Revisiting the functional properties of NPF6.3/NRT1.1/CHL1 in xenopus oocytes

Abstract

Within the Arabidopsis NPF proteins, most of the characterized nitrate transporters are low-affinity transporters, whereas the functional characterization of NPF6.3/NRT1.1 has revealed interesting transport properties: the transport of nitrate and auxin, the eletrogenicity of the nitrate transport and a dual-affinity transport behavior for nitrate depending on external nitrate concentration. However, some of these properties remained controversial and were challenged here. We functionally express WT NPF6.3/NRT1.1 and some of its mutant in Xenopus oocytes and used a combination of uptake experiments using 15 N-labelled nitrate and two-electrode voltage-clamp. In our experimental conditions in xenopus oocytes, in the presence or in the absence of external chloride, NPF6.3/NRT1.1 behaves as a non-electrogenic and pure low-affinity transporter. Moreover, further functional characterization of a NPF6.3/NRT1.1 point mutant, P492L, allowed us to hypothesize that NPF6.3/NRT1.1 is regulated by internal nitrate concentration and that the internal perception site involves the P492 residue.. CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/244467 doi: bioRxiv preprint first posted online Feb. 26, 2018; Characterization of NPF6.3 in xenopus oocytes 2 Most nitrogen enters the food chain by plants. As a consequence, understanding nitrogen uptake and assimilation in plants is of major importance to increase nitrogen use efficiency (NUE). Nitrate is one of the most important nitrogen sources for plants in both natural and agricultural systems. Uptake of nitrate by plant roots has been studied in different species and the following scheme is commonly accepted; there are inducible or constitutive high affinity transport systems (iHATS and cHATS) and low affinity transport systems (iLATS and cLATS) responsible for nitrate transport in low (<0.5 mM) and high external nitrate concentrations (>0.5 mM), respectively. Most of the HATS is supported by members of the NRT2 transporter family (Krapp et al., 2014): NRT2.1, 2.2, 2.4 and 2.5 (Noguero and Lacombe, 2016); whereas LATS is supported by members of the NPF transporter family (Léran et al., 2014; Corratgé-Faillie and Lacombe, 2017). In Arabidopsis thaliana, the founding member of the NPF family, known as NPF6.3/NRT1.1/CHL1 (Tsay et al., 1993), is an exception because it has been demonstrated to behave as a dual affinity transporter (Liu et al., 1999)(Ho and Frommer, 2014) (Parker and Newstead, 2014; Sun et al., 2014; Sun and Zheng, 2015). In addition to nitrate influx, NPF6.3 can also mediate nitrate-efflux from cells (Leran et al., 2013). Moreover, the role of NPF6.3 is not limited to nitrate transport, this protein also plays an important role in nitrate sensing (Bouguyon et al., 2012(Bouguyon et al., , 2015, by mediating auxin transport (Munos et al., 2004; Remans et al., 2006; Ho et al., 2009; Krouk et al., 2010; Gojon et al., 2...

A reevaluation of the contribution of NRT1.1 to nitrate uptake in Arabidopsis under low‐nitrate supply

“…The findings of Wang et al, [17] and Liu et al, [18] have shown that over 75% of high-affinity nitrate uptake in plants (calculated by subtracting the root nitrate uptake rate of nrt1.1 mutants from that of wild-type plants) was contributed by NRT1.1. However, several studies have questioned the contribution made by the NRT1.1 high-affinity function to nitrate uptake with low external nitrate concentrations [21,22]. However, several studies have questioned the contribution made by the NRT1.1 high-affinity function to nitrate uptake with low external nitrate concentrations [21,22].…”

“…Soon after this study, analysis of the nitrate uptake activity in NRT1.1-injected Xenopus oocytes showed that NRT1.1 had both high-and low-affinity nitrate uptake kinetic features, where the shift in affinity was associated with a phosphorylation event on the T101 residue of the NRT1.1 protein [17][18][19][20], providing further evidence that NRT1.1 acts as both a HAT and a LAT. However, several studies have questioned the contribution made by the NRT1.1 high-affinity function to nitrate uptake with low external nitrate concentrations [21,22]. For example, Touraine and Class [23], Muños et al [24], and Remans et al [25] failed to detect reduced HATS influxes in nrt1.1 mutants in low KNO 3 growth medium (compared with wild-type plants) when these plants were precultured with KNO 3 or 1 mM NH 4 NO 3 .…”

The Arabidopsis protein NPF6.2/NRT1.4 is a plasma membrane nitrate transporter and a target of protein kinase CIPK23

“…In our hands, NRT1.4 was electrically silent in oocytes, which could be due to electroneutral nitrate/proton symport by NRT1.4. Chiu et al (2004) reported NRT1.4-dependent nitrate-inward currents by the two-electrode voltage-clamp (TEVC) method, but technical glitches when measuring nitrate fluxes by TEVC in Xenopus oocytes have been reported (Noguero et al, 2018). In summary, our results demonstrate that CIPK23 phosphorylates NRT1.4 at Thr98, and that this modification reduces the transport rate of NRT1.4 in the low-affinity range.…”

Combined Allosteric Responses Explain the Bifurcation in Non-Linear Dynamics of 15N Root Fluxes Under Nutritional Steady-State Conditions for Nitrate

“…In our study, these results are confirmed by the high expression of the BnNRT2.1 gene at low nitrate concentrations and the velocities ratio v app / v out in 15 N fluxes which reveals an increase in one order magnitude in transporters activity from 0.28 (stationary state) to 0.01 mM of external nitrate concentrations ( Figure 6C ). Moreover, a recent study of the functional properties of NPF6.3/NRT1.1 in xenopus ovocytes suggests that the nitrate influx and efflux by this transporter are also controlled by changes in endogenous nitrate concentrations ( Noguero et al, 2018 ). Finally, application of external Glu to uncouple NRT2.1 from NPF6.3/NRT1.1 at the transcriptional and activity levels (exp3) indicated that the NRT2.1 transporter is essential for nitrate translocation and osmotic water flux required for shoot growth in response to nitrate availability.…”