Adaptive Regulation of Nitrate Transceptor NRT1.1 in Fluctuating Soil Nitrate Conditions

摘要

class="head no_bottom_margin" id="sec1title">IntroductionNitrate is an essential mineral nutrient in plants and at the same time acts as a signaling molecule (, ). Its soil concentrations, however, fluctuate in several orders of magnitude from micromolar to millimolar range. To cope with these fluctuations, plants have developed sophisticated sensing and transport systems (). Rigorous molecular studies on ammonium and nitrate uptake have demonstrated the existence and functioning of two distinct uptake systems in plants referred to as high-affinity transport system (HATS) and low-affinity transport system (LATS) (, ). In low nutrient concentration, HATS is ON to scavenge ions and allows plants to maintain a normal uptake rate (, ). In high nutrient concentration, LATS is ON, leading to increased uptake along increasing nitrate gradient (, ). HATS usually follows Michaelis-Menten kinetics and displays saturation characteristics relative to LATS that increase linearly with concentrations. These differences primarily indicate the involvement of distinct sets of genes. Indeed, there are two distinct families of nitrate transporter genes, NRT1 and NRT2, associated with LATS and HATS, respectively (). With an interesting exception, recent studies have revealed that the nitrate transporter NRT1.1 (also known as NPF6.3 or CHL1), which is distinct from most of the members of both HATS and LATS gene family, contributes to both the systems and functions as transceptor (, ), a transporter cum receptor of changes in soil nitrate concentration. The dual-affinity modes of nitrate binding () and a phosphorylation switch allows NRT1.1 protein to control its capacity of switching between high- and low-affinity modes of uptake (). Detailed understanding of this molecular mechanism is essential for improving plant nutrient use efficiency (NUE) (, href="#bib8" rid="bib8" class=" bibr popnode">Gutiérrez, 2012) in a wide range of variation in soil nutrient availabilities, which, however, remains largely unknown.Independent of its transporter function, NRT1.1 also acts as a nitrate sensor, leading to rapid transcriptional regulations of several transporters and assimilatory genes called primary nitrate response (PNR) (href="#bib16" rid="bib16" class=" bibr popnode">Krouk et al., 2006, href="#bib10" rid="bib10" class=" bibr popnode">Ho et al., 2009). In the face of a wide range of variation in extracellular nitrate availabilities, plant adaptation is accompanied by quantifiable changes in PNR mediated by NRT1.1. In vitro and in vivo studies showed a biphasic primary response; at low nitrate concentrations, protein kinase CIPK23 phosphorylates Thr101 of NRT1.1, which allows the maintenance of a low-level primary response relative to the PNR level at high nitrate concentration (href="#bib10" rid="bib10" class=" bibr popnode">Ho et al., 2009). PNR studies in transgenic plants suggest that dual-affinity binding of nitrate and phosphorylation switch jointly allow NRT1.1 to sense a wide range of extracellular nitrate availabilities and are mainly responsible for biphasic adjustment of PNR (href="#bib20" rid="bib20" class=" bibr popnode">Medici and Krouk, 2014; href="#bib14" rid="bib14" class=" bibr popnode">Krouk, 2017). In nrt1.1 loss-of-function mutant plant Arabidopsis thaliana, it has evidently been noted that NRT1.1 regulates the expressions of the dedicated high-affinity transporter nrt2.1. At high nitrate concentrations, the expression of nrt2.1 is not down-regulated when nrt1.1 function is lost, which indicates a critical role of NRT1.1 in the PNR (href="#bib2" rid="bib2" class=" bibr popnode">Bouguyon et al., 2015). However, it remains unknown how the biphasic states of the PNR are regulated by sensing extracellular availabilities of nitrate concentrations.A key question about the biphasic states of NRT1.1 and their connection with dual-affinity nitrate binding and the phosphorylation at Thr101 has a potential structural basis. Recently reported apo- and nitrate-bound crystal structures of Arabidopsis thaliana NRT1.1 revealed a critical role of His 356 in nitrate binding and a phosphorylation-controlled dimerization switch that allows NRT1.1 to retain a dual-affinity mode of nitrate uptake (href="#bib26" rid="bib26" class=" bibr popnode">Sun et al., 2014, href="#bib22" rid="bib22" class=" bibr popnode">Parker and Newstead, 2014). This suggests that assembly and disassembly of the homodimer NRT1.1 controlled by the phosphorylation is responsible for toggling between low- and high-affinity modes of nitrate uptake (href="#bib26" rid="bib26" class=" bibr popnode">Sun et al., 2014). Despite this significant structural analysis, questions remain as to how the post-translational modifications associated with the nitrate sensing enables NRT1.1 to cope with a wide range of nitrate fluctuations. By comparative structural analyses of apo- and nitrate-bound X-ray crystallographic data of Arabidopsis thaliana NRT1.1 (href="#bib22" rid="bib22" class=" bibr popnode">Parker and Newstead, 2014), we report here that the intrinsic local asymmetries between the two protomers of NRT1.1 around the binding and Thr101 sites that are further enhanced by the nitrate binding provide a functional basis for having dual-affinity modes of nitrate binding. These asymmetries poise both the protomers for differential allosteric communications between the binding and phosphorylation sites, thereby regulating the phosphorylation-controlled dimerization of NRT1.1.