Seeds of tobacco (N. tabacum L., cv. K326) were sterilized with 70% ethanol for 2 min and then with 2% sodium hypochlorite solution for a further 15–20 min, rinsed five times with sterile water, and germinated on 1/2 Murashige & Skoog (MS) agar plates for 14 days. Seedlings were transferred to a hydroponic or soil growth system in a growth room (16 h light/8 h dark cycle, 250 µE·m^–2·s^–1 light, 26/22°C light/dark temperature regime, 60% relative humidity). Plastic pots (15 cm × 15 cm × 20 cm) filled with 2.8 L of normal nutrient solution and others filled with 2 kg of soil (80% peat and 20% vermiculite, soil moisture 60%–70%) were used for the plant culture. The normal nutrient solution (for normal growth) contained 1 mM NH₄NO₃, 0.8 mM K₂SO₄, 1 mM KH₂PO₄, 1.5 mM MgSO₄, 2 mM CaCl₂, 3 µM MnSO₄, 1 µM ZnSO₄, 1 µM CuSO₄, 0.1 µM (NH₄)₆Mo₇O₄·4H₂O, 1 µM H₃BO₄, and 20 µM Fe–EDTA. The pH at 6.0–6.3 was adjusted using 1 M KOH.

For the test of growth on NO 3 − as an N source, after the pre-culture of seedlings with the above solution at half-strength or soil for 14 days, plants of a similar size were grown for 25 days in the nutrient solution containing different NO 3 − concentrations (0.5 mM, 2.0 mM, or 5.0 mM; aerated via an electric pump and refreshed every 2 days). The plants grown in the pot soil for 25 days were supplied three times with 100 mL of water containing NO 3 − at a concentration of 0 mM, 0.5 mM, 2 mM, or 5 mM. Four biological replicates were conducted for each treatment.

Homologous sequences of Arabidopsis AtNRT1.1s or AtNPF6s were extracted from the ARAMEMNON database (http://aramemnon.uni-koeln.de/index.ep), tobacco NtNPF6s from a publication by Zhan et al. (2022), and NtNRT1.1 and NtNRT1.2 from Liu et al. (2018). The ClustalW method in the Molecular Evolutionary Genetics Analysis (MEGA) 7.0 software was used to perform a multiple sequence alignment of putative NPF6 peptides for constructing a phylogenetic tree and sequence percent identity, with the following parameters: a gap opening penalty of 15, a gap extension penalty of 0.3, a 25% delay of divergent sequences, and a Gonnet series as the protein weight matrix. The phylogenetic tree and percent identity of NPF6s was constructed with MEGA 7.0 software using the neighbor-joining algorithm. Bootstrap analysis was carried out with 1,000 replicates. Branch lengths (drawn in the horizontal dimension) are proportional to the phylogenetic distances.

The putative open reading frame (ORF) of NtNRT1.1B (i.e., NtNRT1.2 in Liu et al., 2018) was amplified by PCR using the specific primers containing the SalI site (in lowercase letters): NtNRT1.1B-SalI-F, 5′-ATTAgtcgacATGGCACTTCCTGAGACACA-3′, and NtNRT1.1B-SalI-R, 5′-ACTAgtcgacATGACAAACCGGTCCATC-3’. The ORF with the SalI overhang was ligated into the yeast expression vector pYNR (Montanini et al., 2006) after its linearization by SalI. The pYNR-NtNRT1.1B plasmid was transformed into the (Hansenula polymorpha) YNT1 deletion yeast strain △ynt (△ynt, △leu), which is unable to grow on less than 0.5 mM NO 3 − as a sole N source; the pYNR was, respectively, introduced into the yeast strain NCYC495 (△leu) (Pérez et al., 1997) and △ynt (△ynt, △leu) as a positive and negative control. Yeast transformation and complementation were performed, as described in Liu et al. (Liu et al., 2003 note: growth at 37°C). All transformants were first selected on NAAG agar medium (2% glucose, 2% agar form Oxid, 0.17% yeast nitrogen base without amino acids, and ammonium sulfate from Difco, Detroit, MI, USA) containing 5 mM NaNO₃ as the N source. A single colony was picked, suspended in 60 μL of water, serially diluted, and spotted (2 μL) onto the NAAG agar media supplemented with 0.5 mM NO 3 − as the sole N source. The medium pH was adjusted by 1 M HCl or KOH.

The NtNRT1.1B ORF without a stop codon was amplified by a high-fidelity DNA polymerase (NEB, Beijing, China) using primers containing the BamHI site (NtNRT1.1B-BamHI-F, TTggatccATGGCACTTCCTGAGACA and NtNRT1.1B-BamHI-R, gtggatccG ACAATGACAAACC GGTCCAT), cloned into the vector pCF203 [carrying a cauliflower mosaic virus (CaMV) 35S promoter and the GFP gene (Liu et al., 2003)]. For the preparation of the transient expression of NtNRT1.1B::GFP in leaves of tobacco (N. tabacum), an overnight culture (OD₆₀₀ = 0.6) of Agrobacterium GV3101 harboring pCF203-35S-NtNRT1.1B::GFP was centrifuged at 2,000 g for 10 min; the collected bacteria were resuspended in an infiltration medium consisting of 50 mM MES (4-morpholineethanesulfonic acid), 2 mM Na₃PO₄·12H₂O, and 100 μM acetosyringone. Leaves of 19-day tobacco were infiltrated using a 2 mL syringe (without a needle) and grown in a greenhouse for 2–3 days; leaf cells were visualized by confocal microscopy after a 48- to 72-h infiltration.

For staining of the plasma membrane (PM), transfected tobacco leaves were incubated with 20 μM FM™ 4-64 [a molecular probe, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide (Bolte et al., 2004) for 10 min and washed three times with sterile water before visualization. Plant cells were scanned with an energy excitation and emission wavelength at 488 nm and 543 nm by a confocal laser scanning microscope (LSM880, Zeiss, Germany). The PM signal (dyed by FM 4-64) was visualized at between 515 and 640 nm. Brightness and contrast pictures were adjusted using ZEN 2.3 SP1 software. Being a PM-targeted protein marker, the expression of AtDUR3 (Liu et al., 2003) in leaf cells was tested as a reference.

A putative promoter (Pro _NtNRT1.1B ) with a 2,000-bp genomic sequence upstream from the predicted translational start of NtNRT1.1B was amplified by PCR using primers consisting of the HindIII and SalI cloning site (5′-CCCaagcttTGGGAATTTG AAAATTCTACA-3′ and 5′-ACGcgtcgacTTTAGCAAC TTTTCTTGCAC-3′), and cloned into a binary expression vector pBI101-β-glucuronidase (GUS) and -GFP (harboring the kanamycin resistance gene for plant selection), yielding recombination plasmids containing the constructs “Pro _NtNRT1.1B -GUS” and “Pro _NtNRT1.1B -GFP”. These constructs were transformed into tobacco plants using the agrobacterial GV3101-mediated leaf-disk method, as described in Sparkes et al. (2006). Several transgenic lines of Pro _NtNRT1.1B -GUS’ and ‘Pro _NtNRT1.1B -GFP were generated based on kanamycin selection. GUS staining and microscopic (BX51, Olympus, Japan) visualization were performed as stated by Zhang et al. (2019). For the analysis of promoter activity in tissue sections, stained stems were further embedded in paraffin, cut into 10-µm-thick transverse sections, mounted on a glass slide, and visualized under the microscope.

For the observation of GFP localization, roots were mounted in water under a glass coverslip, and GFP signals were scanned with an energy excitation at between 488 and 535 nm by a confocal laser scanning microscope (Olympus FluoView™ FV1000, Japan). The intensity of green fluorescence photographed was quantified using ImageJ software, as described in Zhang et al. (2019). At least 25 individual roots were used for the quantification of the green fluorescence signal intensity in the N treatment experiment.

The NtNRT1.1B ORF was amplified by PCR using primers containing the BamHI site (5′-TTggatccATGGCACTTCCTGAGACA-3′ and 5′-ATggatccTCAATGACAAAC CGGTCCA-3′) and cloned after the 35S promoter in the vector pCF203. To generate transgenic tobacco plants (with a K326 background) with overexpression of NtNRT1.1B, the agrobacterial strain GV3101-mediated leaf-disk method was used, as described above. Several independent homozygous transgenic lines in the T2 generation were obtained on the basis of kanamycin resistance selection and the NtNRT1.1B expression test (see Figure 5A ).

Quantitative reverse transcription (RT)-PCR (qPCR) was performed with total RNA from roots and shoots of both K326 and the transgenic plants created. The procedure for the qPCR experiment followed the same protocol as described in Liu et al. (2018). The relative expression level of NtNRT1.1B was normalized to that of two stable internal reference genes (i.e., α-tubulin and α-actin) (Schmidt and Delaney, 2010). qPCR analysis for each line was conducted with four biological replicates, together with three “no template controls” to check the contamination of reagents. Data were analyzed using the 2^−ΔΔCT method (CT: cycle threshold) (Schmittgen and Livak, 2008). Before qPCR, amplicons of tested genes were obtained by semiquantitative RT-PCR, gel electrophoresed, purified from the gel, and sequenced for confirmation of their specificity and sequence correctness. The following primers were used: NtNRT1.1B-F, 5′-GGTATCTTTGCCACTGTTCA-3′, and NtNRT1.1B-R, 5′-CAGCATCGTCGA ATTG GTC-3′; Ntα-tubulin-F, 5′-GGTATTCAGGTCGGA AATGCA-3′, and Ntα-tubulin-R, 5′-CTTCGTCAATGACAGTAGGCT- 3′; Ntα-Actin-F, 5′-ATGAGAGAG TGCATATCGATTC-3′ and Ntα-Actin-R, 5′-TTAGTATTCCTCGTTA TCATCGT-3′.

In the experiment of NO 3 − influx into roots, NtNRT1.1B-overexpressing tobacco lines and its corresponding WT (K326) were grown as described above. After 3 weeks’ growth in the normal nutrient solution, plants of a similar size were subjected to N starvation for 2 days, and then roots were exposed for 5 or 30 min to an N-free basic solution supplied with 0.1, 1, or 10 mM total N in the form of NO 3 − and/or ¹⁵N- NO 3 − (in 2.8 L solution; 99.72% ¹⁵N abundance in K¹⁵NO₃, Shanghai Research Institute of Chemical Industry, Shanghai, China). For 10 mM total N supply, only 10% N in the form of ¹⁵N- NO 3 − was added to the uptake assay solution; 100% ¹⁵N-labeled NO 3 − was used in the 0.1 or 1 mM nitrate supply. After uptake, the roots were washed four times in 1 mM CaSO₄ for 30 s each, and the roots (after a 5- and 30-min uptake) and shoots (after 30-min uptake) were separately harvested, dried in an oven (at 60°C for 5 days), and ground to a fine powder. Samples (3–4 mg dry matter) were used to determine the ¹⁵N content in the plants via mass spectrometry (DELTA^plusXP; Thermo-Finnigan, Waltham, MA, USA). The ¹⁵N incorporated in the roots or shoots was converted into NO 3 − absorbed by the roots.

For the kinetic study of NO 3 − transport via the NtNRT1.1B pathway, NtNRT1.1B-overexpressing tobacco lines (e.g., L1 and L5) and their WT, K326, were used. The roots of 2-day N-starved tobacco plants or those grown with normal nutrient solution after 14-day germination on a 1/2 MS agar plate and 7-day hydroponic culture were exposed for 3 min to the assay solution containing varying NO 3 − concentrations (in the range of 5,000–7,000 μM; for NO 3 − concentration > 500 μM or >3,000 μM, 50% or 20% ¹⁵ NO 3 − was respectively applied). For the method to determine the ¹⁵N content, see the above statement. The content of NO 3 − in NtNRT1.1B-overexpressing lines. minus that of the WT plants was used to calculate the absorption by NtNRT1.1B. For details of the protocol for N root uptake analysis, refer to the description by Fan et al. (2017).

Plant leaves and roots were separately sampled and weighed, and 100 mg of each sample was extracted for the measurement of nitrate, ammonium, glutamine synthetase (GS), and nitrate reductase (NR), following the methods described by Fan et al. (Fan et al., 2017; Liu et al., 2021). The total N per plant (shoot or root) was determined using the Kjeldahl method, as described in Nelson and Sommers (1973).

After the 4-week pre-culture of NtNRT1.1B-overexpressing tobacco and its WT (K326) with the normal nutrient solution (see above section), plants subjected to N starvation for 2 days or those resupplied with 2 mM NO 3 − after 2 days’ N starvation were used to sample the xylem (Xy) exudate. The upper part of the plants was removed from the middle of the hypocotyl, and the cutting site of the lower part was immediately wiped three times using sterile water-wetted cotton; the xylem exudate was collected over a 0- to 2-, 2- to 5-, 5- to 8-, 8- to 12-, or 12- to 24-h period after removal of the upper part. The xylem exudate collected in the 0- to 2-h period from the plants grown continuously with the normal nutrient solution (containing 1 mM NH₄NO₃ as N source) served as a control sample (CK, Figure 7B ).

Apoplastic washing fluid (AWF) was extracted from tobacco plants grown for 4 weeks on the normal nutrient solution. AWF was extracted from the upper leaves (UL; i.e., leaves not fully opened), mature leaves (ML; i.e., new fully opened leaves, or the third or fourth leaves down from the UL), and lower leaves (LL; i.e., the sixth and seventh leaves down from the UL). The method for the collection of AWF followed a modified infiltration–centrifugation technique, as described in O'Leary et al. (2014), that is, a vacuum pump instead of a syringe was used for leaf infiltration. The content of NO 3 − in the xylem exudate and apoplastic fluid was determined using a continuous flow analyzer (AA3, SEAL, Germany).

Descriptive statistics were applied to show the difference in the activity of promoter and enzyme, biomass, the content of total N, NO 3 − and NH 4 + , as well as gene expression in plants under varied experimental treatments and/or different tobacco lines. Means and standard deviations (SDs) are plotted in Figures 4 – 7 and also in Supplemental Figures 2 , 3 . Statistical analysis was performed using one-way analysis of variance (ANOVA) using the software package SPSS Statistic Client 16.0, SPSS, Beijing, China).

Being an upland crop, tobacco (N. tabacum L.) represents a typical species preferentially using NO 3 − but not NH 4 + as an N source for normal growth and development (Fan et al., 2018). Despite physiological evidence showing the existence in tobacco roots of both high- and low-affinity transport systems, which are suggested to contribute to the effective acquisition of N from soils (Fan et al., 2018), descriptions of the molecular basis of NO 3 − movement in this crop are very limited. To help understand NO 3 − transport/uptake in/by tobacco at a molecular level, based on our previous, preliminary, study on the genetic information of tobacco NtNRTs (Liu et al., 2018), we picked NtNRT1.2 [cloned first in our previous work (Liu et al., 2018) and renamed as NtNRT1.1B (see following section)] as a target to explore its molecular and physiological function in plant nitrate (-N) nutrition. The NtNRT1.2 gene has been reported to contain a putative open reading frame (with 1,785 bp derived from five exons), which encodes a peptide with 594 amino acid residues and exhibits 12 predicted transmembrane domains (TMDs) with large hydrophilic loops between TMD6 and TMD7 (Liu et al., 2018), similar to Arabidopsis AtNRT1.1/1.2.

Recently, based on an updated release of predicted functional gene sequences of tobacco (K326), Zhan et al. (2022) collected 143 putative NPFs and phylogenetically categorized them into eight subfamilies (i.e., NtNPF1–8). However, we have recognized that none of these 143 NtNPFs is identical to the sequence of NtNRT1.1/1.2 described previously by Liu et al. (2018). Thus, we extracted the sequences from the tobacco NtNPF6 subcluster and Arabidopsis AtNRT1s/NPF6s and performed a genetic lineage analysis for the NtNR1.1/1.2. The result indicated that both the previously reported NtNRT1.1 and NtNRT1.2 are/share more homologous to AtNRT1.1 than to AtNRT1.2 (AtNPF4.6) ( Supplemental Figure 1 ). For this reason, we have renamed NtNRT1.1 and NtNRT1.2 here as NtNRT1.1A and NtNRT1.1B, respectively.

To test the functionality of NtNRT1.1B for nitrate transport, a heterologous functional complementation assay in yeast (H. polymorpha) was performed. The NtNRT1.1B ORF was cloned into the yeast expression vector pYNR and transformed into the nitrate transporter-disrupted yeast mutant △ynt (△ynt, △leu) (see Materials and methods), which is unable to grow on less than 0.5 mM NO 3 − as a sole N source (Pérez et al., 1997). The result showed that compared with transformants carrying the empty vector pYNR, yeast cells of △ynt harboring NtNRT1.1B restored the growth on 0.5 mM NO 3 − , comparable to its WT strain, NCYC495 (△leu), transformed with pYNR as a positive control ( Figure 1 ). In addition, as some nitrate and peptide transporters characterized in the NRT1/PTR family are reported to be proton-coupled permeases for the movement of nitrate, potassium, and/or auxin (Wang et al., 2012; Watanabe et al., 2020), the effect of medium pH on NtNRT1.1B-facilitated NO 3 − transport in yeast was examined. However, pYNR-NtNRT1.1B-harboring transformants showed no obvious difference in growth rate at pH levels ranging from 5 to 7 ( Figure 1 ), suggesting that the NtNRT1.1B-encoding protein could mediate NO 3 − import from media across the plasma membrane (PM) of yeast cells independent of pH.

Furthermore, to survey the possibility of nitrate permeation through the cell membrane(s) via the NtNRT1.1B pathway, subcellular localization of NtNRT1.1B was determined using a protein fusion approach, in which a GFP gene (GFP) was C-terminally fused with the NtNRT1.1B ORF to yield NtNRT1.1B::GFP, which was constructed downstream of the CaMV 35S promoter (see Materials and methods). After transient expression of NtNRT1.1B::GFP in tobacco leaf epidermal cells, the GFP signal in the cells was visualized using confocal microscopy (see Materials and methods). NtNRT1.1B::GFP resulted in a green signal, in a fine-line pattern ( Figure 2B ), which overlapped substantially with the red fluorescence of the PM ( Figure 2A ) indicated by the signal derived from a 10-min incubation of the cells with the chemical FM 4-64 (Bolte et al., 2004). This overlap was clearly seen in the yellow of the superposition image that was the result of the merged green and red signals ( Figure 2C ), very similar to that of Arabidopsis high-affinity urea transporter AtDUR3 ( Figures 2E–G ), which is documented to be targeted at the PM (Liu et al., 2003). Thus, the subcellular localization of NtNRT1.1B should mainly reside on the PM of tobacco cells.

The spatiotemporal transcription of a functional gene is an important issue that may reflect its potential biological role in plant growth and development. To illustrate the expression patterns of NtNRT1.1B with regard to tissue/organ specificity and N nutritional response, a promoter activity assay using a GUS/GFP reporter approach was conducted. A 2,000-bp upstream sequence (Pro _NtNRT1.1B ) of NtNRT1.1B ORF was cloned directly before the GUS or GFP gene, and then transformed into tobacco (K326; see Materials and methods). The reporter expression reflecting promoter activity was visualized and/or quantified by (confocal) microscopic analysis (note that for the histochemical detection of GUS, plant tissues were subjected to GUS staining; see Materials and methods) ( Figure 3 ). The blue color derived from the Pro _NtNRT1.1B -tiggered GUS expression occurred clearly in the area of the young (3-week) shoot apex and lateral root primordia, and in the 6-week plant (primary) root and shoot–stem tissue, as well as flower tissues including the receptacle, stigma, anther, and pollen grains ( Figures 3A, B ). A close-up visualization of a cross-section of GUS-stained tissue further revealed a strong appearance of the Pro _NtNRT1.1B activity in the xylem parenchymal cells of the stem vascular stele ( Figure 3C ), and this expression pattern is comparable to an observation of Pro _NtNRT1.1B driven GFP occurrence in the root, where green fluorescence signals were strongly detected in the stele area of the mature zone of the primary root ( Figure 3D ).

To test the stability of the NtNRT1.1B promoter activity in the root, GFP signals were further quantitatively analyzed in the roots of 4-week-old tobacco plants, which were N starved for 1 or 3 days, with N resupplied (in the form of NO 3 − , NH 4 + , or Gln) to the 3-day N-starved plants for 1, 2, or 4 h (see Materials and methods) before measurement. As shown in Figure 4A , the green fluorescence intensity was strongly reduced by medium N depletion (e.g., for 1 or 3 days) in the roots compared with that in the control grown with continuous N provision. Resupply (for 1–4 h) of different N sources, including NtNRT1.1B’s putative substrate, NO 3 − , to the 3-day N-starved tobacco rapidly and markedly enhanced GFP accumulation in the roots relative to that of the control (i.e., the 3-day N-starved tobacco; Figure 4B ). Such N-status-regulated GFP expression patterns triggered by Pro _NtNNRT1.1B activity were confined only in a comparable root tissue area, namely the root stele in the mature zone ( Figure 4A ).

To appreciate the potential biological role of NtNRT1.1B in its native plant, an expression construct of NtNRT1.1B ORF driven by the CaMV 35S promoter was cloned and subsequently introduced into tobacco K326 (WT); three independent NtNRT1.1B overexpression (OE-NtNRT1.1B) homozygous lines were generated for the physiological study (see Materials and methods). Gene expression measurements showed that, in both roots and shoots/leaves, around fourfold higher mRNA abundance of NtNRT1.1B relative to that in the WT was confirmed by quantitative RT-PCR ( Figure 5A ).

For the growth phenotyping and N nutritional physiological assay, transgenic and WT tobacco plants were cultivated hydroponically with nitrate as a sole N source at different concentrations (0.5, 2.0, and 5.0 mM) for 25 days (see Materials and methods). Compared with the WT, OE-NtNRT1.1B lines were bigger and exhibited greater biomass production, particularly for shoots with 35%–46% higher upper part fresh weight under 0.5–5 nitrate provision ( Figures 5B, C ). Accordingly, the content of total N, NO 3 − , and NH 4 + in the shoots of OE-NtNRT1.1B plants was measured to be markedly higher than that of the control ( Figures 5D, E, G ). A similar tendency was also observed in the roots of the transgenic lines relative to the WT ( Figures 5C–E ), with the exception that root NH 4 + content was not significantly different between the WT and OE-NtNRT1.1B lines ( Figure 5G ). Metabolically, measuring the activity of two enzymes required for the first step of NO 3 − or NH 4 + assimilation revealed that NR action in the shoots was proportionally increased at elevated nitrate levels (e.g., ≈ 2–5 mM), and in particular was 20%–41% higher in the NtTR1.2-transgenic plants than in the WT ( Figure 5F ). However, the activity of NR in the roots and of GS in both shoots and roots remained comparably stable in both the transgenic and WT plants ( Figures 5F, H ). An increase in the activity of NR and GS in both roots and shoots with elevated medium N concentrations (0.5–5 mM) agrees with the observation reported by Fan et al. (2018). When plants were grown in pots with soil supplied with nitrate at four rates (0, 0.5, 2, and 5 mM; Supplemental Figure 2A ), results very similar to those achieved by hydroponics, described above, were observed ( Supplemental Figure 2 ).

To determine if the growth improvement in transgenic tobacco was directly linked to NO 3 − uptake/transport strengthened by NtNRT1.1B overexpression, we conducted a short-term root influx study using ¹⁵N- NO 3 − tracer under a nitrate supply at three 10-fold differing levels ( Figure 6A ; see Materials and methods). The measurement of ¹⁵N revealed that the NtNRT1.1B-transgenic tobacco roots could accumulate 15%–20% more NO 3 − (by exposure for 5 min or 30 min of roots to a NO 3 − containing solution) than the WT ( Figure 6A ); approximately 50% more NO 3 − was deposited in the shoots of the transgenic lines than in the shoots of the WT after a 30-min root exposure to 10 mM NO 3 − ( Figure 6A ), although overall NO 3 − accumulation in the shoots was lower than in the roots during this short-term NO 3 − supplementation. Thus, an increase in NtNRT1.1B expression in a constitutive pattern did enhance the root acquisition of NO 3 − from external media.

To further assess a transport property of NtNRT1.1B for NO 3 − across the plant cell membrane, short-term influx into the roots of 7-day hydroponically cultivated NtNRT1.1B-overexpressing plants (line 1) and their WT with or without 2 days’ N starvation, was measured after a 3-min exposure of the roots to the ¹⁵ NO 3 − containing nutrient solution [see Materials and methods; note: by 3 min uptake, an increase in¹⁵N in shoots was not detectable, indicating no movement of root-absorbed ¹⁵N to the upper parts of the plant (data not shown)]. At a range of sub-millimolar concentrations (5–500 µM tested), NO 3 − root influx showed that NtNRT1.1B did facilitate an import of NO 3 − into the roots, and followed Michaelis–Menten kinetics ( Figure 6B ). The root absorption via NtNRT1.1B was saturated at ≈ 500 μM NO 3 − , and exhibited maximal transport activity, i.e., V _max, at 29.38 ± 1.57 or 19.54 ± 0.75 μmol NO 3 −  g^–1 dry weight (DW) h^–1 as well as a transport affinity constant, K _m, at 29.05 ± 3.34 or 45.12 ± 9.07 μM for NO 3 − under root N starvation or N provision ( Figure 6B ), respectively. This kinetic property of NtNRT1.1B for NO 3 − transport was very similarly detected in another transgenic line ( Supplemental Figure 3 ). Although the measured values of K _m and V _max of NtNRT1.1B for NO 3 − are numerically different on N depletion or N supply in the growth medium, which may affect plant internal N status and/or metabolism, such K _m or V _max data fall into a similar value range. At higher NO 3 − concentrations (i.e., 1,000–7,000 µM), root NO 3 − accumulation values derived by subtracting the value in NtNRT1.1B-overexpressing plants from that in WT plants at the same external NO 3 − concentration could not fit any possible mechanism equation ( Figure 6B ). Thus, NtNRT1.1B could be considered as a high-affinity component for nitrate permease in tobacco plants.

As NtNRT1.1B represents a homolog of nitrate transporters and its promoter activity could be clearly detected in the stem xylem area and root stele ( Figures 3C, D ), we speculated that NtNRT1.1B might favor nitrate translocation from the roots to the upper parts of the plant. To test this hypothesis, the content of NO 3 − in the root xylem exudate and leaf AWF was measured. Four-week-old plants starved of N for 2 days or resupplied with 3 mM NO 3 − after 2 days’ N starvation were excised at the stem 2–3 cm above the root base for xylem exudate collection, and differently aged leaves were sampled for AWF extraction (see Materials and methods). Under 2-days’ N deprivation, NO 3 − in the root exudate was detectable with gradually reduced concentrations over the period of a continuous xylem sap collection (0–12 h), but there was no significant difference between WT and transgenic plants ( Figure 7A ). Following resupplementation of NO 3 − to the 2-day N-starved roots, NO 3 − concentration in the root exudate was 15%–20% higher in the OE-NtNRT1.1B lines than in the WT (except for the sample collected in the first 2 hours) ( Figure 7B ); this NtNRT1.1B overexpression-related elevation of NO 3 − in xylem saps was also clearly observed in the control treatment (CK, the plants grown with the normal nutrient solution containing 1 mM NH₄NO₃) ( Figure 7B ). Furthermore, preparation of the leaf AWF allowed us to determine a generally increased concentration of NO 3 − in the out-cell-space fluid of mature leaves ( Figure 7C ), in which NtNRT1.1B-transformed lines even showed a threefold higher concentration of NO 3 − in their AWF than the WT ( Figure 7C ). These data may indicate at least a significant effect or role for NtNRT1.1B in facilitating the long-distance translocation of nitrate between the roots and the upper parts of plants.