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Nitrate transporter 1.1 alleviates lead toxicity in Arabidopsis by preventing rhizosphere acidification

Exposure to Pb2+ increases NRT1.1-mediated uptake of nitrate in Arabidopsis and is associated with decreased uptake of Pb into the plant, which is a consequence of decreased acidification in the rhizosphere.


Introduction
Consumption of lead (Pb) can result in a variety of healthrelated ailments ranging from learning disabilities to cardiovascular disease (Chiodo et al., 2007;Navas-Acien et al., 2008). Crops can take up Pb from the soil and, under certain conditions, high levels can accumulate in the edible parts of crops (Jin et al., 2005a(Jin et al., , 2005b. The contamination of soil by Pb has continuously increased over recent decades as a result of various anthropogenic activities, such as waste disposal and the use of sewage water for irrigation (Clemens, 2006;Pourrut et al., 2011). Consequently, minimizing Pb in soil, particularly in an agricultural context, is a subject of increasing concern for researchers across a number of different scientific disciplines. Using plant-based technology to remove Pb from contaminated soils (phytoremediation) has gained a great deal of attention because of its low costs and environmental friendliness (Tangahu et al., 2011;Selamat et al., 2014). However, there are few natural hyperaccumulator plants for Pb and most of those that have been reported grow slowly and have small biomass, which limits their efficiency in cleaning contaminated soil (Ernst, 2000;van der Ent et al., 2013). Using a biological engineering approach to increase Pb accumulation in non-food crops with rapid growth and large biomass may be an alternative strategy to hyperaccumulating plants. A clear understanding of the factors that affect Pb uptake by plants is a prerequisite for ensuring that such a strategy is successful.
The amount of Pb taken up by plant roots is closely associated with its bioavailability in the growth medium (Kopittke et al., 2008;Uzu et al., 2009). The bioavailability of Pb in soil is affected by numerous factors including the cation exchange capacity (CEC), texture, redox potential, clay mineralogy, organic content, and pH of the soil, and also by the levels of other elements that are present (i.e. phosphorus and sulfur) (Jin et al., 2005b). Among these factors, soil acidity has been recognized as one of the most important (Fischer et al., 2014), because as pH decreases, the solubility of Pb increases in the form of free or solvated ions and ion pairs. Nutrient management, particularly nitrogen fertilization, significantly affects the pH of soil (Thomson et al., 1993). Nitrogen is taken up by plants mainly in the form of ammonium (NH 4 + ) and nitrates (NO 3 − ). Physiologically, when NO 3 − is taken up by plants, there is a simultaneous uptake of protons (H + ) resulting in an increase in the pH of the rhizosphere. Conversely, when NH 4 + is taken up, H + is released into the rhizosphere, resulting in a decrease in the pH (Marschner, 1995). In this context, the uptake of NO 3 − by roots may decrease the amount of Pb 2+ acquired by plants by consumption of rhizospheric H + , which thus lowers the level of soluble Pb 2+ , whereas the opposite may be true for uptake of NH 4 + .
Owing to the fact that Pb is a non-essential element for plants, its entry into root cells may rely on transporters/channels for various bivalent nutrient cations (Pourrut et al., 2011). In addition to H + , our previous studies have shown that decreased uptake of the anion NO 3 − by roots is accompanied by decreased uptake of several cations, including Ca 2+ , K + , Fe 2+ , and Cd 2+ (Luo et al., 2012;Mao et al., 2014), indicating that the uptake of NO 3 − may be non-specifically coupled with the uptake of cations. Hence, the uptake of nitrate may favor the uptake of Pb 2+ . However, this is in contrast with the effect noted above that nitrate uptake may decrease Pb bioavailability in the rhizosphere. Thus, it is not clear how NO 3 − uptake by roots affects Pb 2+ uptake. The pathways of NO 3 uptake by plant roots are complex. It has been reported that six NO 3 transporters (NRTs) are involved in uptake in Arabidopsis (Wang et al., 2012(Wang et al., , 2018Léran et al., 2014). NRT1.1 is one of the most common NRTs with dual-affinity, being involved in both high-and low-affinity uptake Ye et al., 2019), NRT1.2 is a low-affinity transporter , whilst NRT2.1, NRT2.2, NRT2.4, and NRT2.5 only take part in high-affinity uptake (Cerezo et al., 2001;Li et al., 2007;Kiba et al., 2012;Lezhneva et al., 2014). These NRTs often show different responses to various stressors (Krapp et al., 2011;Mao et al., 2014;Fang et al., 2016). Given the presumed association between NO 3 − and Pb 2+ uptake, it is clearly important to determine whether the activities of the different NRTs affect the uptake of Pb.
In the present study, we investigated the interaction between NRT-mediated NO 3 uptake and Pb 2+ exposure in Arabidopsis. Our results revealed a new mechanism by which the plants responded to Pb 2+ stress: exposure to Pb specifically induced NRT1.1-mediated NO 3 uptake, which consequently prevented acidification of the rhizosphere and thus decreased entry of Pb into the plants by decreasing its bioavailability. Our findings may provide a new strategy for manipulating Pb levels in plants, such as improving the efficiency of removal of Pb from contaminated soils and minimizing the accumulation of Pb in food crops.

Cultivation conditions
The experiments were performed on plants grown on agar medium in sterile square Petri dishes (10 × 10 cm). Briefly, the seeds were surfacesterilized and sown on basal agar medium containing 1% sucrose (w/v). The nutrient composition of the basal agar medium had a NO 3 − /NH 4 + ratio of 3:1 and was as follows: 750 μM NaH 2 PO 4 , 500 μM MgSO 4 , 375 μM K 2 SO 4 , 2.25 mM KNO 3 , 375 μM (NH 4 ) 2 SO 4 , 1 mM CaCl 2 , 10 μM H 3 BO 3 , 0.5 μM MnSO 4 , 0.5 μM ZnSO 4 , 0.1 μM CuSO 4 , 0.1 μM (NH 4 ) 6 Mo 7 O 24 , and 25 μM Fe-EDTA. Because nrt1.1 mutants are hypersensitive to low pH , the agar medium was adjusted to pH 6.5. For treatment with Pb, 4-d-old seedlings were transferred to Petri dishes with fresh basal medium with or without 300 μM lead acetate, Pb(CH 3 COO) 2 . Each dish contained 15 seedlings. To examine the effects of N source on the response to Pb 2+ , we used basal medium adjusted to contain different NO 3 − :NH 4 + ratios. The total N concentration fixed at 3.0 mM and the ratio was adjusted using KNO 3 and (NH 4 ) 2 SO 4 . The resulting differences in K concentrations were balanced by adjusting the K 2 SO 4 concentration.

Measurement of Pb 2+ concentrations
After 7 d of Pb 2+ treatment, the seedlings were washed with pure water, divided into shoots and roots, and dried at 70 °C for 48 h. The dried samples were then wet-digested as previously described (Guan et al., 2018), and diluted with ultrapure water. The concentrations of lead were analysed using a Microwave Plasma-Atomic Emission Spectrometer (4210 MP-AES, Agilent Technologies). Four biological replicates per treatment were analysed, each of which consisted of root or shoot tissues from 90 seedlings.

GFP expression analysis
Seedlings of the pNRT1.1::NRT1.1-GFP (green fluorescent protein) transgenic plants were cultured on complete nutrient agar medium with or without 300 μM Pb(CH 3 COO) 2 for 3 d. The distribution and intensity of the green fluorescence in their roots were then observed under a microscope (Eclipse NI; Nikon) and imaged using a camera attached to the microscope. Four replicate seedlings per treatment were analysed.

Measurement of NO 3 uptake
The net fluxes of NO 3 were measured along the root axis in the meristematic, elongation, and maturation zones using a non-invasive microelectrode ion flux measurement system (SIET IPA-2, Applicable Electronics, Inc., Forestdale, MA, USA) , which can directly record transmembrane ion influx and efflux in a non-contact manner by detecting the diffusion potentials outside of the membrane (Shabala and Newman, 1997). Seedlings were pre-cultured in complete nutrient medium with or without 300 μM Pb(CH 3 COO) 2 for 3 d, and were then transferred to 5-cm diameter plates with a nutrient solution containing 750 μM NaH 2 PO 4 , 500 μM MgSO 4 , 375 μM K 2 SO 4 , 2.25 mM KNO 3 , 375 μM (NH 4 ) 2 SO 4 , 1000 μM CaCl 2 , and 0.05% MES buffer (w/v), with a pH of 5.8. using NMT The NO 3 − fluxes of the roots were measured The flux of NO 3 in each section of the roots was then measured for 30 min using an electrode as described by Hawkins et al. (2008). Six replicate seedlings per treatment were analysed.

Measurement of relative expression of NRT genes
Root samples of ~100 mg (FW) were collected and frozen in liquid nitrogen. Total RNA was extracted using RNAisoPlus (Takara) and was then used to synthesize first-strand cDNA using a PrimeScript RT reagent kit (Takara). The cDNA was mixed with an SYBR Green RT-PCR kit (Takara) and the corresponding primers in a 25-μl reaction system; the primers are listed in Supplementary Table S1. All quantitative reverse-transcription (qRT-)PCR analyses were performed in an Opticon 2 Real-Time PCR System (MJ Research, MA, USA). Relative transcript levels were determined and corrected with efficiency calculations as described in Fang et al. (2016), and normalizeding to the geometric mean of expression of UBQ10 and EF1α for each sample (Vandesompele et al., 2002). Four biological replicates per treatment were analysed, each of which consisted of 75-90 seedlings.

Determination of lipid peroxidation
We measured malondialdehyde (MDA), a product of lipid peroxidation, according to the method described by Zhang et al. (2016). Samples of ~0.1 g of root or shoot tissue were frozen in liquid nitrogen and homogenized in 1.5 ml of 10% trichloroacetic acid (TCA) at 4 °C. After centrifugation at 15 000 g for 10 min, the supernatant was collected and incubated with 1 ml of 0.6% thiobarbituric acid (TBA) (dissolved in 10% TCA) at 100 °C for 20 min. The absorbance was measured at 440, 532, and 600 nm using an ELISA (SpectraMax 13X, Melceemlar Devices, USA). A total of 4-5 biological replicates per treatment were analysed, each of which consisted of 45-90 seedlings.

Determination of pH in the agar rooting medium
The pH of the agar rooting medium was determined based on the method described by Hachiya et al. (2012). The rooting medium was collected into a 50-ml centrifuge tube, which was frozen at -20 °C overnight and thawed at room temperature to free the aqueous phase from the agar. The mixture was then filtered at room temperature and the pH of the supernatant was determined using a desktop pH electrode (Sartorius). Measurements were taken on agar from five replicate plates per treatment, each of which contained 15 seedlings.

Pb 2+ stress stimulates NO 3 uptake by roots
Abiotic stress often negatively affects NO 3 − uptake by the roots (Mao et al., 2014;Bai et al., 2017). In the present study, we first evaluated the effect of Pb 2+ exposure on NO 3 − uptake in the roots of Arabidopsis Columbia-0 (Col-0) using a non-invasive technique. The transmembrane NO 3 − fluxes of the roots were measured along the root axis in the meristematic, elongation, and maturation zones. Interestingly, although Pb 2+ is a toxic metal for plants, 3 d of exposure to 300 μM Pb 2+ in the agar medium increased the rate of net NO 3 − influx in all zones (Fig.  1). The net NO 3 − influx in the mature root zone was greater than that in the other two zones, and exposure to Pb 2+ resulted in about twice the uptake of NO 3 − compared to the controls. To investigate the molecular basis underlying the stimulation of root NO 3 − uptake in response to Pb 2+ exposure, we examined the expression of six NRT genes that are involved in NO 3 − uptake by roots. We found that only the expression of NRT1.1 was significantly increased after 3 d of 300 μM Pb 2+ exposure ( Fig. 2A) whilst the expression of the other five genes, NRT1.2, NRT2.1,NRT2.2,NRT2.4,and NRT2.5 were not significantly affected ( Fig. 2B-E). We then examined the time-course of NRT1.1 expression in response to exposure to Pb 2+ and found that there was limited induction during the first 12 h of exposure, but after that it increased significantly ( Supplementary Fig. S1). These results indicated that the stimulation of NO 3 − uptake by Pb 2+ may have been mediated by NRT1.1. Consistent with this, GFP fluorescence was observed in the roots of pNRT1.1::NRT1.1-GFP transgenic plants (which are in the chl1-5 background, an NRT1.1-null mutant) and indicated that exposure to Pb 2+ caused a clear increase in NRT1.1-GFP protein levels ( Fig. 2F). We then compared the rate of net NO 3 − influx by the roots of wild-type Col-0 plants and two NRT1.1-null mutants, chl1-5 and nrt1.1-1. Exposure to Pb 2+ had no effect on influx in the nrt1.1 mutant in any of the three root zones that we examined (Fig. 3), which was in contrast with the results for the Col-0 plants. These results demonstrated that induction of NRT1.1 activity was responsible for the increase in root NO 3 − uptake in the presence of exposure to Pb 2+ .

Pb 2+ resistance in Arabidopsis is specifically associated with NRT1.1
We investigated the association between NRTs and Pb resistance in Arabidopsis. Because NRT1.1 appeared likely to be responsible for the stimulation of NO 3 − uptake by Pb 2+ , we first examined the effects of its loss of function on resistance to Pb stress. After 7 d of exposure to 300 μM Pb 2+ , the inhibition of root growth in the NRT1.1-null mutants chl1-5, nrt1.1-1, and chl1-6, was greater than that in their corresponding wild-type plants (Fig. 4A, B, Supplementary Fig. S2). In addition, reductions in root elongation over time in response to exposure to Pb 2+ were greater in the NRT1.1-null mutants compared to the wild-types ( Supplementary Fig. S3) and there was a significant interaction between NRT1.1 function and exposure to Pb 2+ (P<0.05). These results indicated that NRT1.1 is required for Pb 2+ resistance, and further evidence for this was provided by Measurements of root growth in pNRT1.1::NRT1.1-GFP transgenic plants, which showed that the inhibition of root elongation in the NRT1.1-null mutants could be rectified by complementation with NRT1.1 (Supplementary Fig. S4). Lipid peroxidation is often used as an indicator of stress effects and therefore we measured the amounts of MDA (a product of this process; Hodges et al., 1999) in the roots and shoots. After 7 d of exposure to 300 μM Pb 2+ , the MDA levels in both the roots and shoots of the chl1-5 and nrt1.1-1 mutants were greatly increased, whereas there was no significant effect in the Col-0 plants (Fig. 4C). This provided further support for NRT1.1 being required for Pb 2+ resistance in Arabidopsis.
NRT1.1 also functions as a NO 3 sensor, and this is independent of its uptake activity (Ho et al., 2009;Bouguyon et al., 2015). We therefore examined Pb 2+ resistance in the chl1-9 mutant, which is defective in terms of NO 3 uptake but has a normal NO 3 sensing function (Ho et al., 2009). We found that chl1-9 had a similar Pb 2+ resistance phenotype in terms of root growth to the other nrt1.1-knockout mutants ( Supplementary  Fig. S5), indicating that NO 3 − uptake rather than sensing was the likely mechanism behind NRT1.1-conferred Pb 2+ resistance. We also checked the Pb 2+ resistance of the nlp7-knockout mutant, which shares common features with the nrt1.1-knockout mutant in terms of loss of many NO 3 sensing functions, but it retains normal root NO 3 uptake activity (Castaings et al., 2009;Marchive et al., 2013). As expected, the Pb 2+ resistance of nlp7 was similar to that of the wild-type plants ( Supplementary Fig.  S5). These results provided further support for NO 3 − -uptake activity rather than NO 3 − -sensing activity being involved in NRT1.1-mediated Pb 2+ resistance.
We then examined associations between Pb toxicity and other NRTs and found that the nrt1. 2, nrt2.1, nrt2.2, nrt2.4, and nrt2.5 mutants all had similar root elongation to their corresponding wild-types ( Supplementary Fig. S6). Owing to the fact that NRT2.1, NRT2.2, NRT2.4, and NRT2.5 are high-affinity NO 3 − uptake transporters, we also evaluated the effects of Pb 2+ exposure on root growth in the corresponding NRT-null mutants under growth conditions with 0.2 mM NO 3 − . We found that root elongation in nrt2. 1, nrt2.2, nrt2.4, and nrt2.5 was similar to that of the corresponding wild-type plants ( Supplementary Fig. S7). We also examined Pb sensitivity in the nrt2.1 nrt2.2 double-mutant, which has only 20-40% of the high-affinity NO 3 − uptake of the wild-type (Li et al., 2007;Lezhneva et al., 2014). Again, we found that this double-mutant also had similar root elongation compared to the wild-type in growth medium containing 0.2 mM NO 3 − ( Supplementary Fig. S8). These results suggested that NRT1.2, NRT2.1, NRT2.2, NRT2.4, and NRT2.5 are not involved in Pb 2+ resistance in Arabidopsis, or at least they do not act like NRT1.1 in conferring resistance.

The level of Pb accumulated in Arabidopsis is specifically controlled by NRT1.1
We investigated how NRT1.1 affected the levels of Pb accumulated in plants. After 7 d of exposure to 300 μM Pb 2+ , the concentrations in the roots of the chl1-5 and nrt1.1-1 mutants were ~3-fold higher than that in the Col-0 plants (Fig. 5). In addition, the mutants also had ~80% higher concentrations in their shoots. Similar differences was also found between the  Supplementary Fig. S10). These results indicated that NRT1.1 negatively regulated the Pb concentrations in the plant tissues, providing further evidence that NRT1.1 is required for resistance to Pb 2+ exposure in Arabidopsis. It is  Effects of Pb 2+ stress on net NO 3 − influx in different root sections of Arabidopsis wild-type Col-0, and the nrt1.1-1 and chl1-5 mutants. Seedlings at 4 d old were transferred to medium with or without 300 μM Pb(CH 3 COO) 2 and net NO 3 − flux was measured after 3 d using a non-invasive microelectrode ion flux measurement system (Fig. 1). Measurements were taken in the meristematic (A), elongation (B), and maturation zones. Data are means (±SE) of four biological replicates. Different letters indicate significant differences between means as determined using two-way ANOVA followed by Tukey's multiple comparisons test (P<0.05). Significant interactions between Pb treatment and genotype are indicated by an asterisk (*P<0.05). (This figure is available in colour at JXB online.) worth noting that in all the plant lines examined, the Pb concentrations in the shoots were much lower than that in the roots. This was probably because Pb has extremely low mobility in plants (Pourrut et al., 2011), and hence most will remain in the root tissues after being absorbed from the growth medium.
We also examined whether the other NRTs had a role in affecting the accumulation of Pb, and found that the nrt1. 2, nrt2.1, nrt2.2, nrt2.4, and nrt2.5 mutants had similar Pb concentrations in their roots and shoots as their corresponding wild-types when grown in both sufficient (2.25 mM) or low-NO 3 − (0.2 mM) conditions ( Supplementary Fig. S11). in addition, the nrt2.1 nrt2.2 double-mutant also had similar Pb concentrations to the Col-0 plants when grown with 0.2 mM NO 3 − (Supplementary Fig. S8). Thus, the NRT1.2, NRT2.1, NRT2.2, NRT2.4, and NRT2.5 transports do not play a role in affecting the Pb in Arabidopsis, leading us to conclude that NRT1.1 negatively regulates the Pb in a relatively specific manner.

NRT1.1-mediated reduction of Pb 2+ uptake is associated with increased pH in the rhizosphere
As noted in the Introduction, uptake of NO 3 − may nonspecifically couple with the uptake of cations and thus favor uptake of Pb 2+ , whilst the simultaneous uptake of H + will increase the pH of the rhizosphere and thus reduce the bioavailability of Pb 2+ . Given that we observed a decrease in uptake of Pb 2+ , we measured the pH in the agar growth medium, which was initially set to 6.5. After 7 d of treatment with 300 μM Pb 2+ , the pH in the media of the NRT1.1-knockout mutants decreased to ~4.0, whilst the pH in the Col-0 medium was ~5.0 (Fig. 6A).Computer modelling using GEOCHEM-PC (Parker et al., 1995) indicated that the activity of Pb 2+ in our growth medium was sharply reduced when the pH increased ( Supplementary Fig. S12). It is therefore likely that the higher concentrations of Pb accumulated in the nrt1.1-knockout mutants probably resulted from the increased Pb 2+ availability that occurred because of the lower pH in the rhizosphere. We then used MES to buffer the pH in the growth media. After 7 d of Pb 2+ treatment, the presence of 0.05% MES (w/v) substantially reduced the differences in pH between the Col-0 rooting medium and those of the two nrt1.1 mutants ( Supplementary Fig. S13). This was associated with significantly reduced concentrations of Pb in both the roots and shoots of the mutants (Fig. 6C), suggesting that there was an interaction between NRT1.1 and the pH of the rhizosphere in the uptake of Pb. Thus, the results provided direct evidence that the negative effect of NRT1.1 on Pb uptake was associated with pH-determined Pb availability in the rhizosphere. Given that the toxicity of Pb is closely associated with the concentration that is accumulated in plant tissues (Pourrut et al., 2011), we evaluated the effect of NRT1.1 on plant resistance to exposure in the presence of MES. As expected, the MES treatment almost completely removed the Pb 2+ -related inhibition of root growth in the nrt1.1 mutants (Fig. 6B). Taken together, the results indicated that upon exposure to Pb 2+ , up-regulation of NRT1.1 in the roots reduced acidification of the rhizosphere and hence reduced Pb 2+ uptake, thus creating a mechanism whereby the plants increased their resistance to Pb 2+ toxicity.

An efficient contribution of NRT1.1 to Pb 2+ resistance requires the co-supply of NO 3 and NH 4 +
As the resistance to entry of Pb 2+ into the plants associated with NRT1.1 depended on alterations in the pH of the rhizosphere, we wanted to assess how this was affected by the form in which nitrogen was supplied. We therefore fed Col-0, nrt1.1-1, and chl1-5 plants with different ratios of NO 3 to NH 4 + (N:A ratio). When NH 4 + was supplied as the sole N source, the root growth of Col-0 plants was greatly inhibited by exposure to Pb 2+ , and they had similar root elongation to the nrt1.1 mutants (Fig. 7A). This was associated with equally strong acidification of the rhizosphere (Fig. 7B), which would have resulted in high Pb 2+ availability ( Supplementary Fig. S12). When NO 3 was supplied together with NH 4 + , the root growth of the Col-0 plants improved progressively as the N:A ratio increased, whilst in the nrt1.1 mutants there was only an initial small increase in growth at an N:A ratio of 1:3. The variations in root growth were associated with corresponding changes in the pH of the growth media (Fig. 7B). When NO 3 was supplied as the sole N source, exposure to Pb 2+ had little effect on the root growth of any of any of the plants and this was associated with high pH in the growth media. These results indicated that an efficient contribution of NRT1.1 to Pb 2+ resistance in Arabidopsis required the co-supply of NO 3 and NH 4 + . We then measured the Pb concentrations in the plants and found that in growth media with either NH 4 + or NO 3 − supplied as the sole N source there were no differences between Col-0 and the mutants in either the roots or shoots (Fig. 7C, D). However, when NH 4 + and NO 3 − were co-supplied, both the mutants had higher Pb concentrations in their roots and shoots compared with the Col-0 plants, and this correlated with the differences in pH in the growth media and with the root growth phenotypes. These results further indicated that the presence of both NO 3 and NH 4 + in the growth medium enhances the contribution of NRT1.1 to Pb 2+ resistance in Arabidopsis.
We also examined the effects of Pb 2+ exposure on the expression of NRT1.1 and on the influx of NO 3 − into roots in response to different N:A ratios. In Col-0, both the expression of NRT1.1 and the influx of NO 3 − were similar between control and Pb 2+ -treated plants when either NH 4 + or NO 3 − was supplied as the sole N source, but both were up-regulated by exposure to Pb 2+ when NH 4 + and NO 3 − were co-supplied (Supplementary Figs S14, S15).Furthermore, two-way ANOVA of the root NO 3 − influx data confirmed that the NRT1.1 transporter was required for the up-regulation of NO 3 − influx that resulted from exposure to Pb 2+ in the growth media containing both NH 4 + and NO 3 − .These results suggested that co-supply of NH 4 + and NO 3 − ensured the induction of NRT1.1-mediated NO 3 − uptake in response to exposure to Pb 2+ .

Discussion
The removal of heavy metals from contaminated soils and minimizing their accumulation in food crops are two important challenges that span scientific disciplines (Alexander et al., 2006;Tangahu et al., 2011). Identification of the mechanisms that control the entry of these pollutants into plant tissues is essential if we wish to address these challenges by using biotechnology to engineer modifications of relevant metabolic pathways. In this study, we have demonstrated the existence of a mechanism by which Arabidopsis prevents uptake of Pb 2+ by its roots, namely that exposure to Pb 2+ stimulates NRT1.1mediated uptake of NO 3 − and this results in removal of H + from the rhizosphere, which in turn lowers the bioavailability of Pb 2+ , thus reducing its uptake into the plant.
Pb 2+ can cause damage to plants at both the physiological and molecular levels (Pourrut et al., 2011). The presence of high levels of Pb 2+ can lead to inhibition in photosynthesis, Fig. 5. Pb concentrations in Arabidopsis wild-type Col-0, and the nrt1.1-1 and chl1-5 mutants exposed to Pb 2+ stress. Seedlings at 4 d old were transferred to medium with or without 300 μM Pb(CH 3 COO) 2 and measurements were taken after 7 d. Data are means (±SE) of four biological replicates. Different letters indicate significant differences between means as determined using two-way ANOVA followed by Tukey's multiple comparisons test (P<0.05). (This figure is available in colour at JXB online.) an increase in the ATP/ADP ratio, detrimental changes in the cell cycle, breaks in single-and double-strands of DNA, and the accumulation of reactive oxygen species (ROS) (Verma and Dubey, 2003;Patra et al., 2004;Romanowska et al., 2005Romanowska et al., , 2006Gichner et al., 2008). Thus, Pb 2+ stress might be expected to have an adverse effect on NO 3 − uptake by roots; however, we found that exposure to Pb 2+ stimulated NO 3 − uptake in Arabidopsis roots under our experimental growth conditions (Fig. 1). Although several nitrate transporters (NRTs) are involved in NO 3 − uptake in Arabidopsis, we found that Pb 2+ only stimulated NRT1.1, as evidenced by its increased expression in seedlings and increased levels of transcripts in the roots (Fig.  2). Furthermore, the loss of function of NRT1.1 in nrt1.1 mutants abolished the effect of Pb 2+ on root NO 3 − uptake (Fig. 3). These results raised the question of how NRT1.1-controlled NO 3 − uptake was induced by exposure to Pb 2+ . Time-course analysis showed that the induction of NRT1.1 was initially slow and significant increases were not detected until after12 h of exposure ( Supplementary Fig. S1), suggesting that induction was probably the result of the progression of Pb toxicity. Several studies have shown that Pb 2+ stress results in a significant increases in the level of endogenous abscisic acid (ABA) in plants (Parys et al., 1998;Atici et al., 2005;Cenkci et al., 2010), and ABA has been shown to significantly up-regulate the expression of NRT1.1 in roots (Kiba et al., 2011). This may provide a mechanism by which Pb 2+ stimulates NRT1.1-controlled uptake ofNO 3 − but further research is needed. Comparison of growth responses to Pb 2+ exposure in various NRT-related mutants also indicated that only the NRT1.1 transporter was required for plant resistance to Pb 2+ (Fig. 4, Supplementary Figs S2, S3). NRT1.1 was initially identified as a NO 3 transporter responsible for root NO 3 uptake (Tsay et al., 1993), but it has also been shown to function as a NO 3 sensor and an auxin transporter, and these two functions are independent of its uptake activity (Ho et al., 2009;Krouk et al., 2010;Bouguyon et al., 2015). Consequently, NRT1.1 has been shown to be involved in many physiological processes, including the regulation of root growth (Guo et al., 2001;Fig. 6. Effects of pH on Pb 2+ toxicity in Arabidopsis wild-type Col-0, and the nrt1.1-1 and chl1-5 mutants. Seedlings at 4 d old were transferred to medium with or without 300 μM Pb(CH 3 COO) 2 and with or without the pH buffer MES, and measurements were taken after 7 d. The initial pH of the growth medium was 6.5. (A) The pH of the growth medium with Pb 2+ and without pH-buffer. (B) Representative images of plants grown with pH buffer and with or without Pb 2+ . (C) Pb concentrations in roots and shoots of plants grown with Pb 2+ and with or without pH buffer. Data are means (±SE) of 4-5 biological replicates. Different letters indicate significant differences between means as determined using either one-or two-way ANOVA (depending on whether one or two different variables were considered) followed by Tukey's multiple comparisons test (P<0.05). Significant interactions between Pb treatment and genotype are indicated by an asterisk (*P<0.05). (This figure is available in colour at JXB online.) Remans et al., 2006), control of the expression of the gene for another NO 3 − transporter, NRT2.1 (Muños et al., 2004;Ho et al., 2009;Bouguyon et al., 2015), and regulation of resistance to NH 4 + , Cd 2+ , and H + stress (Hachiya et al., 2011;Mao et al., 2014;Fang et al., 2016). Most of these NRT1.1associated processes are dependent on the presence of NO 3 − in the growth medium, as they act through either NO 3 − uptake activity or the NO 3 − sensing function (Wang et al., 2012). In our current study, we found that the NRT1.1-conferred resistance to Pb 2+ probably depends on the NO 3 − -uptake activity ( Supplementary Fig. S14). Thus, the induction of NRT1.1mediated NO 3 − -uptake as a result of exposure to Pb 2+ (Fig. 1) may be part of the response mechanisms to enhance Pb resistance. As noted above, an increase in endogenous ABA in plants may be a factor leading to NRT1.1 induction under Pb 2+ stress. In relation to this, found that the ABA-hypersensitive triplemutants abi1 hab1 abi2 and abi1 hab1 pp2ca (Fujii et al., 2009) had a greater resistance to Pb 2+ stress than the wild-type plants ( Supplementary Fig. S16). Measurement of Pb concentrations showed that the effect of NRT1.1 resulted from a reduction in the entry of Pb 2+ into the plants (Fig. 5). The amount of Pb 2+ taken up by roots is highly associated with its availability in the rhizosphere, which is significantly affected by pH (Fischer et al., 2014). Here, we found that the loss function of NRT1.1 in nrt1.1-knockout mutants resulted in a lower pH in the growth medium (Fig. 6A), which favored an increase in Pb 2+ activity, and thus increasing its uptake by the plants. This effect was greatly reduced when the pH-buffer MES was present in the growth medium (Fig. 6B, C), leading us to conclude that the reduction in uptake of Pb 2+ associated with NRT1.1 occurred because of the consumption of H + by root cells during NO 3 − uptake, which increased the pH of the rhizosphere and thus decreased Pb 2+ solubility. This was supported by the observation that the H + concentration of the growth medium was highly correlated with the Pb concentrations in the tissues of wild-type and mutant plants supplied with various N:A ratios ( Supplementary Fig. S17).
NO 3 − uptake activity is a common function for NRTs (Wang et al., 2012;Léran et al., 2014), but we found that only NRT1.1-mediated uptake played a role in plant resistance to Pb 2+ , whilst the other NRTs that we examined had little effect. Our previous study had shown that efficient prevention of acidification in the rhizosphere requires a significant consumption of H + , which can be achieved through adequate NO 3 uptake by the roots . Thus, insufficient uptake of NO 3 may lead to insufficient consumption of H + that is inadequate to decrease Pb 2+ solubility in the rhizosphere. In the current study, we found that knockout of NRT1.1 resulted in a decrease in NO 3 − uptake rate of >50% in the presence of Pb 2+ (Fig. 3), which suggested that the other five NRTs (i.e. NRT1. 2,NRT2.1,NRT2.2,NRT2.4,and NRT2.5) are responsible for <50% of the total root NO 3 uptake. The AtNRT2 transporters have functional redundancy for high-affinity nitrate uptake (Li et al., 2007;Lezhneva et al., 2014); however, the nrt2.1nrt2.2 double-mutant had similar root elongation and Pb concentrations as wild-type plants when grown in low-nitrate medium ( Supplementary Fig. S8). Hence, relatively low levels of NO 3 uptake by the other five NRTs may explain why they were not as effective as NRT1.1 in inhibiting Pb 2+ uptake. It is worth noting that Pb 2+ can be easily precipitated by phosphates, which may have affected the phosphate availability under our growth conditions. Lowering the pH would increase the solubility of Pb-phosphate precipitates in the medium (Sauvé et al., 1998) and hence the lower pH in the rhizosphere of the nrt1.1-knockout mutants compared with that of the wild-type would favor greater solubilization of phosphate precipitates. This suggests that it is more likely that the inhibition of root growth in the mutants was the result of greater Pb 2+ solubility rather than insufficient phosphate availability.
Interestingly, co-supply of NH 4 + and NO 3 − in the growth medium was required to ensure the induction of NRT1.1mediated NO 3 − uptake in response to Pb 2+ exposure (Supplementary Figs S14, S15). Although the NRT1.1mediated process of resistance depended on NO 3 uptake, the presence of NH 4 + was also required, as evidenced by the fact that there were no differences in root growth and the pH in the growth medium between the wild-type and mutants when NO 3 was the sole N source (Fig. 7A, B). As previously noted, NO 3 − uptake by roots consumes H + , whereas NH 4 + uptake produces H + (Marschner, 1995), and thus if a plant only takes up NO 3 and not NH 4 + then the pH in the rooting medium would increase. In addition, if a plant is fed with NO 3 as its sole N source, feedback inhibition would limit the increase in pH in the rooting medium to a level that the plant could tolerate (Imsande, 1986;Helali et al., 2010), because beyond that the plant would be damaged. This may be the reason why the pH of the growth medium for both the wild-type and the nrt1.1 mutants increased and reached a maximum of ~6.0 as the N:A ratio increased and NO 3 became the sole N source (Fig. 7B). This led to a similar root growth between these plants in the presence of Pb 2+ (Fig. 7A). When NO 3 and NH 4 + were both present in the growth medium, the loss of function of NRT1.1 may have resulted in H + consumption through NO 3 uptake mediated by the other NRTs, but this was not sufficient to counteract H + production by NH 4 + uptake, hence leading to greater acidification in the rhizosphere. This may explain why only the co-supply of NO 3 and NH 4 + could result in higher Pb accumulation and lower Pb 2+ resistance in the nrt1.1 mutants compared to the wild-type. It may be possible to use biotechnological modifications of NRT1.1 activity to effectively manipulate Pb levels in plants because NO 3 and NH 4 + are often both present in agricultural soils.
In summary, in this study we found that NRT1.1 negatively regulates Pb levels in Arabidopsis by preventing acidification of the rhizosphere through consumption of H + in the process of NO 3 uptake, which has the effect of reducing the bioavailability of Pb 2+ . Induction of NRT1.1 may therefore be considered as a regulatory mechanism used by Arabidopsis to cope with Pb 2+ stress.