which is suppressed in an nrt1.1 mutant. This indicates that NRT2.4 and NRT2.5 are subjected to repression by NRT1.1-mediated NO3- sensing, and not to feedback repression by reduced N metabolites. We further show that key regulators of NRT2 transporters, such as HHO1, HRS1, PP2C, LBD39, BT1, and BT2, are also regulated by NRT1.1-mediated NO3- sensing, and that several of them are involved in NO3- repression of NRT2.1, NRT2.4, and NRT2.5. Finally, we provide evidence that it is the phosphorylated form of NRT1.1 at the T101 residue, which is most active in triggering the NRT1.1-mediated NO3- regulation of all these genes. Altogether, these data led us to propose a regulatory model for high-affinity NO3- uptake in Arabidopsis, highlighting several NO3- transduction cascades downstream of the phosphorylated form of the NRT1.1 transceptor.

Genome-wide analysis in response to nitrogen and carbon identifies regulators for root AtNRT2 transporters.

In Arabidopsis (Arabidopsis thaliana), the High-Affinity Transport System (HATS) for root nitrate (NO3-) uptake depends mainly on four NRT2 NO3- transporters, namely NRT2.1, NRT2.2, NRT2.4, and NRT2.5. The HATS is the target of many regulations to coordinate nitrogen (N) acquisition with the N status of the plant and with carbon (C) assimilation through photosynthesis. At the molecular level, C and N signaling pathways control gene expression of the NRT2 transporters. Although several regulators of these transporters have been identified in response to either N or C signals, the response of NRT2 gene expression to the interaction of these signals has never been specifically investigated, and the underlying molecular mechanisms remain largely unknown. To address this question we used an original systems biology approach to model a regulatory gene network targeting NRT2.1, NRT2.2, NRT2.4, and NRT2.5 in response to N/C signals. Our systems analysis of the data identified three transcription factors, TGA3, MYC1, and bHLH093. Functional analysis of mutants combined with yeast one-hybrid experiments confirmed that all three transcription factors are regulators of NRT2.4 or NRT2.5 in response to N or C signals. These results reveal a role for TGA3, MYC1, and bHLH093 in controlling the expression of root NRT2 transporter genes.

NRT2.1 C-terminus phosphorylation prevents root high affinity nitrate uptake activity in Arabidopsis thaliana.

In Arabidopsis thaliana, NRT2.1 codes for a main component of the root nitrate high-affinity transport system. Previous studies revealed that post-translational regulation of NRT2.1 plays an important role in the control of root nitrate uptake and that one mechanism could correspond to NRT2.1 C-terminus processing. To further investigate this hypothesis, we produced transgenic plants with truncated forms of NRT2.1. This revealed an essential sequence for NRT2.1 activity, located between the residues 494 and 513. Using a phospho-proteomic approach, we found that this sequence contains one phosphorylation site, at serine 501, which can inactivate NRT2.1 function when mimicking the constitutive phosphorylation of this residue in transgenic plants. This phenotype could neither be explained by changes in abundance of NRT2.1 and NAR2.1, a partner protein of NRT2.1, nor by a lack of interaction between these two proteins. Finally, the relative level of serine 501 phosphorylation was found to be increased by ammonium nitrate in wild-type plants, leading to the inactivation of NRT2.1 and to a decrease in high affinity nitrate transport into roots. Altogether, these observations reveal a new and essential mechanism for the regulation of NRT2.1 activity.

Redox metabolism: the hidden player in carbon and nitrogen signaling?

While decades of research have considered redox metabolism as purely defensive, recent results show that reactive oxygen species (ROS) are necessary for growth and development. Close relationships have been found between the regulation of nitrogen metabolism and ROS in response to both carbon and nitrogen availability. Root nitrate uptake and nitrogen metabolism have been shown to be regulated by a signal from the oxidative pentose phosphate pathway (OPPP) in response to carbon signaling. As a major source of NADP(H), the OPPP is critical to maintaining redox balance under stress situations. Furthermore, recent results suggest that at least part of the regulation of the root nitrate transporter by nitrogen signaling is also linked to the redox status of the plant. This leads to the question of whether there is a more general role of redox metabolism in the regulation of nitrogen metabolism by carbon and nitrogen. This review highlights the role of the OPPP in carbon signaling and redox metabolism, and the interaction between redox and nitrogen metabolism. We discuss how redox metabolism could be an important player in the regulation of nitrogen metabolism in response to carbon/nitrogen interaction and the implications for plant adaptation to extreme environments and future crop development.