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

We report the characterization and identification of molecular elements involved in the signalling pathways repressing NRT2 transporters and root nitrate uptake in response to nitrate. 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.