The Plant Journal (2015) 83, 466–479

doi: 10.1111/tpj.12901

The Arabidopsis root stele transporter NPF2.3 contributes to nitrate translocation to shoots under salt stress Christelle Taochy1,†, Isabelle Gaillard1, Emilie Ipotesi1, Ronald Oomen1,‡, Nathalie Leonhardt2, Sabine Zimmermann1, Jean-Beno^ıt Peltier1, Wojciech Szponarski1, Thierry Simonneau3, Herve Sentenac1, Remy Gibrat1 and Jean-Christophe Boyer1,* 1 Biochimie et Physiologie Mole culaire des Plantes, Institut de Biologie Inte grative des Plantes, UMR 5004 CNRS, UMR 0386 INRA/Montpellier SupAgro/Universite de Montpellier, F–34060 Montpellier, France, 2 Laboratoire de Biologie du De veloppement des Plantes, Institut de Biologie Environnementale et Biotechnologie, Laboratoire des Echanges Membranaires et Signalisation, UMR 7265 CNRS/CEA/Universite Aix-Marseille II, F-13108 St Paul lez Durance, France, 3 Laboratoire d’Ecophysiologie des Plantes sous Stress Environnementaux, Institut de Biologie Inte grative des Plantes, UMR 0759 INRA/Montpellier SupAgro, F-34060, Montpellier, France Received 5 March 2015; revised 28 May 2015; accepted 1 June 2015; published online 8 June 2015. *For correspondence (e-mail [email protected]) † Present address: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Qld 4072, Australia. ‡ Present address: Cell Biology Laboratory, Vilmorin, SA 30210, Ledenon, France.

SUMMARY In most plants, NO
3 constitutes the major source of nitrogen, and its assimilation into amino acids is mainly achieved in shoots. Furthermore, recent reports have revealed that reduction of NO
3 translocation from roots to shoots is involved in plant acclimation to abiotic stress. NPF2.3, a member of the NAXT (nitrate excretion transporter) sub-group of the NRT1/PTR family (NPF) from Arabidopsis, is expressed in root pericycle cells, where it is targeted to the plasma membrane. Transport assays using NPF2.3-enriched Lactococcus lactis membranes showed that this protein is endowed with NO
3 transport activity, displaying a strong selectivity for NO
3 against Cl
. In response to salt stress, NO
3 translocation to shoots is reduced, at least partly because expression of the root stele NO
3 transporter gene NPF7.3 is decreased. In contrast, NPF2.3 expression was maintained under these conditions. A loss-of-function mutation in NPF2.3 resulted in decreased root-to-shoot NO
3 translocation and reduced shoot NO
3 content in plants grown under salt stress. Also, the mutant displayed impaired shoot biomass production when plants were grown under mild salt stress. These mutant phenotypes were dependent on the presence of Na+ in the external medium. Our data indicate that NPF2.3 is a constitutively expressed transporter whose contribution to NO
3 translocation to the shoots is quantitatively and physiologically significant under salinity. Keywords: Arabidopsis thaliana, nitrate, ion transporter, root-to-shoot translocation, salt stress, NPF family, NAXT, Lactococcus lactis.

INTRODUCTION Soil nitrate is the major source of nitrogen for plants in temperate climates (Miller and Cramer, 2004). In many herbaceous species, its assimilation into amino acids is mainly achieved in leaves (Smirnoff and Stewart, 1985; Andrews, 1986). Nitrate uptake from the soil and its distribution between roots and shoots are highly complex functions involving coordinated activity of a large set of influx or efflux transport systems (Dechorgnat et al., 2011; Wang et al., 2012). In addition to providing NO
3 ions to all plant tissues for assimilation or storage, NO
3 transport activity plays a role 466

in plant responses to various forms of environmental stress. An example is the role that NPF2.7/NAXT1 (nitrate excretion transporter 1, At3g45650) plays in plant responses to external and cytosolic acidification. Stress-triggered cytosolic acidification in roots results in NO
3 secretion into the external medium, a process that has been proposed to provide an electrical shunt favoring H+ excretion by proton pumps active at the plasmalemma, which thereby prevents a toxic decrease in cytosolic pH. At the molecular level, evidence has been obtained that NO
3 secretion from root periphery cells into the external medium upon acidification stress is © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd

A nitrate xylem loader under salt stress 467 mainly mediated by NPF2.7 (Segonzac et al., 2007). This transporter is one of a group of seven NAXT proteins, whose members are all expected to mediate NO
3 transport based on their high level of sequence similarity. NAXT transporters belong to the NRT1/PTR family (53 members in Arabidopsis; Tsay et al., 2007), for which a unified nomenclature using the family name NPF (NRT1/PTR family) has recently been proposed (Leran et al., 2014). Most NPF transporters characterized to date show NO
3 or peptide transport activity (Leran et al., 2014, 2015). Some members display permeability for other substrates such as auxin, abscisic acid, glucosinolate, nitrite, jasmonoyl isoleucine or gibberelin in addition to, or in the absence of, permeability to NO
3 (Leran et al., 2014; Pike et al., 2014; Saito et al., 2015). Another example of the role of NO
3 transport activity in plant acclimation to abiotic stress has been provided by molecular analyses of NO
3 translocation to the shoots (Li et al., 2010; Chen et al., 2012). So far, three NO
3 transporters from the NRT1/PTR family, NPF7.3/NRT1.5 (At1g32450, Lin et al., 2008), NPF7.2/NRT1.8 (At4g21680, Li et al., 2010) and NPF6.3/NRT1.1 (At1g12110, Leran et al., 2013), have been shown to contribute to NO
3 transport in the xylem vasculature. Both NPF6.3 and NPF7.3 mediate NO
3 secretion into the xylem sap, whereas NPF7.2 mediates NO
3 retrieval from the xylem sap. Various stresses (Cd2+, salt and osmotic stress) trigger antagonistic effects on expression of NPF7.2 and NPF7.3, with the former being increased and the latter decreased. This regulation promotes NO
3 re-allocation from shoots to roots, and such reallocation has been proposed to be a general response of plants to various stresses (Gojon and Gaymard, 2010; Li et al., 2010; Chen et al., 2012; Zhang et al., 2014). Despite these recent advances, membrane transport systems responsible for NO
3 translocation to shoots in plants grown under environmental stress remain poorly characterized at the molecular level. For instance, when NPF7.3 expression is strongly repressed under salt stress (Chen et al., 2012), other transport systems must be involved in NO
3 loading into the xylem sap. Here, we report that a NAXT member, NPF2.3 (At3g45680), is a NO
3 transporter that is essentially expressed in the root pericycle. We show that NPF2.3 contributes to NO
3 secretion into the root xylem sap under salt stress. This transporter may play a role in plant acclimation to mild salinity as it allows increased biomass production under such conditions. RESULTS NPF2.3 is a member of the NAXT sub-family within the NRT1/PTR family NPF2.3 belongs to the seven-member NAXT group of the 53-member NRT1/PTR family (NPF) from Arabidopsis thaliana (Figure 1). NAXT genes are organized as a cluster on

chromosome 3. NPF2.3 shares 62–76% protein sequence identity with other NAXT members. In particular, its sequence shows 66% identity with that of NPF2.7/NAXT1 (Figure S1), the first characterized member of the NAXT family (Segonzac et al., 2007). For comparison, NPF2.3 shows only 34% identity with NPF2.13 (At1g69870), its closest NPF relative outside the NAXT group. A phylogenetic tree of the NPF2 sub-family of NPF members from various monocots and dicots shows that this sub-family may be divided into two clades, NPF2a and NPF2b (Figure S2). The NPF2a clade comprises all the NAXT members from Arabidopsis (Figure 1), but no other proteins from this species. It is thus proposed that all members of this clade are NAXT-like proteins. Interestingly, these proteins are more highly represented in dicots (two to nine members per species) than in monocots (a single member in rice and Brachypodium, and none in maize). As previously observed for NPF2.7 (Segonzac et al., 2007), NPF2.3 gene expression is not regulated by external NO
3 availability (Figure S3). NPF2.3 mediates nitrate but not chloride transport NPF2.3-mediated transport activity was initially investigated in the Xenopus oocyte expression system. Localization of an NPF2.3–GFP fusion protein to the plasma membrane of oocytes injected with the corresponding cRNA indicated that NPF2.3 is targeted to the membrane of these cells (Figure S4a,b). Oocytes were thereafter injected with NPF2.3 cRNA. Unfortunately, neither direct measurements of NO
3 uptake or efflux using 15 NO
3 (Figure S4c,d) nor current recordings using the two-electrode voltageclamp technique (Figure S5) unambiguously revealed NPF2.3-mediated transport activity, even under conditions where the NPF7.3 NO
3 transporter was shown to be active (Lin et al., 2008). An alternative heterologous expression system involving the Gram-positive bacterium Lactococcus lactis (Kunji et al., 2005; Frelet-Barrand et al., 2010a), was therefore used. L. lactis cells were transformed with a pNZ8148-based construct expressing NPF2.3 tagged with AcV5 (Lawrence et al., 2003) and expressed under the control of the nisininducible nisA promoter (Zhou et al., 2006). The NPF2.3– AcV5 protein was successfully detected in the L. lactis membrane by Western blot analyses using an anti-AcV5 antibody (Figure 2a). Its apparent molecular weight (52 kDa) was lower than its theoretical molecular weight (61 kDa). This difference was also observed after expression of NPF2.3–AcV5 in a cell-free translation system using wheat germ lysate (Figure 2b). Such shifts in apparent molecular weights are fairly common for membrane proteins (Rath et al., 2009). Nitrate transport was assessed in proteoliposomes reconstituted by mixing soybean lipids (Glycine max) and

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 83, 466–479

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Figure 1. NPF2.3 and the NAXT sub-group in the Arabidopsis NRT1/PTR family. An unrooted phylogenetic tree of Arabidopsis proteins from the NRT1/PTR family (NPF). Polypeptide sequence alignment of the 53 Arabidopsis NPF members was performed using Clustal Omega (Sievers et al., 2011), and the tree was generated with PHYML software (Guindon and Gascuel, 2003) using the maximumlikelihood method and 1000 bootstrap replicates. Bootstrap values (as percentages) are indicated at the corresponding nodes. When available, previous names used in the literature are displayed in parentheses. Transport systems with functional data are shown in bold.

membrane proteins from L. lactis cells expressing NPF2.3– AcV5. Two types of control vesicles were used for comparison: proteoliposomes prepared from soybean lipids and membrane proteins from L. lactis cells transformed with the empty vector, and liposomes prepared from soybean lipids only. These three types of large unilamellar vesicles (LUVs) are referred to as NPF2.3-PLUV, PLUV and LUV, respectively. Nitrate transport into the vesicles (JNO3) was determined as previously described (Pouliquin et al., 1999; Gibrat and Grignon, 2003; Segonzac et al., 2007). Briefly, a positive-inside electrical diffusion potential (Em) was generated by imposing a steep inward cation concentration

gradient across the vesicle membrane (which contained a cation ionophore), and quantitatively monitored using the oxonol VI membrane potential fluorescent probe. The depolarizing effect of addition to the external medium of permeant anions such as NO
3 or Cl
allowed determination of the corresponding fluxes (JNO3 or JCl). Compared to control PLUV and LUV vesicles, NPF2.3-PLUV vesicles displayed a strong NO
3 transport rate, with a pH optimum at 6.5 (Figure 2c). Moreover, NPF2.3-PLUV vesicles exhibited a threefold higher JNO3 than JCl, whereas PLUV vesicles did not significantly discriminate between NO
3 and Cl
(Figure 2d). Taken together, these data indicate that

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 83, 466–479

A nitrate xylem loader under salt stress 469 Ctrl

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Figure 2. NPF2.3 expressed in L. lactis displays nitrate transport activity. (a, b) NPF2.3 polypeptide production and immunodetection of AcV5-tagged NPF2.3 polypeptides using an anti-AcV5 tag antibody. (a) NPF2.3 production in L. lactis. Western blot analysis was performed on membrane fractions prepared from L. lactis cells transformed either with a vector harboring an NPF2.3–AcV5 expression cassette (NPF2.3 lane) or the control empty vector (Ctrl lane). (b) NPF2.3 production in vitro. Western blot analysis of cell-free expression products in a wheat germ lysate. A PCR fragment encompassing the NPF2.3-AcV5 sequence under control of the T7 RNA polymerase promotor was added to the coupled transcription/translation mix (NPF2.3 lane) or omitted (Ctrl lane). (c, d) Functional characterization of transport activity of NPF2.3 polypeptides produced in L. lactis and reconstituted into proteoliposomes. Anion transport activity in liposomes or proteoliposomes was assayed using the oxonol VI membrane potential fluorescent probe. Values are means  SE (n = 3). Liposomes (large unilamellar vesicles, LUVs) were prepared from soybean lipids, and proteoliposomes were prepared from soybean lipids and membrane fractions purified from L. lactis cells expressing NPF2.3– AcV5 (NPF2.3-PLUV) or expressing an empty plasmid (PLUV). (c) NO
3 transport rate (JNO3) for LUV, PLUV and NPF2.3-PLUV as a function of external pH. (d) Comparison of NO
3 and Cl
transport rates in NPF2.3-PLUV and PLUV at pH 6.5.

NPF2.3 reconstitution in LUVs resulted in electrogenic passive transport activity (driven by the electrical field and providing an electrical shunt short-circuiting the net flux of cations) that showed selectivity for NO
3 compared with Cl
. NPF2.3 is expressed in root pericycle cells, and NPF2.3 localizes to the plasma membrane Transgenic plants expressing the GUS or GFP reporter gene under the transcriptional control of the NPF2.3 promoter were generated and named PNPF2.3:GUS and PNPF2.3: GFP, respectively. GFP images of 1-week-old PNPF2.3:GFP

seedlings revealed that NPF2.3 is essentially expressed in roots (Figure 3a–c). GUS staining in PNPF2.3:GUS roots was localized in the stele of the mature primary and secondary roots, but root tips were not stained (Figure 3d). In crosssections of roots from 6-week-old plants, GUS staining was found at the periphery of the central vascular cylinder (Figure 3e). Confocal microscopy analysis of PNPF2.3:GFP roots from 14-day-old plants revealed that GFP fluorescence was restricted to pericycle cells (Figure 3f–h). Confocal images of roots from a transgenic line expressing an NPF2.3–GFP translational fusion protein (PNPF2.3: NPF2.3-GFP construct) provided further evidence that NPF2.3 is expressed in root pericycle cells, but also suggested that the protein is targeted to the plasma membrane (Figure 3i–k). The plasma membrane localization of NPF2.3 was confirmed in Arabidopsis protoplasts transiently expressing the NPF2.3–GFP fusion protein under the control of the CaMV 35S promoter (Figure 3l–o). Disruption of NPF2.3 decreases the xylem flux of NO
3 to the shoots under salt stress An NPF2.3 T-DNA insertion line with the insertion in the NPF2.3 coding sequence (SALK_008188, here named npf2.3-1) was obtained in the homozygous state. The T-DNA insertion is localized at the start of exon 4 (Figure 4a). Complementation tests were performed using homozygous transgenic plants (npf2.3C) created by transformation of the npf2.3-1 mutant with a construct designed to express a GFP-tagged version of NPF2.3 under the control of its own promoter. Production of NPF2.3 full-length transcripts was abolished in the npf2.3-1 mutant and restored in the complemented line (Figure 4b) to a level comparable to that of the NPF2.3 transcript in wild-type control plants (Figure S6). The hypothesis that NPF2.3 plays a role in NO
3 translocation to the shoots was checked by collecting and analyzing xylem sap from excised root systems. Measuring both the exudation rate and the concentration of NO
3 in the exudate allowed computation of the root pressure-driven flux of NO
3 translocated by the xylem sap to the shoots (NO
3 translocation rate). Under control conditions, the exudation rates (Figure 4c), NO
3 concentrations in the exudate (Figure 4d) and thus NO
3 translocation rates (Figure 4e) were similar in mutant, wild-type and complemented plants. Absence of an npf2.3-1 phenotype under control conditions could not be ascribed to compensatory effects resulting from transcriptional de-regulation of the transporter genes NPF6.3, NPF7.2 and NPF7.3 involved in NO
3 transport in xylem tissues (Figure S6, t = 0). Salt stress has been shown to affect the shoot/root NO
3 ratio and expression of transport systems involved in NO
3 translocation to the shoots (Li et al., 2010; Chen et al., 2012). We therefore decided to investigate the effect of salt

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 83, 466–479

A nitrate xylem loader under salt stress 473 Shoot NO3– content

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2 1 0 1 2 3 4 5 6 7 Days of recovery Figure 6. NPF2.3 knockout mutation results in reduced shoot NO
3 contents under Na+ stress. (a) Plants were grown on standard hydroponic solution for 5 weeks, and then transferred onto a medium of the same composition (Ctrl) or supplemented with either 25 mM Na2SO4 (Na 50) or 25 mM K2SO4 (K 50). Shoot NO
3 contents were assayed 3 days after the transfer. Values are means  SE (n = 8–10). The results are representative of four independent biological experiments. (b) Recovery from salt stress. After the 3 day Ctrl and Na 50 treatments, plants were transferred onto standard solution without Na+ addition (recovery treatment, day 0). Shoot NO
3 contents were then periodically measured (left panel). Relative gene expression levels for NPF2.3, NPF7.2 and NPF7.3 were monitored as described in Figure 5 by quantitative RT-PCR (right panel). In the left panel, full and dotted lines indicate plants subjected to the 3 day pre-treatment in the presence (Na+) or absence (Ctrl) of Na+, respectively. Values are means  SE (n = 8–10 plants). The results are representative of two independent biological experiments. Asterisks indicate statistically significant differences between the wild-type and npf2.3-1 mutant plants (Student’s t test: *P < 0.05, **P < 0.01). DW, dry weight.

were measured 3 and 8 days later. In all the plants previously subjected to salt stress, removal of Na2SO4 from the external solution resulted in gradual restoration of NO
3 content in leaves, such that the difference in shoot NO
3 content between the npf2.3-1 mutant and control plants

was abolished after 8 days of growth in the absence of Na+ (Figure 6b, left panel). These results indicate that the sensitivity of the npf2.3-1 mutant to Na+ stress, in terms of shoot NO
3 content, is reversible. During recovery from salt stress, the NPF2.3 transcript level remained rather constant, i.e. it was similar at the end of the Na+ treatment (day 0 of recovery in Figure 6b, right panel) and after Na+ removal from the medium during the recovery period. In contrast, the 25 mM Na2SO4 treatment resulted in a strong increase in NPF7.2 transcripts and a strong decrease in NPF7.3 transcripts (day 0 in Figure 6b), in agreement with other data (Figures 5c and S6). Withdrawal of Na+ from the external solution (recovery treatment) resulted in changes in transcript levels in the opposite direction: a rapid decrease in the NPF7.2 transcript level and an increase in the NPF7.3 transcript level (Figure 6b, right panel). Thus, the marked transcriptional responses of NPF7.2 and NPF7.3 to salinity appear to be rapidly reversible. NPF2.3 disruption decreases plant tolerance to mild salinity Tolerance to salt stress may be assessed by comparing plant biomass production in the absence of salinity and under mild salinity conditions compatible with completion of the plant cycle (Munns and James, 2003; Munns and Tester, 2008). Wild-type, npf2.3-1 mutant and npf2.3C complemented plants were grown in soil up to the bolting stage (end of the vegetative cycle; approximately 10 weeks after sowing) under controlled conditions in an automated phenotyping chamber (Granier et al., 2006), allowing soil humidity to be maintained at a constant value (0.35 g water per g dry soil). Na2SO4 was added into the soil when the plants were 3 weeks old (six-leaf stage); at this time, Na+ soil content was increased from approximately 0 to 32 or 42 lmol Na+ per g dry soil. Shoot biomass was determined at the bolting stage, approximately 7 weeks after onset of the Na+ treatments (Figure 7). Growth of the wild-type plants was not significantly affected by these conditions, indicating that, in soil-grown plants, these salt treatments corresponded to a mild salt stress. Growing plants under standard conditions did not result in a significant difference in shoot biomass production between wild-type and npf2.3-1 plants. However, when grown on salt-treated soils, npf2.3-1 plants showed a significant decrease in shoot biomass (by 15% at the highest Na2SO4 concentration tested, compared to wildtype plants). Similar results were obtained after treatments with NaCl (Figure S10a). These data indicate that NPF2.3 contributes to sustaining plant growth under moderate salinity. Finally, measurements of shoot biomass in plants subjected to water deficit did not provide any evidence that NPF2.3 plays a role in plant acclimation to water stress (Figure S10b).

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 83, 466–479

A nitrate xylem loader under salt stress 471

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Figure 4. NPF2.3 disruption results in decreased NO
3 loading into the xylem sap upon Li+ stress. (a) Schematic representation of the NPF2.3 gene in the npf2.3-1 mutant line. Gray rectangles represent exons. Lines represent introns. The T-DNA insertion site is indicated by the arrow. (b) Absence of NPF2.3 transcripts in the npf2.3-1 mutant as assessed by RTPCR analysis of total RNA isolated from roots. RNA was extracted from wild-type plants (WT), npf2.3-1 mutant plants and complemented plants (npf2.3C) grown under standard hydroponic conditions for 5 weeks. Bands are from the same gel. EF1a expression was used as control. (c–f) Five-week-old plants grown on standard hydroponic solution were transferred for 24 h to the same medium supplemented with 5 mM LiCl (+Li) and without LiCl (Ctrl). Plants were de-topped, and exudates were collected for 1.5 h. Ion fluxes to leaves were obtained by multiplying their concentrations in the exudates by the sap exudation rates. Values are means  SE (n = 10–15). Asterisks indicate statistically significant differences between the wild-type and npf2.3-1 mutant plants (Student’s t test: *P < 0.05, **P < 0.01). DW, dry weight. These results are representative of two independent biological experiments. (c) Sap exudation rate. (d) NO
3 concentrations in the exudates. (e) NO
3 translocation rates. (f) Li+ translocation rates.

K+ translocation rate was also lower in the npf2.3-1 mutant (Figure S7a). However, no significant difference in the translocation rate of Li+ (Figure 4f) and other cations (Ca2+ and Mg2+) (Figure S7b) was observed between the three

genotypes (wild type, npf2.3-1 and npf2.3C). A decrease in K+ translocation to the shoots has also been reported in nrt1.5-2, another mutant that is impaired in NO
3 secretion into the xylem sap (Lin et al., 2008). Such data are consistent with the hypothesis of strong interactions between NO
3 secretion and K+ secretion into the xylem sap (Lin et al., 2008), possibly due to electrical constraints, e.g. electro-neutrality of the sap or control of membrane potential at the sites of secretion. Short-term 15 NO
3 transport studies in planta (performed as described in Methods S1) showed that the npf2.3-1 mutant is impaired in root-to-shoot NO
3 translocation in the presence of 50 mM Na2SO4 in the external solution (by 20%) (Figure S8a). 15 NO
3 uptake experiments indicated that this defect cannot be attributed to impaired NO
3 absorption (Figure S8b). NPF2.3 expression is barely affected by salt stress NPF2.3 transcript accumulation was barely affected by the Li+ treatment performed above (Figure 5a). A 24 h treat
ment with 100 mM Na+, introduced either as SO2
4 or as Cl salt, was found to result in a strong increase in expression of the RD29A gene, a transcriptional marker of osmotic (high-salt) stress (Yamaguchi-Shinozaki and Shinozaki, 1994). However, NPF2.3 transcript levels were not substantially affected by these salt treatments (Figure 5a). Similar treatments with lower Na+ concentrations (25 and 50 mM Na+) had almost no effect on NPF2.3 gene expression (Figure 5b). In a further test, monitoring gene expression for 48 h revealed that the level of NPF2.3 transcript remained reasonably constant in the presence of 100 mM external Na+ (Figure 5c). This is in strong contrast to a sharp increase in NPF7.2 expression (Li et al., 2010) and a sharp decrease in NPF7.3 expression (Lin et al., 2008) (Figure 5c). NPF7.2 and NPF7.3 are two salt stress-regulated NO
3 transporter genes identified as being involved in the distribution of NO
3 between roots and shoots under salt stress (Chen et al., 2012; Zhang et al., 2014). At the protein level, salt stress did not alter the localization of the NPF2.3 protein, which remained localized in pericycle cells (Figure 5d,e). In order to detect possible regulation of NPF2.3 at the protein level, western blot analyses using an anti-GFP antibody were performed on plasma membrane fractions isolated from roots of the npf2.3C line expressing the NPF2.3–GFP protein fusion. However, despite several attempts, no signal was detected in western blots, probably because NPF2.3 is expressed only in the pericycle, a very minor proportion of the entire root system. NPF2.3 disruption decreases shoot NO
3 content under Na+ stress In a first set of experiments, shoot NO
3 contents were measured in 5-week-old plants grown in soil and sub-

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 83, 466–479

472 Christelle Taochy et al. NPF2.3 gene expression

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50

Time (h) Figure 5. NPF2.3 expression in roots is barely affected by salt stress, in contrast to NPF7.2 and NPF7.3 expression. Wild-type plants were grown for 4 weeks under hydroponic conditions on standard medium, and then transferred onto a medium of the same composition (Ctrl treatment) or supplemented with salts as indicated. Gene transcript accumulation was assayed by quantitative RT-PCR using the PDF2 and TIP41 housekeeping genes for normalization. (a) NPF2.3 and RD29A gene expression 24 h after transfer to medium supplemented with 5 mM LiCl, 50 mM Na2SO4 or 100 mM NaCl (added to the standard solution). The gene expression fold change represents the amount of transcript (number of copies per ng RNA) observed under the experimental condition relative to that for the same transcript measured 24 h after transfer to control media (Ctrl). RD29A was used as a control gene whose expression is increased upon osmotic (high-salt) stress. Values are means  SE (three pools of six plants each). (b) NPF2.3 relative gene expression in roots of wild-type plants after 24 h treatment with Na2SO4 at the indicated Na+ concentrations. Values are relative to those obtained in the absence of salt stress (0 Na+). Values are means  SE (three pools of six plants each). (c) Kinetics of NPF2.3, NPF7.2 and NPF7.3 transcript accumulation in response to addition of 50 mM Na2SO4 into the medium. The gene expression fold change represents the amount of transcript (number of copies per ng RNA) at the indicated time divided by the amount of the same transcript at t = 0. Values are means  SE (three pools of six plants each). (d, e) NPF2.3 protein localization in roots under Na+ treatment. Plants transformed with a PNPF2.3:NPF2.3-GFP construct (see Figure 3) were grown in vitro on MS/2 medium for 14 days, and then transferred to MS/2 medium supplemented with 50 mM Na2SO4. GFP fluorescence was observed by laser confocal microscopy 24 h after the transfer. Scale bars = 50 lm. (d) Transverse optical section (data acquired through successive 1 lm depth optical longitudinal sections, Z-stack). (e) Longitudinal optical section for the same root as in (d).

jected to NaCl or Na2SO4 treatments. Initial tests indicated that when the ‘theoretical’ concentration of Na+ in the soil water was set to 85 mM for both salts, NaCl displayed higher toxicity than Na2SO4 (Figure S9a). Therefore, a lower Na+ concentration (50 mM) was subsequently used for NaCl, so that neither of the two salt treatments lead to apparent deleterious effects during the course of the experiment. Within 2 days, both the NaCl and Na2SO4 treatments led to decreased shoot NO
3 contents in all plants, when compared with plants grown under control conditions (in the absence of added NaCl and Na2SO4) (Figure S9b). However, the decrease was more pronounced in the npf2.3-1 mutant compared with wild-type and npf2.3C plants. In contrast, the npf2.3-1 and wild-type plants displayed similar Na+ shoot contents (Figure S9c). A second set of experiments was performed using hydroponically grown plants (Figure 6). Shoot NO
3 contents of wild-type, mutant and complemented genotypes were assayed after a 3 day salt treatment with 25 mM Na2SO4 or 25 mM K2SO4 (50 mM Na+ or K+) (Figure 6a). Addition of either salt to the external medium resulted in a decrease in shoot NO
3 content in all plants, in agreement with previous reports (Chen et al., 2012; Debouba et al., 2013) and the data shown in Figure S9(b,d). However, as shown in Figure 6(a), when the solution was supplemented with 50 mM Na+, the salt-induced decrease in shoot NO
3 content was more severe in the npf2.3-1 mutant plants than in the control wild-type plants (by 24%). This defect in mutant plants could not be ascribed to changes in the expression levels of the transporter genes NPF6.3, NPF7.2 and NPF7.3 (Figure S6, t = 72 h), and was not observed at lower (25 mM) or higher (100 mM) Na+ concentrations (Figure S9d). However, it was observed at 25 mM Na+ after a longer treatment (8 days instead of 3 days). When K2SO4 was substituted for Na2SO4 at the same concentration (25 mM) in the 3 day treatment, shoot NO
3 contents in mutant and control plants were no longer different (Figure 6a). Thus, the defect in NO
3 allocation to the shoots displayed by the mutant plants upon salt stress depends on both the duration and intensity of the stress, and may be ascribed to the presence of Na+ in the medium rather than that of K+ or the accompanying anion (SO2
4 ). The ability of the npf2.3-1 mutant to recover wild-type levels of shoot NO
3 contents when the plants are no longer facing salt stress conditions was assessed. As mentioned above, after 3 days of growth on 25 mM Na2SO4, npf2.3-1 mutant plants showed approximately 25% lower shoot NO
3 content than wild-type plants (Figure 6b, left panel, at day 0 of the recovery treatment). Then salt-treated plants from the same batch as well as control (untreated) plants were transferred to standard culture medium (without addition of Na+), and shoot NO
3 contents

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 83, 466–479

A nitrate xylem loader under salt stress 479 Saito, H., Oikawa, T., Hamamoto, S. et al. (2015) The jasmonate-responsive GTR1 transporter is required for gibberellin-mediated stamen development in Arabidopsis. Nat. Commun. 6, 6095. Segonzac, C., Boyer, J.C., Ipotesi, E., Szponarski, W., Tillard, P., Touraine, B., Sommerer, N., Rossignol, M. and Gibrat, R. (2007) Nitrate efflux at the root plasma membrane: identification of an Arabidopsis excretion transporter. Plant Cell, 19, 3760–3777. Sievers, F., Wilm, A., Dineen, D. et al. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539. Smirnoff, N. and Stewart, G.R. (1985) Nitrate assimilation and translocation by higher plants: comparative physiology and ecological consequences. Physiol. Plant. 64, 133–140. Tsay, Y.F., Chiu, C.C., Tsai, C.B., Ho, C.H. and Hsu, P.K. (2007) Nitrate transporters and peptide transporters. FEBS Lett. 581, 2290–2300.

Wang, Y.Y., Hsu, P.K. and Tsay, Y.F. (2012) Uptake, allocation and signaling of nitrate. Trends Plant Sci. 17, 458–467. Yamaguchi-Shinozaki, K. and Shinozaki, K. (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell, 6, 251–264. Zhang, H.X., Hodson, J.N., Williams, J.P. and Blumwald, E. (2001) Engineering salt-tolerant Brassica plants: characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation. Proc. Natl Acad. Sci. USA, 98, 12832–12836. Zhang, G.B., Yi, H.Y. and Gong, J.M. (2014) The Arabidopsis ethylene/jasmonic acid-NRT signaling module coordinates nitrate reallocation and the trade-off between growth and environmental adaptation. Plant Cell, 26, 3984–3998. Zhou, X.X., Li, W.F., Ma, G.X. and Pan, Y.J. (2006) The nisin-controlled gene expression system: construction, application and improvements. Biotechnol. Adv. 24, 285–295.

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 83, 466–479

474 Christelle Taochy et al. npf2.3-1 phenotype results from the presence of Na+ in the culture medium in a time- and dose-dependent manner, is specific to Na+ compared to K+, and is reversible when salt stress is removed. Thus, we show that NPF2.3 plays a significant physiological role in NO
3 translocation from roots to shoots when the plant is subjected to salt stress.

Shoot biomass (mg DW)

340 320 300 280

**

260 240

**

WT npf2.3-1 npf2.3C

220

Different transporters contribute to the tuning of NO
3 translocation to the shoots

200 0

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Na+ ( mol g–1 dry soil) Figure 7. NPF2.3 knockout mutation results in reduced shoot biomass under moderate salt stress. Plants were grown in soil in an automated phenotyping growth chamber. Evapo-transpiration was automatically compensated for by daily addition of equivalent amounts of water in order to maintain soil humidity at a constant value (0.35 g water per g dry soil) throughout the experiment. After 3 weeks (six-leaf stage), Na2SO4 was added to the soil at the indicated final concentrations. Shoots were sampled at the bolting stage (initiation of the floral stem; 10-week-old plants), dried and weighed. Values are means  SE (n = 12–18). Asterisks indicate statistically significant differences between wild-type and mutant plants (Student’s t test: **P < 0.01). The results are representative of two independent biological experiments.

DISCUSSION A constitutively expressed NO3 transporter contributing to NO
3 secretion into the root xylem sap Analysis of NPF2.3-mediated NO
3 transport activity was successfully performed using L. lactis but not the Xenopus oocyte expression system. It is worth noting that, in a systematic screening approach reported recently (Leran et al., 2015), only two transporters (NPF5.5 and NPF5.10) of 21 previously uncharacterized Arabidopsis NPF proteins were found to mediate 15 NO
3 net uptake in oocytes. This may indicate that some of the tested transporters are strictly dedicated to NO
3 secretion, without any capacity to mediate NO
3 uptake, or are permeable to substrates other than NO
3 , and/or are pH-sensitive and do not display transport activity at the tested pH. However, it may also indicate that the Xenopus oocyte expression system, for as yet unknown reasons, is not suited for functional characterization of some NPF members, as suggested by our results for NPF2.3. Characterization of a mutant plant line disrupted in the NPF2.3 gene, npf2.3-1, provided a direct indication that the root stele NPF2.3 transporter plays a role in NO
3 translocation to the shoots. Indeed, the absence of NPF2.3 resulted in decreased NO
3 loading into the xylem sap, shoot NO
3 content and biomass production. However, such alterations were only observed in mutant plants subjected to salt stress, and not in plants grown under standard optimal conditions, even though NPF2.3 was expressed at similar levels under both conditions. This salt stress-dependent

In addition to NPF2.3, two other transporters have been shown to be involved in the tuning of NO
3 translocation from roots to shoots under salt stress: NPF7.2 and NPF7.3 (Chen et al., 2012). NPF7.3, like NPF2.3, is expressed in root pericycle cells and mediates NO
3 secretion into the xylem sap (Lin et al., 2008). When plants are grown under standard conditions, NPF7.3 strongly contributes to NO
3 translocation to the shoots (Lin et al., 2008). Characterization of npf7.3 knockout mutant plants has shown that, in plants grown under standard conditions, absence of NPF7.3 expression results in a strong decrease in NO
3 translocation to the shoots, by up to approximately 50% (Lin et al., 2008). NPF7.2 is expressed in the xylem parenchyma cells that abut xylem vessels (Li et al., 2010). In contrast to NPF7.3 and NPF2.3, NPF7.2 plays a role in NO
3 retrieval from the root xylem vessels. Absence of NPF7.2 activity results in a large increase in NO
3 concentration in the xylem sap (approximately 40%) (Li et al., 2010). NPF2.3 displayed constitutive expression, with the level of its transcript being similar in plants grown under optimal conditions and when subjected to various levels of salt stress. These results raise the question of why our experiments did not detect any effect of the npf2.3-1 loss-of-function mutation on NO
3 secretion to the shoots when the plants were grown under optimal conditions. This could not be ascribed to compensatory changes in expression levels of the other NO
3 transporter genes known to contribute to NO
3 translocation to the shoots. The simplest hypothesis is that NPF2.3 contributes to NO
3 secretion to the shoots in plants grown under standard conditions, but this contribution is redundant in the presence of other secretion transporters such as NPF7.3. Upon salt stress, the expression levels of NPF7.2 and NPF7.3 are increased and decreased, respectively (Li et al., 2010; Chen et al., 2012; this work), whereas NPF2.3 expression remains fairly constant. In this situation, NPF2.3 may be responsible for approximately 25% of the NO
3 translocation rate into the xylem sap to the shoots (Figure 4). Under these conditions, the contribution of NPF7.3 is likely to be very low, as its expression is rapidly and strongly repressed in the presence of Na+ (Chen et al., 2012), by up to 20-fold in our experimental system (Figure 5c). In other words, these data indicate that NPF2.3 is a constitutively expressed NO
3 transporter of the root stele whose relative

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 83, 466–479

A nitrate xylem loader under salt stress 475 contribution to NO
3 translocation to the shoots appears to be quantitatively significant only under salinity, perhaps due to the strong salt-triggered decrease in NPF7.3-mediated transport. The relative contribution of NPF2.3 to NO
3 translocation in the presence or absence of salt stress may also involve regulation of this transporter at the post-transcriptional/translational level. Interestingly, like Arabidopsis NPF2.3, the Vitis vinifera NAXT-like transporter vvNPF2.1/vvNAXT1 is not transcriptionally regulated by salt, but is expressed at significantly higher levels in roots of a salt-tolerant than a salt-sensitive rootstock (Henderson et al., 2014). NO
3 translocation to the shoots and acclimation to salinity Leaf meristems and photosynthetic tissues are particularly sensitive to salt stress (Munns, 2002; Munns and Tester, 2008). Hence, control of Na+ translocation from the roots to the shoots by the xylem sap plays a central role in plant tolerance to salinity, especially in poorly tolerant species such as Arabidopsis. With regarding to this control, it should be noted that disruption of the gene encoding the NPF7.3 transporter has been shown to lead to both decreased Na+ translocation to the shoots and increased tolerance to salinity (Chen et al., 2012). Thus, in addition to contributing to NO
3 re-allocation to roots, the salinityinduced decrease in NPF7.3 expression probably prevents detrimental Na+ allocation to shoots. In contrast, NPF2.3 mediates NO
3 translocation to shoots without inducing any significant increase in shoot Na+ content (Figure S9c). This absence of stimulation of Na+ accumulation in shoots is likely to reduce the detrimental effects of salinity on plant growth. In addition, functional data indicate that NPF2.3 is incompetent for Cl
transport, which may be beneficial for plants grown in the presence of NaCl. Evidence is available that NPF7.2/NPF7.3-mediated reduction of NO
3 translocation to shoots and preferential allocation of this anion to roots plays an important role in plant acclimation to abiotic stress, including salinity (Gojon and Gaymard, 2010; Li et al., 2010; Chen et al., 2012; Zhang et al., 2014). Coordinated responses of NPF7.2 and NPF7.3 expression to salt stress are further demonstrated in the present study. Although NPF2.3 is expressed at similar levels in the presence or absence of salt, its role in NO
3 translocation to leaves under salt stress raises the question of whether, like NPF7.2 and NPF7.3, this transporter contributes to plant tolerance to salinity. The npf2.3-1 mutant displayed significantly reduced shoot biomass upon mild salt stress. A possible explanation for this salinity-dependent phenotype is that NPF2.3 mediates a basal root-to-shoot NO
3 translocation activity that may mostly benefit plants facing salt stress of moderate intensity, where NPF7.2/ NPF7.3-mediated re-allocation of NO
3 from shoots to roots is already effective but still limited.

In most studies on plant responses to salt stress reported so far, plants were exposed to high salt concentrations (≥100 mM; Claeys et al., 2014). In contrast, plant acclimation to moderate saline conditions, involving salt levels often encountered during the normal plant life cycle, is poorly studied at the molecular level, even though plants often experience stresses that are not immediately life-threatening but that do affect growth and productivity (Claeys et al., 2014). By contributing to plant growth maintenance under mild salinity, NPF2.3 appears to be involved in such acclimation. However, the limited extent of npf2.3-1 phenotypes indicates that other genes are probably also involved. Our data reveal that, in Arabidopsis, NO
3 distribution between roots and shoots under abiotic stress does not only rely on tuning of the antagonistic activities of NPF7.2 and NPF7.3. Indeed, plants constitutively express at least one transporter, NPF2.3, whose activity results in NO
3 supply to leaves in plants under salt stress. We propose that, in the context of the reduction of NO
3 translocation to shoots upon salinity, Arabidopsis plants require a dedicated xylem loading system that involves NPF2.3, to allocate sufficient levels of NO
3 to leaves of plants under mild salt stress. The identification of NPF2.3 as a contributor to NO
3 translocation to shoots provides further evidence for the importance of NO
3 transport systems in the acclimation to salinity stress. EXPERIMENTAL PROCEDURES Primer sequences The sequences of the oligonucleotides used in this study are listed in Table S1.

Plant growth conditions and mutant line genotyping All plant material was derived from the Arabidopsis thaliana ecotype Col-0. GFP and GUS microscopy observations were performed on 1–2-week-old plants grown in vitro on twofold diluted Murashige and Skoog (MS/2) culture medium (Murashige and Skoog, 1962), under controlled environmental conditions (16/8 h light/dark cycle at 21°C/18°C, 125 lmol photons m
2 sec
1 light intensity, 70% relative humidity). Gene expression analyses, 15 NO
3 translocation and sap flux measurements were performed on plants grown hydroponically for 4–6 weeks on standard medium as described previously (Lejay et al., 1999), under controlled environmental conditions (8/16 h light/dark cycle at 23°C/20°C, 250 lmol photons m
2 sec
1 light intensity, 70% humidity). The standard medium contains 1 mM KH2PO4, 1 mM MgSO4, 250 lM K2SO4, 250 lM CaCl2, 100 lM Na-FeEDTA, 50 lM KCl, 30 lM H3BO3, 5 lM MnSO4, 1 lM ZnSO4, 1 lM CuSO4 and 0,7 lM MoNaO4. Unless stated otherwise, the source of NO
3 was 1 mM KNO3. The hydroponic solution was changed twice a week. For NO
3 and Na+ content measurements in soil-grown plants, plants were grown in loam for 5 weeks, under the same controlled environmental conditions as described for hydroponics. The humidity of the substrate was regularly checked and re-adjusted to 3.5 g water per g dry loam. In experiments aimed at assessing

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 83, 466–479

476 Christelle Taochy et al. biomass production, plants were grown up to the bolting stage in soil (a mix of loamy soil and organic compost at a ratio of 1:1, v/v) in a PHENOPSIS phenotyping automation chamber (Granier et al., 2006) under controlled environmental conditions (8/16 h light/dark cycle at 21°C/20°C, 180 lmol photons m
2 sec
1 light intensity, 70% relative humidity). Evapo-transpirational water loss was automatically compensated for by daily addition of equivalent amounts of nutrient solution (modified 1/10th strength Hoagland solution) in order to maintain soil humidity at a constant value (0.35 g water per g dry soil, taken as non-limiting for growth; Granier et al., 2006). The npf2.3-1 (SALK_008188) T-DNA insertion mutant in the NPF2.3 (At3g45680) locus was obtained from the SIGnAL Collection at the Salk Institute (Alonso et al., 2003). Heterozygous seeds were obtained from the European Arabidopsis Stock Center (http://arabidopsis.info/), and selected to the homozygous stage. The location of the T-DNA insertion was confirmed by sequencing the PCR product amplified from npf2.3-1 genomic DNA using primers F3-19 and LBB1.

Molecular cloning and plant transformation The NPF2.3 ORF was amplified by Reverse Transcriptase-PCR from total root RNA extracted from Arabidopsis plants grown in standard hydroponics using primers B613gL and B613gR. The PCR fragment was digested using BamHI, and cloned into the BamHI site of the p35S:GFP vector (Clontech, https://www.clon tech.com/). In the resulting plasmid, pG613, the stop codon of NPF2.3 is replaced by a serine codon, leading to a C-terminal translational fusion between the NPF2.3 ORF and the downstream GFP sequence. This plasmid was used for transient expression in Arabidopsis protoplasts to investigate the subcellular localization of NPF2.3. To obtain the PNPF2.3:GUS transcriptional fusion, the 1469 bp sequence upstream of the NPF2.3 initiation codon was amplified from genomic DNA using primers P3-1500S and P3-1Rev, and the PCR fragment was inserted into the XbaI/NcoI sites of plant transformation vector pCAMBIA1305.1 (http://www.cambia.org/daisy/ cambia/585). The resulting plasmid, pCSGN3, harbors the GUS coding sequence under the transcriptional control of the NPF2.3 promoter sequence. In order to facilitate cloning of transcriptional and translational NPF2.3–GFP fusions and to introduce a factor Xa protease site between the NPF2.3 and GFP coding sequences (allowing removal of the GFP tag from protein fusions), a pCAMBIA1300 vector was modified as follows. A DNA fragment containing GFP and NOS terminator sequences was amplified from vector P35S:GFP using primers FGFP and RNOS, and the PCR fragment was cloned into the BamHI/EcoRI sites of pCAMBIA1300. The resulting plasmid, pCambGFP, harbors a BamHI site (for cloning of translational GFP fusions), followed by the factor Xa sequence, a SacI site (for cloning of transcriptional fusions) and the GFP sequence. To obtain the PNPF2.3:GFP transcriptional fusion, the NPF2.3 promoter sequence was amplified from pCSGN3 using primers FP21500S and RPN2-S, and the PCR fragment was cloned into the SalI/SacI sites of pCambGFP. The resulting plasmid, pP2GFP, was used to transform wild-type plants. To obtain the PNPF2.3:NPF2.3-GFP translational fusion, the NPF2.3 genomic sequence with its promoter region was amplified from genomic DNA using primers FP2-1500S and B613gR, and the PCR fragment was cloned into the SalI/BamHI sites of pCambGFP. The resulting plasmid (pPN2GFP) was used to transform the npf2.3-1 mutant. This genotype was named npf2.3C and used as a complemented line in this study.

Arabidopsis plants were transformed using the floral-dip immersion protocol described by Clough and Bent (1998). For heterologous expression of NPF2.3 in L. lactis, the NPF2.3 ORF was amplified from pG613 using primers FLN2P and RN2V5, and the PCR fragment was cloned into the PstI/SacI sites of the L. lactis expression vector pNZ8148 (Mierau and Kleerebezem, 2005) to create plasmid pLN2V5A.

GUS and GFP analyses GUS analyses were performed as described by Jefferson et al. (1987). Transient expression of the P35S:NPF2.3-GFP construct was achieved by transforming protoplasts from Arabidopsis cells with pG613 (see above) as described by Leon et al. (2002). Transformed protoplasts, as well as roots from transgenic plants expressing GFP fusions (PNPF2.3:NPF2.3-GFP or PNPF2.3:GFP), were observed by laser confocal microscopy using a Zeiss Axiovert 200M LSM 510 Meta NLO microscope (http://www.zeiss.com). GFP was excited using an argon laser at 488 nm, and fluorescence signal was collected through a 500–530 nm band-pass filter. Autofluorescence was detected through a 560 nm long-pass filter. Image acquisition and treatments were performed using LSM 5 IMAGE BROWSER software (Zeiss). Images of whole-plant GFP fluorescence were obtained using an Olympus SZX2-16 fluorescence stereomicroscope equipped with an Olympus DP-72 digital camera (https://www.olympus-lifescience.com). GFP was excited using an X-Cite lamp at 470  20 nm, and fluorescence was detected through a 500–530 nm band-pass filter.

Quantitative real-time PCR analysis Total RNAs were extracted using an RNeasy plant mini kit (Qiagen, https://www.qiagen.com) from roots of plants grown hydroponically for approximately 4 weeks. First-strand cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen, https://www.lifetechnologies.com), according to the manufacturer’s instructions. The primer pairs used were NPF2.3F2-19 and NPF2.3 R2-108 for NPF2.3 (located in the 30 UTR); NRT1.5-F and NRT1.5-R for NPF7.3 (spanning intron 3); NRT1.8-F and NRT1.8-R for NPF7.2 (spanning intron 4); RD29a-F and RD29a-R for RD29a (spanning intron 2). For normalization, three reference genes were selected on the basis of their expression stability in roots and leaves under our conditions (Czechowski et al., 2005): TIP41-like (At4g34270), EF1a (At5g60390) and PDF2 (At1g13320). PCR reactions were performed on a LightCycler 480 (Roche, http://www.roche.com) using SYBR Green PCR reagent (Roche) as described by Cuellar et al. (2013). Reactions were performed in triplicate for three independent biological samples.

NPF2.3 protein expression in a cell-free system and in L. lactis The AcV5 epitope-tagged NPF2.3 protein was expressed in vitro using the eukaryotic cell-free system included in the RTS 100 Wheat Germ CECF kit (5 Prime, https://www.5prime.com). The DNA template for coupled in vitro transcription/translation was obtained by amplifying the NPF2.3–AcV5 sequence from the pLN2V5A Lactococcus plasmid by two-step PCR using a protocol and oligonucleotides provided in the Wheat Germ LinTempGenSet kit (5 Prime). In vitro expression was performed according to the manufacturer’s instructions. For expression of the NPF2.3–AcV5 sequence in L. lactis, plasmid pLN2V5A was used to transform L. lactis strain NZ9000 (NIZO

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 83, 466–479

A nitrate xylem loader under salt stress 477 Food Research, http://www.nizo.com). The methodology used for L. lactis experiments, including heterologous gene expression and isolation of membrane proteins, was essentially as described by Frelet-Barrand et al. (2010b), except for the following modifications. Nisin was obtained from Sigma (https://www.sigmaaldrich. com), and optimal induction was achieved by addition of 100 ng ml
1 nisin for 4 h at 30°C. Cells were disrupted by four successive passages at 1.4 kbar in a Cell-D cell disruptor (Constant Systems Ltd, http://www.constantsystems.com). Immunodetection of the NPF2.3–AcV5 fusion protein was performed as described by Segonzac et al. (2007), except that primary and secondary antibodies were used at 1:4000 and 1:100 000 dilutions, respectively. Chemiluminescent signals were acquired using a Fuji LAS-3000 CCD imager (http://www.gelifesciences.com).

Functional characterization of NPF2.3 activity in reconstituted proteoliposomes In vitro passive NO
3 and Cl
fluxes, i.e. driven by the transmembrane electrical gradient (Em), were measured on proteoliposomes reconstituted at a lipid/protein ratio of 200:1 (w/w) (Grouzis et al., 1997) from L. lactis membrane proteins. Vesicles were positively polarized by inward Li+ diffusion gradients in the presence of ionophore ETH149 (http://www.sigmaaldrich.com), and Em was monitored using the lipophilic fluorescent anion oxonol VI (Molecular Probes, http://www.lifetechnologies.com). Quantitative comparison of the Em dissipation kinetics in the absence or presence of 100 mM NO
3 (or Cl
) in the external solution allowed determination of passive NO
3 (or Cl
) flux (JNO3) as described by Pouliquin et al. (1999), except that the assays were performed on a Wallac Victor 2 microplate reader (Perkin-Elmer, http://www.gmi-inc.com).

Root xylem sap analyses Root xylem sap was collected as described by Javot et al. (2003). Five-week-old hydroponically grown plants were de-topped, leaving the entire root systems remaining in the original culture solution. In order to allow determination of sap volume, the hypocotyl was carefully threaded into a graduated glass micropipette. Lowviscosity dental paste (President Microsystem Light; Coltene, http://www.coltene.com) was used to ensure that the system was watertight. After 1.5 h of spontaneous exudation, root systems were collected for root dry weight measurements.

Ion content analyses Cation contents were determined by absorption spectrometry as described previously (Nublat et al., 2001) using a SpectrAA 220 FS spectrometer (Varian, https://www.varian.com) or by inductively coupled plasma atomic emission spectroscopy. NO
3 content was measured by colorimetric assays following reduction into NO
3 using either a cadmium column included in an analytical chain in continuous flux as described by Munos et al. (2004), or using vanadium (III) chloride as described by Miranda et al. (2001), except that absorbance was measured using a Wallac Victor 2 microplate reader (Perkin-Elmer).

In silico analyses Sequence alignments and phylogenic tree construction were performed using online software available at http://mobyle.pasteur.fr/ cgi-bin/portal.py. Multiple protein sequence alignment of all NPF (NRT1/PTR) protein sequences was performed using Clustal Omega (Sievers et al., 2011). The alignment was further edited manually using CLC SEQUENCE VIEWER version 7.0 (http://www. clcbio.com/). The maximum-likelihood phylogeny was calculated

by alignment using the PhYML algorithm (Guindon and Gascuel, 2003), using the BLOSOM 62 scoring matrix (Henikoff and Henikoff, 1992) and a bootstrap analysis of 1000 replicates. The graphical output of the unrooted phylogram was generated using DENDROSCOPE 3 software (Huson and Scornavacca, 2012).

ACKNOWLEDGEMENTS We are grateful to Alain Gojon for critical reading of the manuscript and Bernard Carroll (School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Australia) for English language corrections and useful comments. Pascal Tillard (Stable Isotopes Platform) and Genevieve Conejero (Montpellier RIO Imaging Platform) are acknowledged. We thank Myriam Dauzat and Christine Granier for help with the PHENOPSIS robot, and Anne-Alienor Very for sharing her expertise on the Xenopus system. Anne Chevallier and Annie Frelet-Barrand are acknowledged for providing us with starting material and helpful advice on the Lactococcus system. This work was supported by a grant from the French Agence Nationale de la Recherche (Naxtress ANR project, 2010-BLAN-1713-02). C.T. is the recepient of a Contrat Jeune Scientifique grant from the Institut National pour la Recherche Agronomique.

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article. Figure S1. Multiple protein sequence alignment of NPF2.3, NPF2.7 and three other members of the NRT1/PTR family known to be expressed in roots and involved in nitrate long-distance transport. Figure S2. Arabidopsis thaliana NPF2.3 (AtNPF2.3) belongs to the NPF2a clade of the NPF2 sub-family of NPF transporters in flowering plants. Figure S3. NPF2.3 gene expression is not regulated by external NO
3 availability. Figure S4. 1 5NO
3 flux analysis does not provide evidence for NPF2.3-mediated NO
3 transport in Xenopus oocytes expressing NPF2.3. Figure S5. Whole-cell currents recorded in Xenopus oocytes expressing NPF2.3 reveal activation of endogenous conductance and do not provide evidence for NPF2.3-mediated NO
3 transport. Figure S6. NPF2.3 knockout mutation does not affect transcript levels of NO
3 transporters known to play a role in NO
3 fluxes in xylem vessels. Figure S7. NPF2.3 disruption results in decreased translocation rates of NO
3 and K+ but not of Li+, Ca2+ and Mg2+. Figure S8. The deficit in root-to-shoot NO
3 translocation in npf2.31 under salt stress does not result from impaired root NO
3 uptake. Figure S9. Disruption of NPF2.3 results in lower shoot NO
3 contents under salt stress in both soil-grown and hydroponically grown plants but does not alter shoot Na+ contents. Figure S10. NPF2.3 knockout mutation alters shoot biomass under salt stress but not under water stress. Table S1. Sequences of oligonucleotides used in this study. Methods S1. Root-to-shoot nitrate translocation analysis using 15 NO
3 .

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