Multiple mechanisms of nitrate sensing by <italic>Arabidopsis</italic> nitrate transceptor NRT1.1

In Arabidopsis the plasmamembrane nitrate transceptor (transporter/receptor) NRT1.1 governs many physiological and developmental responses to nitrate. Alongside facilitating nitrate uptake, NRT1.1 regulates the expression levels of many nitrate assimilation pathway genes, modulates root system architecture, relieves seed dormancy and protects plants from ammonium toxicity. Here, we assess the functional and phenotypic consequences of point mutations in two key residues of NRT1.1 (P492 and T101). We show that the point mutations differentially affect several of the NRT1.1-dependent responses to nitrate, namely the repression of lateral root development at low nitrate concentrations, and the short-term upregulation of the nitrate-uptake gene NRT2.1, and its longer-term downregulation, at high nitrate concentrations. We also show that these mutations have differential effects on genome-wide gene expression. Our findings indicate that NRT1.1 activates four separate signalling mechanisms, which have independent structural bases in the protein. In particular, we present evidence to suggest that the phosphorylated and non-phosphorylated forms of NRT1.1 at T101 have distinct signalling functions, and that the nitrate-dependent regulation of root development depends on the phosphorylated form. Our findings add to the evidence that NRT1.1 is able to trigger independent signalling pathways in Arabidopsis in response to different environmental conditions. Plants are sessile organisms that face dramatic fluctuations of external mineral nutrient availability. Thus, they have developed sophisticated nutrient sensing systems, which activate physiological and developmental responses that prevent nutrient deficiency or toxicity. To date, most of nutrient sensing systems remain unknown at the molecular level1. One exception is the sensing of nitrate (NO3 ), which relies largely on ‘transceptors’, that is membrane NO3 − carriers fulfilling a dual transport/sensing function2–6. The most documented NO3 − transceptor is NRT1.1 of Arabidopsis thaliana7–9, also called CHL1 (ref. 6) or NPF6.3 (ref. 10). Initially characterized as a dual-affinity transporter involved in root uptake and root-to-shoot translocation of NO3 − (refs 11,12), NRT1.1 was later shown to mediate an impressive number of responses to NO3 − (ref. 8), among which three have been extensively investigated. First, NRT1.1 is required for the ‘primary nitrate response’ (PNR), that is the short-term regulation of gene expression upon first NO3 − supply. This includes the fast induction of many genes of the NO3 − assimilation pathway such as NRT2.1, which encodes a main component of the root NO3 − uptake system13–15. Second, NRT1.1 is also responsible in the longer term for an opposite response of some of these genes (for example NRT2.1) that are feedback repressed by high nitrogen provision13,16. Third, NRT1.1 was shown to promote root branching in response to NO3 −