Ammonium Transporter (BcAMT1.2) Mediates the Interaction of Ammonium and Nitrate in Brassica campestris

The provision of ammonium (NH₄⁺) and nitrate (NO₃⁻) mixture increases the total nitrogen (N) than the supply of sole NH₄⁺ or NO₃^– with the same concentration of total N; thus, the mixture contributes to better growth in Brassica campestris. However, the underlying mechanisms remain unknown. In this study, we analyzed NH₄⁺ and NO₃^– fluxes using a scanning ion-selective electrode technique to detect under different N forms and levels in B. campestris roots. We observed that the total N influxes with NH₄⁺ and NO₃⁻ mixture were 1.25- and 3.53-fold higher than those with either sole NH₄⁺ or NO₃⁻. Furthermore, NH₄⁺ and NO₃^– might interact with each other under coexistence. NO₃^– had a positive effect on net NH₄⁺ influx, whereas NH₄⁺ had a negative influence on net NO₃^– influx. The ammonium transporter (AMT) played a key role in NH₄⁺ absorption and transport. Based on expression analysis, BcAMT1.2 differed from other BcAMT1s in being upregulated by NH₄⁺ or NO₃⁻. According to sequence analysis and functional complementation in yeast mutant 31019b, AMT1.2 from B. campestris may be a functional AMT. According to the expression pattern of BcAMT1.2, β-glucuronidase activity, and the cellular location of its promoter, BcAMT1.2 may be responsible for NH₄⁺ transport. Following the overexpression of BcAMT1.2 in Arabidopsis, BcAMT1.2-overexpressing lines grew better than wildtype lines at low NH₄⁺ concentration. In the mixture of NH₄⁺ and NO₃^–, NH₄⁺ influxes and NO₃^– effluxes were induced in BcAMT1.2-overexpressing lines. Furthermore, transcripts of N assimilation genes (AtGLN1.2, AtGLN2, and AtGLT1) were significantly upregulated, in particular, AtGLN1.2 and AtGLT1 were increased by 2.85–8.88 times in roots, and AtGLN1.2 and AtGLN2 were increased by 2.67–4.61 times in leaves. Collectively, these results indicated that BcAMT1.2 may mediate in NH₄⁺ fluxes under the coexistence of NH₄⁺ and NO₃^– in B. campestris.

Introduction

The efficiency and availability of nitrogen (N) have decisive influences on plant growth and crop productivity (Hachiya and Sakakibara, 2017). For most plants, nitrate (NO₃^–) and ammonium (NH₄⁺) are major sources of inorganic N. In C₃ plants, NO₃^– reduction is inhibited by elevated carbon dioxide (CO₂), whereas NH₄⁺ assimilation is affected little (Bloom et al., 2010). NH₄⁺ is believed to be a preferable N source for the future when global levels of CO₂ are predicted to increase (Hachiya and Sakakibara, 2017). However, NH₄⁺ at millimolar concentrations in the soil solution or hydroponic culture causes growth suppression and chlorosis (ammonium toxicity) in plants, unlike NO₃^– at the same concentration (Miller and Cramer, 2004).

Extensive studies suggest that a mixture of NO₃^– and NH₄⁺ nutrition stimulates plant growth beyond that observed with NO₃^– or NH₄⁺ alone (Britto and Kronzucker, 2001). The use of the mixture enhances N-use efficiency and improves crop productivity (Wang and Shen, 2011; Hachiya et al., 2012). The mixture greatly improves plant growth and population productivity in maize, especially in high planting density (Wang et al., 2019). When NO₃^– and NH₄⁺ co-exist, NH₄⁺ responses are altered by NO₃^– and vice versa (Hachiya and Sakakibara, 2017). Previous researchers have investigated the interaction between NH₄⁺ and NO₃^– fluxes. Compared with the influx with sole NH₄⁺, net NH₄⁺ influx has been shown to increase with a mixture of NH₄⁺ and NO₃^– in rice using an N labeling technique (Kronzucker et al., 1999); and a similar effect has been observed in Brassica napus (Babourina et al., 2007), Populus popularis (Luo et al., 2013), and Triticum aestivum (Zhong et al., 2015) using the microelectrode technique, whereas a negative effect has been observed in tea (Ruan et al., 2016). Similarly, NH₄⁺ affects NO₃^– fluxes (Kronzucker et al., 1999; Zhong et al., 2015; Ruan et al., 2016). Therefore, the interaction between NH₄⁺ and NO₃^– may depend on plant species or N conditions.

Under natural conditions, plant growth and development are typically limited by N availability; thus, plants have evolved different transport and signaling mechanisms to adapt to different N sources (Kiba and Krapp, 2016). NH₄⁺ and NO₃^– fluxes are mediated by specific genes for ammonium transporters (AMTs) and nitrate transporters (NRTs), respectively (Nacry et al., 2013). In Arabidopsis, NRTs include 72 members belonging to four families: nitrate transporter 1/peptide transporter family (NRT1/PTR), NRT2, chloride channels (CLC), and slow anion channel-associated 1/slow anion channel homologs (SLAC1/SLAH) (Krapp et al., 2014). Some of these genes are related to NO₃^– uptake, xylem loading, and efflux systems (Krapp et al., 2014). AMTs generally contain AMT1 and AMT2 subfamilies (Loque and von Wirén, 2004; McDonald and Ward, 2016). In Arabidopsis, AtAMT1.1, AtAMT1.2, AtAMT1.3, and AtAMT1.5 are expressed in roots (Yuan et al., 2007), and play different roles during NH₄⁺ assimilation (Yuan et al., 2007). AtAMT1.1, AtAMT1.3 and AtAMT1.5 contribute to NH₄⁺ absorption from the soil, whereas AtAMT1.2 mediates NH₄⁺ uptake via the apoplastic transport route (Yuan et al., 2007), and exclusively regulates NH₄⁺ flux into the vasculature (Straub et al., 2017). Furthermore, plant cells eliminate the activity of AMT1.1 (Lanquar et al., 2009) or AMT1.3 (Wang et al., 2013) to avoid excessive NH₄⁺ accumulation.

AMTs transcript levels are affected by the N status of plants. N deficiency strongly induces AMT1.1, AMT1.3, and AMT1.5 transcription (Yuan et al., 2007; Camañes et al., 2009), whereas that of AMT1.2 is not affected to a large extent (Pearson et al., 2002). When NH₄⁺ is resupplied to N-deficient plants, AMT1.1, AMT1.3, and AMT1.5 genes are downregulated (Yuan et al., 2007); whereas AMT1.2 is upregulated (Pearson et al., 2002; Yuan et al., 2007). Furthermore, AMTs transcript levels are subjected to control by NO₃^– (Camañes et al., 2009). However, AMT homologs in different species are often not similarly regulated, which may reflect the different nutritional needs of particular species (Loque and von Wirén, 2004).

Flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee) is a prominent vegetable in South China due to the taste and nutrient content of its flower stalk, and it has the largest growing area and yield in South China (Song et al., 2012). In our previous study, we showed that NH₄⁺ and NO₃^– mixtures were more beneficial to B. campestris qualities than sole N source, and they improved N-use efficiency (Song et al., 2012). However, there is no information regarding the interactions between NH₄⁺ and NO₃^– and how this affects N uptake at physiological, morphological, and molecular levels. In this study, we examined the characteristics of NH₄⁺ and NO₃^– fluxes and their interactions in B. campestris using the scanning ion-selective electrode technique (SIET). Regarding the analysis of AMT1s transcripts, we observed that the expression pattern of BcAMT1.2 differed from those of other BcAMT1s in B. campestris. Furthermore, the GUS activity of BcAMT1.2_pro::GUS and used reverse genetic approaches in Arabidopsis suggested to elucidate the physiological roles of BcAMT1.2 in response to the coexistence of NH₄⁺ and NO₃^–. Altogether, these results indicated that BcAMT1.2 participated in the interaction between NH₄⁺ and NO₃^– in B. campestris.

Materials and Methods

Plant Materials and Culture Conditions

The flowering Chinese cabbage variety “Youlv80”, which was provided by the Guangzhou Academy of Agriculture Sciences (Guangdong Province, China), was used in this study. Experiments were carried out in a controlled-environment growth chamber programmed for 16 h light/8 h dark and a 25/23°C day/night cycle, relative humidity of 70%, and light intensity of 150 μmol m^–2 s^–1. Seeds were sterilized in 2.5% (w/v) NaClO for 10 min, washed five times with sterile distilled water, and cultured on vertical 0.7% agar plates (17.5 cm long × 16 cm wide × 3 cm high). The agar medium contained 1/2 no-N basal modified MS salt (pH 5.8), supplemented with 4 mmol L^–1 NaNO₃ as the N source. On the 6^th day of germination, the seedlings were hydroponically cultured in 1/2 MS as an N-deficient treatment for 7 d. The nutrient solution was replaced every 2 days and continually aerated by air pumps. After N starvation, the seedlings were harvested to measure ion fluxes or other treatments.

Measurement of NH₄⁺ and NO₃^– Ion Fluxes on the Surface of B. campestris Roots

To monitor net fluxes of NH₄⁺ and NO₃^– in B. campestris roots in response to different N treatments, the primary roots were selected and immersed in measuring solutions with different treatment [A. 0.25 mmol L^–1 NH₄⁺: 0.1 mmol L^–1 CaCl₂, 0.3 mmol L^–1 2-(N-morpholino) ethanesulfonic acid hydrate buffer (MES) (pH5.8, same as below), and 0.25 mmol L^–1 NH₄Cl; B. 1.0 mmol L^–1 NH₄⁺: 0.1 mmol L^–1 CaCl₂, 0.3 mmol L^–1 MES, and 1.0 mmol L^–1 NH₄Cl; C. 0.25 mmol L^–1 NO₃^–: 0.1 mmol L^–1 CaCl₂, 0.3 mmol L^–1 MES, and 0.25 mmol L^–1 NaNO₃; D. 1.0 mmol L^–1 NO₃^–: 0.1 mmol L^–1 CaCl₂, 0.3 mmol L^–1 MES, and 1.0 mmol L^–1 NaNO_3; E. NH₄⁺ + NO₃^–: 0.1 mmol L^–1 CaCl₂, 0.3 mmol L^–1 MES, 0.25 mmol L^–1 NH₄Cl, and 0.75 mmol L^–1 NaNO₃]. Prior to analysis, B. campestris roots were transferred to Petri dishes containing 10 mL of measuring solution and equilibrated for 10 min. The equilibrated roots were moved to another Petri dish containing fresh measuring solution to measure NH₄⁺ or NO₃^– flux. Ion flux was measured using SIET (MA01002 system; Younger USA Science and Technology Limited Liability Company, Amherst, MA, USA), which was conducted on-site at Xuyue Science and Technology Company Limited (Beijing, China). The SIET system and its application process for ion flux detection have been previously described in detail (Zhong et al., 2015; Ruan et al., 2016).

To determine the regions along the root where the maximal ion influxes of NH₄⁺ or NO₃^– occurred, a preliminary experiment was conducted, in which an initial measurement was performed at different points from the root tip (1, 2, 4, 10, 15, 20, 25, 30, and 35 mm). Based on this experiment, we selected 20 and 30 mm from the root apex as the measurement site of NH₄⁺ and NO₃^– influxes (Supplementary Figure S2). The recording rate of ion flux was one reading every 6 s and this lasted for 10 min in each root. Six similar seedlings per treatment were measured.

To evaluate the interaction of NH₄⁺ and NO₃^– fluxes, the roots of B. campestris were soaked in measurement solutions. The effect of NO₃^– on NH₄⁺ flux [F (with NO₃^–): 0.1 mmol L^–1 CaCl₂, 0.3 mmol L^–1 MES, 0.1 mmol L^–1 NH₄Cl, and 1 mmol L^–1 NaNO₃; G (without NO₃^–): 0.1 mmol L^–1 CaCl₂, 0.3 mmol L^–1 MES, 0.1 mmol L^–1 NH₄Cl]. The NH₄⁺ flux was measured using SIET for 3 min after equilibration in measuring solution for 10 min. Thereafter, 1.0 mmol L^–1 NH₄Cl was added to the measuring solution, which was mixed thoroughly by expelling and drawing it into a pipette during the first 1–2 min. NO₃^– flux was measured using SIET for 17 min. The effect of NH₄⁺ on NO₃^– flux [H (with NH₄⁺): 0.1 mmol L^–1 CaCl₂, 0.3 mmol L^–1 MES, 0.1 mmol L^–1 NaNO₃, with 1 mmol L^–1 NH₄Cl; I (without NH₄⁺): 0.1 mmol L^–1 CaCl₂, 0.3 mmol L^–1 MES, 0.1 mmol L^–1 NaNO₃]. NO₃^– flux was measured utilizing SIET for 3 min after equilibrated in measurement solution for 10 min. Thereafter, 1.0 mmol L^–1 NaNO₃ was added to the measuring solution. The test process was the same as that described above. Six biological replicates were used for each measurement.

Analysis of AMTs and NRTs Transcripts in Roots

B. campestris seedlings that had been N-starved for 7 d were subjected to different N treatments. The treatments were as follows: (1) exposure to different N levels: 0, 0.25, and 1.0 mmol L^–1 NaNO₃/NH₄Cl were added, then roots were harvested after 20 min during the N-resupply treatments; (2) effect of NH₄⁺ on NO₃^–: 1 mmol L^–1 NH₄Cl was added into the solution with or without NaNO₃, then roots were harvested at 0, 10, and 20 min after adding NH₄Cl; (3) effect of NO₃^– on NH₄⁺: 1 mmol L^–1 NaNO₃ was added into the solution with or without NH₄Cl, then roots were harvested at 0, 10, and 20 min after adding NaNO₃. All samples were immediately frozen in liquid nitrogen and stored at –80°C for quantitative real-time polymerase chain reaction (qPCR).

qPCR

Total RNA was extracted from samples using an Eastep^® Super Total RNA Extraction Kit (Promega, Beijing, China) and was reverse transcribed using a PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa Bio, Dalian, China). The qPCR was performed in a LightCycler 480 Real-Time PCR System (Roche, Basel, Switzerland), using SYBR^® Premix Ex Taq™ (TaKaRa Bio). The primer pairs used are listed in Supplementary Table S1. GAPDH was used as an internal control. Three biological replicates were used to calculate relative gene expression levels.

BcAMT1.2 Cloning and Sequence Analysis

Based on the AMT1.2 sequence of Brassica rapa (retrieved from GenBank, accessions no. XM_009113156.2), primers (Supplementary Table S1) were designed to amplify the full-length of BcAMT1.2 by PCR using the cDNA of B. campestris as the template. The PCR product was cloned into binary vector pCAMBIA3301 (Dingguo Biotechnology, Beijing, China) that carried two CaMV 35S promoters (35S_pro) and phosphinothricin resistance marker genes and was sequenced. Based on the deduced amino acid sequence, transmembrane motifs, subcellular localization, and signature motifs were predicted using Protter (http://wlab.ethz.ch/protter/), Softberry (http://www.softberry.com), and Weblogo (http://weblogo.berkeley.edu/logo.cgi/), respectively. The multiple sequence alignment of 32 AMT proteins from plants was performed using the ClustalW method and a phylogenetic tree was constructed using MEGA 6.0 based on the neighbor-joining algorithm. Bootstrap analysis was carried out with 1000 replicates. The accession numbers of the amino acid sequences of the AMTs are listed in Supplementary Table S2.

Heterologous Expression of BcAMT1.2 in Yeast

The open reading frame (ORF) of BcAMT1.2 was amplified by PCR using the primers (Supplementary Table S1) and constructed into pYES2 vector (Waryong Biotechnology, Beijing, China). As described by Yuan et al. (2007), pYES2 and pYES2-BcAMT1.2 plasmids were transformed into yeast mutant cells 31019b (Δmep1, Δmep2, Δmep3, and ura3). Growth complementation assays were performed on a solid yeast N base medium at pH 5.8 and were supplemented with 2% galactose and 2 mmol L^–1 arginine or NH₄Cl as the sole N source. Yeast cells were incubated at 30°C for 3 days.

BcAMT1.2::GUS Constructs Used for Arabidopsis Transformation and β-Glucuronidase (GUS) Assays

The BcAMT1.2::GUS construct, containing 1519 bp of BcAMT1.2 promoter cloned by our lab, was amplified by PCR from the DNA of B. campestris using special primers (Supplementary Table S1). They were ligated into the pCAMBIA1391 vector which harbored GUS, without a promoter (Dingguo Biotechnology), yielding a pCAMBIA1391-BcAMT1.2_pro::GUS construct. Via Agrobacterium tumefaciens-mediated transformation, BcAMT1.2_pro::GUS transgenic plants were generated in a wildtype (Col-0) background. Second generation (T₂) seeds were germinated on a medium containing 1/2 modified MS, 4 mmol L^–1 NaNO₃ and 0.7% agar for 14 d (growth conditions as described above). Some seedlings were subjected to N-free MS treatment for 4 d, and transferred to either the nutrition of N-free MS or the one of N-free MS containing 0.25 mmol L^–1 NH₄⁺/NO₃^–, and incubated with gentle shaking for 2 h. Histochemical GUS assays were performed as described by Yao et al. (2008). After histochemical staining, seedlings were cleared in 70% ethanol. The images were examined under a digital microscope (VHX-5000; Keyence, Osaka, Japan).

Generation of BcAMT1.2-Overexpressing Arabidopsis Transgenic Lines

Wildtype Arabidopsis (Col-0) was transformed with Agrobacterium GV3101 harboring the pCAMBIA3301-35S_pro::BcAMT1.2 construct. Several transformants were screened by Basta on soil and subjected to PCR analysis using bar primers and qPCR tests of leaves using special BcAMT1.2 primers (Supplementary Table S1). Independent homozygous BcAMT1.2-transformed lines were generated in the T₄ generation.

Plant Culture for Growth Test, NH₄⁺ Content, Ion Fluxes, and Gene Expression

For the growth test, surface-sterilized Arabidopsis seeds were germinated on a 1/2 MS agar-medium (containing 4 mmol L^–1 NaNO₃ as N source) for 4 d and the seedlings were transferred to vertical plates containing 0.25 mmol L^–1 NH₄Cl for 10 d. Ten seedlings were used for the measurements of biomass and primary root length. Then, seedlings were mixed to measure the NH₄⁺ content; 3 biological replicates were used for each line. The measurement of NH₄⁺ content has been previously described by Ivančič and Degobbis (1984).

For the ion flux test, surface-sterilized Arabidopsis seeds were germinated on a 1/2 MS agar-medium (containing 4 mmol L^–1 NaNO₃ as N source) for 4 d and transferred to an N-free 1/2 MS agar-medium for 7 d. Arabidopsis roots were transferred to a measuring solution (0.1 mmol L^–1 CaCl₂, 0.3 mmol L^–1 MES, 0.25 mmol L^–1 NH₄Cl, and 0.75 mmol L^–1 NaNO₃) and NO₃^– and NH₄⁺ fluxes were measured using SIET. Six similar seedlings per treatment were selected to measure ion flux.

Arabidopsis seeds were pre-cultured for 4 d (as described above for the ion flux test) and transferred to a 1/2 MS agar-medium (containing 0.25 mmol L^–1 NH₄Cl + 0.75 mmol L^–1 NaNO₃) for 10 d. Shoots and roots were harvested to isolate total RNA for qPCR analysis and measure the content of NH₄⁺ and NO₃^–, as described by Ivančič and Degobbis (1984) and Downes (1978), respectively. Three biological replicates were used for each measurement. The wildtype was used as control in the above tests.

Statistical Analysis

Microsoft Excel (Microsoft Corporation, USA) and SPSS 17 (SPSS Incorporation, Chicago, USA) were used to analyze the data. An one-way ANOVA was performed. SigmaPlot 11.1 (Jandel Scientific Software, San Rafael, CA, USA) was utilized to draw figures for data presentation. For gene expression analysis, Hem I software (Heatmap Illustrator, version 1.0) (Deng et al., 2014) was used to generate hierarchical cluster heat maps.

Results

Net Fluxes of NO₃^– and NH₄⁺ in Response to Treatment With Different N Forms and Levels

After 7 d N-starvation, B. campestris roots were immersed in measuring solutions containing different N forms (1 mmol L^–1 NH₄Cl, 1 mmol L^–1 NaNO₃, 0.25 mmol L^–1 NH₄Cl + 0.75 mmol L^–1 NaNO₃) to monitor net NO₃^– and NH₄⁺ fluxes. Net NO₃^– and NH₄⁺ flux curves are shown in Figures 1A–C. Net NO₃^– fluxes fluctuated gently in sole NO₃^– (Figure 1A) or mixed N (Figure 1C). In contrast, net NH₄⁺ fluxes increased transitorily, then decreased gradually and subsequently increased in sole NH₄⁺ (Figure 1B), whereas net NH₄⁺ fluxes changed stably in the mixed N treatment (0.25 mmol L^–1 NH₄Cl + 0.75 mmol L^–1 NaNO₃) (Figure 1C). Compared with fluxes in sole N source, NO₃^– fluxes were decreased in mixed N forms and NH₄⁺ fluxes were close to the fluxes of sole NH₄⁺ (1 mmol L^–1 NH₄Cl) which did not decrease with increasing NH₄⁺ concentration (Figures 1A–C). Thus, the mixed N treatment significantly enhanced total N fluxes (Figure 1D) under the same total N conditions (i.e. 3.53-fold for sole NO₃^–, 1.25-fold for sole NH₄⁺).

FIGURE 1

Figure 1 Net fluxes of NO₃^– and NH₄⁺ on root surfaces of Brassica campestris in response to treatments with different N forms. (A) Net NO₃^– fluxes under 1 mmol L^–1 NO₃^–; (B) net NH₄⁺ fluxes under 1 mmol L^–1 NH₄⁺; (C) net NO₃^– and NH₄⁺ fluxes under mixture of 0.25 mmol L^–1 NH₄⁺ and 0.75 mmol L^–1 NO₃^–; (D) total N fluxes under different N forms. Net influxes are suggested by negative values, whereas net effluxes are indicated by positive values. The data represent mean ± SE (n = 6). Different letters indicate significant differences at P < 0.05.

To eliminate the effect of N concentration on N fluxes, we measured that net NO₃^– and NH₄⁺ fluxes under different N levels. The influx rates of NH₄⁺ or NO₃^– increased significantly with an increase in N concentration, NH₄⁺ and NO₃^– influx rates in 1 mmol L^–1 N were 2.66-fold and 1.33-fold of those in 0.25 mmol L^–1, respectively (Figures 2A, B). In addition, NH₄⁺ influx rates were 1.42 and 2.88 times higher than those of NO₃^– at N levels of 0.25 and 1 mmol L^–1, respectively. This indicated that the roots of B. campestris showed a preference for NH₄⁺ over NO₃^–.

FIGURE 2

Figure 2 NO₃^– and NH₄⁺ net fluxes and expressions of BcAMTs and BcNRTs in B. campestris roots in response to treatments with different N levels. (A) Net NH₄⁺ fluxes in different NH₄⁺ levels (0.25, and 1 mmol L^–1 NH₄⁺). (B) Net NO₃^– fluxes in different NO₃^– levels (0.25, and 1 mmol L^–1 NO₃^–). (C, D) BcAMTs and BcNRTs expression in different N levels, respectively (0.25, and 1 mmol L^–1 NH₄⁺/NO₃^–). GAPDH was used as internal control. The data represent the mean ± SE (n = 6 in A–B, n = 3 in C–D). Significant differences (P < 0.05) between treatments are indicated by different letters.

The absorption of NH₄⁺ and NO₃^– are mediated by AMTs and NRTs, respectively. To investigate how the expression of the N transporter genes was affected in roots in response to the addition of NH₄⁺ or NO₃^–, we measured the mRNA levels of four BcAMT genes (BcAMT1.1, BcAMT1.2, BcAMT1.3, and BcAMT1.5) and five BcNRT genes (BcNRT1.1, BcNRT1.8, BcNRT2.1, BcNRT3.1, and BcNAXT1) using qPCR. After a 7-d period of N-starvation, the addition of different N levels had significant effects on the expression levels of BcAMT and BcNRT genes. Compared with the expression levels at nitrogen starvation (0 mmol L^–1 N), the expression levels of BcAMT1.1, BcAMT1.3, and BcAMT1.5 decreased in response to NH₄⁺ (0.25 and 1 mmol L^–1) and 0.25 mmol L^–1 NO₃^–, but they increased in response to 1 mM NO₃^– treatment (i.e. 1.30–1.88 times) (Figure 2C). In contrast, BcAMT1.2 expression increased significantly under 1 mM NH₄⁺ (i.e. 2.30 times higher), and it was also significantly enhanced with an increase in NO₃^– concentration (i.e. 2.01 and 6.51 times higher in response to 0.25 mmol L^–1 and 1 mmol L^–1 NO₃^– treatment, respectively) (Figure 2C). BcAMT1s expression levels were increased by supplying 1 mmol L^–1 NO₃^–, with the expression of BcAMT1.1, BcAMT1.2, BcAMT1.3, and BcAMT1.5 being 2.36, 2.83, 3.12, and 2.41 times higher, respectively, than that with the same NH₄⁺ concentration (Figure 2C). In contrast to 0.25 mmol L^–1 NH₄⁺, adding NO₃^– enhanced BcAMT1.2 expression levels (Supplementary Figure S3A).

Compared with the expression in nitrogen starvation, BcNRT1.1 expression was lower following treatment with 0.25 mmol L^–1 NH₄⁺, although it did not appear to be affected by treatment with 1 mmol L^–1 NH₄⁺. In contrast, although the expression of other BcNRTs was not affected by treatment with 0.25 mmol L^–1 NH₄⁺, the expression was significantly enhanced in response to treatment with 1 mmol L^–1 NH₄⁺ (Figure 2D). Except for BcNRT1.1, the expression of other BcNRTs increased gradually with the concentration of NO₃^– (Figure 2D). BcNRTs expression was increased by supplying 1 mmol L^–1 NO₃^–, with the expression of BcNRT1.1, BcNRT1.8, BcNRT2.1, BcNRT3.1, and BcNAXT1 being 2.74, 2.03, 3.06, 2.68, and 1.20 times higher than that with the same NH₄⁺ concentration, respectively (Figures 2C, D). In contrast to treatment with 1 mmol L^–1 NO₃^–, adding a mixture of 0.25 mmol L^–1 NH₄⁺ and 1 mmol L^–1 NO₃^– decreased the expression levels of BcNRT1.8, BcNRT2.1, BcNRT3.1, and BcNAXT1 (Supplementary Figure S3B).

Interactions Between NH₄⁺ and NO₃^– in Roots of B. Campestris

To elucidate the interaction between NH₄⁺ and NO₃^–, we undertook dynamic monitoring of NH₄⁺ fluxes after adding NH₄⁺ to the bathing solution either with or without NO₃^–. Before adding NH₄⁺, net NH₄⁺ influxes of bathing solution with NO₃^– were higher than that of bathing solution without NO₃^– (Figure 3A). Regardless of whether the bathing solution contained NO₃^– or not, net NH₄⁺ influxes rates after adding NH₄⁺ increased markedly for 30 to 90 s (t1 stage), then decreased quickly for 180 s (t2 stage), then increased gradually (t3 stage), followed by a slow relaxation to the stable level (t4 stage) (Figure 3A). With the exception of several time points in the t2 stage, net NH₄⁺ influxes of the solution with NO₃^– was higher than that of the solution without NO₃^–. There was no obvious difference between NH₄⁺ flux rates in the bathing solution with or without Na⁺, indicating that adding Na⁺ had no obvious effect on NH₄⁺ flux in this study (Supplementary Figure S4). It indicated that NO₃^– influenced NH₄⁺ flux rates.

FIGURE 3

Figure 3 Interaction between NO₃^– and NH₄⁺ fluxes on root surfaces of B. campestris. (A) Influence of NO₃^– on net NH₄⁺ fluxes after adding 1 mmol L^–1 NH₄⁺ to the bathing solution with or without 1 mmol L^–1 NO₃^–. (B) Influence of NH₄⁺ on net NO₃^– fluxes after adding 1 mmol L^–1 NO₃^– to the bathing solution with or without 1 mmol L^–1 NH₄⁺. Changes in net NH₄⁺/NO₃^– fluxes in roots at 30 s intervals are presented. The vertical arrow indicates the point at which 1 mmol L^–1 NH₄⁺ or NO₃^– was added. t1–t4 represent the stages of net NH₄⁺/NO₃^– fluxes after adding NH₄⁺/NO₃^– to the bathing solution. The data represent the mean ± SE (n = 4–6) during the measurement period.

Before adding NO₃^–, NO₃^– fluxes of the bathing solution without NH₄⁺ showed net influxes, whereas those with NH₄⁺ showed net effluxes (Figure 3B). Net NO₃^– influx began to increase rapidly for 60 s (t1 stage) after adding NO₃^– and decreased gradually for 330–420 s (t2 stage). Subsequently, net NO₃^– influx rates increased slowly for approximately 210 s (t3 stage) and remained stable (t4 stage). During the stages t1 and t2, net NO₃^– influx rates of the bathing solution with NH₄⁺ were lower than those for the bathing solution without NH₄⁺. There was no obvious difference between the bathing solution with and without NH₄⁺ during the stages t3 and t4, indicating that NH₄⁺ affected net NO₃^– influxes.

BcAMTs and BcNRTs Expression in Response to Treatment With Adding NH₄⁺ or NO₃^– in B. campestris Roots

Compared with the expression in N deficiency, adding NH₄⁺ without NO₃^– markedly reduced the expression levels of BcAMT1.1, BcAMT1.3, and BcAMT1.5, whereas it induced the expression of BcAMT1.2 after 20 min (Figure 4A). Moreover, adding NH₄⁺ with NO₃^–, resulted in a sharp increase in the expression of BcAMT1.2 and a weak transient increase in the expression of BcAMT1.1, BcAMT1.3, and BcAMT1.5 (Figure 4A). Sole NO₃^– treatment increased the expression of BcAMT1.2 and BcAMT1.5 and decreased that of BcAMT1.1 and BcAMT1.3 (Figures 4A, B), whereas adding NO₃^– to the nutrient solution containing NH₄⁺ resulted in a decrease in the transcript levels of four BcAMT1s (Figure 4B).

FIGURE 4

Figure 4 The expression of AMTs and NRTs in B. campestris roots in response to adding NH₄⁺ or NO₃^– to different N nutrient. (A) AMTs and NRTs expression in seedlings by adding NH₄⁺ to the nutrient without or with NO₃^–. (B) AMTs and NRTs by adding NO₃^– to nutrient solution without or with NH₄⁺. After 7 d N-starvation, seedlings were transferred to different nutrients. –N: Nitrogen starvation; –N + NH₄⁺: adding 1 mmol L^–1 NH₄⁺ for 10 min and 20 min; +NO₃^–: adding 1 mmol L^–1 NH₄⁺ for 10 min and 20 min; +NO₃^–: adding 1 mmol L^–1 NO₃^–; +NO₃^– + NH₄⁺: after adding 1 mmol L^–1 NO₃^– for 10 min, adding 1 mmol L^–1 NH₄⁺ for 10 min and 20 min; –N + NO₃^–: adding 1 mmol L^–1 NO₃^– for 10 min and 20 min; +NH₄⁺: adding 1 mmol L^–1 NH₄⁺; +NH₄⁺ + NO₃^–: after adding 1 mmol L^–1 NH₄⁺ for 10 min, adding 1 mmol L^–1 NO₃^– for 10 min and 20 min. GAPDH was used as internal control. The heat map represents the values of normalization for different genes.

In terms of NRTs expression, sole NH₄⁺ treatment resulted in a slight increase in the expression of BcNRT1.1, BcNRT1.8, BcNRT2.1, and BcNRT3.1, and clearly increased the expression of BcNAXT1 compared with N starvation (Figure 4A), whereas sole NO₃^– treatment resulted in a marked increase of five NRTs transcripts (Figure 4B). However, the effect of adding NO₃^– was more pronounced than that obtained with the combined addition of NO₃^– and NH₄⁺ (Figure 4A). The transcript levels of five NRTs were upregulated in response to the addition of NO₃^–, whereas BcNRT1.1, BcNRT2.1, and BcNRT1.8 expression levels were clearly downregulated by adding NH₄⁺ and slightly upregulated by the subsequent addition of NO₃^– (Figure 4B). However, the expression levels were lower than those obtained in response to the N mixture in which NO₃^– was added for 10 min and NH₄⁺ was added for another 10–20 min (Figure 4A).

Most BcNRTs transcripts were induced by NO₃^– and inhibited by NH₄⁺, whereas BcAMT1s transcripts were inhibited by NH₄⁺ except for BcAMT1.2, which was induced by adding NH₄⁺ and the effect was strengthened by adding NO₃^–. Regarding the analysis of AMT1s and NRTs transcripts, we speculated that BcAMT1.2 might play an important role in the coexistence of NO₃^– and NH₄⁺.

Cloning of a Putative ORF Encoding an AMT1.2 Homolog From B. campestris

To isolate the AMT1.2 gene from B. campestris, we designed primers based on the sequence of AMT1.2 from B. rapa (accession no. XM_009113156.1) (Supplementary Table S1), we obtained the homologous sequence using cDNA from B. campestris, designated BcAMT1.2 (GenBank accession no. MF966937.1). The complete ORF of BcAMT1.2 consisted of 1539 nucleotides and encoded a 54.94 kD polypeptide. Phylogenetic analysis of AMT1 and AMT2 subfamily members from other plant species showed that BcAMT1.2 belonged to the AMT1 cluster (Figure 5A), shared high sequence identity with Populus trichocarpa and Arabidopsis AMT1.2, and shared 99% identity with B. rapa AMT1.2 (Figure 5B). It was predicted to be a member protein exhibiting nine transmembrane domains with an N-terminus outside and C-terminus inside the cytoplasm (Figure 5B). The sequence of BcAMT1.2 contained the signature motif “²¹⁰DFAGSGVVHMVGGIAGLWGALIEGPR²³⁵” near the 5^th transmembrane domain (Figure 5B). The subcellular location in onion cells also showed that BcAMT1.2 was located in the plasma membrane (Supplementary Figure S5).

FIGURE 5

Figure 5 Sequence analysis of BcAMT1.2 and functional complementation in yeast mutant 31019b cells by BcAMT1.2. (A) Phylogenetic tree of AMT homologs. It was constructed by the Neighbor-Joining method in MEGA 6.0. Bootstrap values were derived from 1000 replications, and evolutionary distances were estimated in terms of the number of amino acid substitutions per site. The numbers at the nodes are bootstrap values. Accession numbers of protein sequences of AMTs from given plant species are listed in Supplementary Table S2. At, Arabidopsis thaliana; Bc, Brassica campestris; Br, Brassica rapa; Os, Oryza sativa; Ptr, Populus trichocarpa. BcAMT1.2 was represented by a black box. (B) Amino acid sequence alignment of AMT1.2 from B. campestris and B. rapa. The alignment was performed using ClustalW. Amino acids are presented as capital letters; residues are shown in white letters on black if two sequences have identical residues at the aligned positions. Thick lines below sequences show the positions of potential transmembrane α-helices (TMs) as predicted using Protter (http://wlab.ethz.ch/protter/). The sequences marked signature motif indicated a motif specific to the AMT1 sub-family identified using Weblogo (http://weblogo.berkeley.edu/logo.cgi/). (C) Functional complementation in yeast mutant 31019b cells by BcAMT1.2. pYES2: empty vector was used as negative control, pYES2-BcAMT1.2: BcAMT1.2 ORFs was cloned into pYES2 vector. Yeast cell suspensions were adjusted to an optical density at 600 nm of 1.0 (dilution 1), and serially diluted by factors of 10. For each dilution, 3 μL of the yeast cell suspensions were spotted on yeast N base medium with 2 mmol L^–1 NH₄⁺ or arginine.

To investigate whether BcAMT1.2 is a functional ammonium transporter, we recombined the ORF of BcAMT1.2 into pYES2 vector, and transformed this into yeast mutants 31019b. Negative control cells transformed into pYES2 did not grow normally on a solid medium with 2 mmol L^–1 NH₄⁺ as the only N source, whereas recombinant strains harboring pYES2-BcAMT1.2 grew normally (Figure 5C). This indicated that BcAMT1.2 may be a functional ammonium transporter. BcAMT1.2 was constitutively expressed throughout the growth period, mainly in roots and leaves, whereas the expression in stems and flowers was lower (Supplementary Figure S6A). In roots and leaves, BcAMT1.2 expression decreased significantly as N starvation progressed (Supplementary Figures S6B, C).

We subsequently investigated that histochemical staining for BcAMT1.2_pro::GUS transformants that were treated with NH₄⁺, NO₃^–, or N-deficiency and stained for GUS activity. In leaves and roots, GUS activity was greater in response to treatment with NH₄⁺ or NO₃^– (Figures 6C–F) than that with N-deficiency (Figures 6A, B). GUS was mainly expressed in the vascular tissues of roots and shoots (Figures 6A–F). Two lines showed a similar pattern in response to N-deficiency and a low concentration of NH₄⁺ or NO₃^– after N-deficiency.

FIGURE 6

Figure 6 Histochemical staining for GUS activity in Arabidopsis seedlings transformed with BcAMT1.2pro::GUS. (A, B) Seedlings of two lines of transforming with BcAMT1.2pro::GUS in Arabidposis (Line1, Line2) were subjected to N-deficiency for 4 d. (C, D) Seedlings were subjected to 0.25 mmol L^–1 NH₄⁺ for 2 h after N-deficiency. (E, F) Seedlings were subjected to 0.25 mmol L^–1 NO₃^– for 2 h after N-deficiency. (B), (D), and (F) were the magnification of root zones in a rectangular box of (A), (C), and (E), respectively.

Heterologous Expression of BcAMT1.2 in Arabidopsis

To gain an insight into the possible function of BcAMT1.2 in NH₄⁺ transportation and utilization in plants, BcAMT1.2 was overexpressed in the Arabidopsis wildtype line (Col-0), which was supplied with 0.25 mmol L^–1 NH₄⁺ as the sole N source. Several independent homozygous lines harboring BcAMT1.2 were constructed and the expression of BcAMT1.2 in Arabidopsis was confirmed by qPCR (Figure 7A). These seedlings were grown for 10 d on vertical agar plates containing 0.25 mmol L^–1 NH₄Cl after a 4-d pre-culture on 4 mmol L^–1 NaNO₃. The growth phenotype of transgenic lines showed that the overexpression of BcAMT1.2 could promote the growth of Arabidopsis seedlings at a low concentration of NH₄⁺ (Figure 7B). Compared with the biomass in the wildtype, three BcAMT1.2-overexpressing (BcAMT1.2-ox) lines significantly increased the biomass of shoots and roots (Figure 7C), and the length of primary root (Figure 7D). Furthermore, NH₄⁺ content was increased by 17.9–32.0% in BcAMT1.2-ox lines (Figure 7E).

FIGURE 7

Figure 7 Overexpression of BcAMT1.2 in Arabidopsis promoted plant growth on 0.25 mmol L^–1 NH₄⁺. (A) Detection of BcAMT1.2 expression in several transgenic Arabidopsis lines. The qPCR was performed on total RNA extracted from the leaves of 2-week-old T₁ seedlings; Arabidopsis ACTIN2 was used as internal control. (B) Growth phenotype of T₄ transgenic lines and wildtype on low NH₄⁺. Seedlings were grown vertically on solid medium containing 0.25 mmol L^–1 NH₄⁺ for 10 d after a 4-d pre-culture on 4 mmol L^–1 NO₃^–. Three independent transgenic lines of overexpressed BcAMT1.2 were used. (C) Biomass fresh weight of shoots and roots. (D) The length of primary roots. (E) NH₄⁺ content of the whole plants. Each value represents the mean ± SE (n = 3 in A, C and n = 10 in D, E). Different lowercase letters indicate significant differences at P < 0.05.

Ion Fluxes of Overexpression BcAMT1.2 Lines in Arabidopsis Under Coexistence of NH₄⁺ and NO₃^–

To examine how BcAMT1.2-ox lines affected the absorption of NH₄⁺ and NO₃^–, we measured ion flux rates of Arabidopsis seedlings in response to the mixture of N (0.25 mmol L^–1 NH₄⁺ and 0.75 mmol L^–1 NO₃^–) using SIET. BcAMT1.2-ox lines OX-6 and OX-9 showed larger net NH₄⁺ influxes than the wildtype, and but had little difference in the last minutes of the experiment (Figure 8A). BcAMT1.2-ox lines influenced NO₃^– flux, which was changed significantly from net influxes to net effluxes in the BcAMT1.2-ox line (Figure 8B). During the test process, BcAMT1.2-ox lines increased 32.8–45.7% in net NH₄⁺ influx and 2.50–2.72-fold in net NO₃^– efflux in response to a mixture of NH₄⁺ and NO₃^– (Figures 8A–C). These observations indicated that overexpression of BcAMT1.2 increased NH₄⁺ influxes and NO₃^– effluxes in Arabidopsis. The results of NO₃^– content showed a similar tendency (Figure 8D); however, BcAMT1.2-ox lines had little influence on NH₄⁺ content and even reduced it (Figure 8D).

FIGURE 8

Figure 8 Net ion fluxes and content of NH₄⁺, NO₃^–, and expression of N assimilation genes in Arabidopsis wildtype and BcAMT1.2-ox lines (OX-6, OX-9) under a mixture of 0.25 mmol L^–1 NH₄⁺ and 0.75 mmol L^–1 NO₃^–. (A, B) Net fluxes NH₄⁺ and NO₃^– of wildtype and BcAMT1.2-ox lines. (C) Mean values of NH₄⁺ and NO₃^– net fluxes during the whole test time from (A) and (B). (D) The content of NH₄⁺ and NO₃^– in wildtype and BcAMT1.2-ox lines. (E, F) The expression levels of N assimilation genes in roots and leaves, respectively. Arabidopsis ACTIN2 was used as internal control. Each value represents the mean ± SE (n = 6 in A–D, n = 3 in E–F). Different lowercase letters indicate significant differences at P < 0.05.

To understand if the overexpression of BcAMT1.2 will affect N assimilation, we investigated the expression levels of five N assimilation genes in Arabidopsis under a mixture of NH₄⁺ and NO₃^–. GLN, GDH and GLT encode glutamine synthetase (GS), glutamate dehydrogenase (GDH), and NADH-dependent glutamate synthase (GOGAT), respectively. In roots, the transcript levels of AtGLN1.2 and AtGLT1 were 5.73–8.88-fold and 2.85–3.83-fold higher in BcAMT1.2-ox lines than those in the wildtype (Figure 8E), respectively; in leaves, AtGLN1.2 and AtGLN2 transcript levels were 2.67–2.76-fold and 2.71–4.61-fold higher in both BcAMT1.2-ox lines than those in the wildtype, respectively (Figure 8F). Other genes were affected little, either significantly or inconsistently, between two BcAMT1.2-ox lines (Figures 8E, F). Elevated transcription of N assimilation genes (i.e. GLN1.2, GLN2, and GLT1) might be physiologically crucial for the plants to effectively assimilate and utilize the higher levels of NH₄⁺ induced by overexpressing BcAMT1.2, to retain NH₄⁺ at a relatively stable level.

Discussion

Characteristics of NH₄⁺, NO₃^– Fluxes, and Related Genes Expression in the Roots of B. Campestris

Compared with the growth with a sole N source, a mixture of NO₃^– and NH₄⁺ accelerates plant growth (Supplementary Figure S1) (Wang and Shen, 2011; Song et al., 2012). Plants often show a preference for the uptake of NH₄⁺ or NO₃^– (Song et al., 2016). Previous studies have shown that molecule-specific activities associated with net NO₃^– and NH₄⁺ fluxes can be evaluated non-invasively using SIET (Xu et al., 2006). In this study, we observed that the total N influx of the NH₄⁺ and NO₃^– mixture was higher than that of sole NH₄⁺ or NO₃^– at the same N amount (Figures 1A–D), which is consistent with previous studies on wheat (Zhong et al., 2015) and tea (Ruan et al., 2016). However, it is contrary to the results reported by Arkon et al. (2012), who show a significant decrease of total N uptake in B. napus by an NH₄⁺ and NO₃^– mixture. NH₄⁺ or NO₃^– uptake is affected by the depolarization of electrical membrane potential which increases with the increase in NH₄⁺ or NO₃^– concentration, reaches the peak and changes to be steady, according to the Michaelis-Menten equation (Wang et al., 1994). We observed similar results in Figures 2A, B. However, at the same concentration, the net influx of NH₄⁺ was greater than that of NO₃^– in the roots of B. campestris (Figures 1A–D; Figures 2A, B), and at the concentrations of 0.25 mmol L^–1 and 1 mmol L^–1, net NH₄⁺ uptake was 1.42-fold and 2.88-fold higher than net NO₃^– uptake, respectively (Figures 2A, B). This indicated that B. campestris exhibited a preference for NH₄⁺ over NO₃^–. Previous studies have made similar observations (Zhong et al., 2015; Ruan et al., 2016). Indeed, many plants use NH₄⁺ as their preferred N form (Socci and Templer, 2011) and most plants prefer to absorb NH₄⁺ rather than NO₃^– when NH₄⁺ and NO₃^– are supplied at the same concentration (Zhong et al., 2015; Ruan et al., 2016). Arkon et al. (2012) reported that N uptake and plant growth in B. napus are no significantly affected by adding NH₄⁺ or mixed N during the first 24–72 h, whereas causes N uptake and plant growth to decrease after 15 days of treatment compared with NO₃^– treatment. This may be associated with ammonium toxicity (Arkon et al., 2012; Hachiya et al., 2012; Hachiya and Sakakibara, 2017). Therefore, B. campestris plant prefers NH₄⁺ to NO₃^– on the premise that ammonium toxicity cannot affect plant cells in a short time.

In plants, the absorption of NH₄⁺ or NO₃^– is mainly regulated by AMT or NRT genes, respectively (Glass et al., 2002), and their expression levels are regulated by N status and forms (Gazzarrini et al., 1999; Yuan et al., 2007). In this study, compared with the transcripts in N-deficiency, BcAMT1.1, BcAMT1.3, and BcAMT1.5 transcripts were repressed by adding NH₄⁺ and affected slightly by NO₃^–, whereas BcAMT1.2 expression was induced by both NH₄⁺ and NO₃^– (Figure 2C). The response of BcAMT1.1, BcAMT1.3, and BcAMT1.5 to NH₄⁺ was similar to the results in Arabidopsis (Gazzarrini et al., 1999; Yuan et al., 2007). Those of BcAMT1.2 to NH₄⁺ and NO₃^– were consistent with previous results (Pearson et al., 2002; Yusuf and Deepa, 2017). BcNRTs transcripts were more affected by NO₃^– than NH₄⁺, as they were upregulated with an increase in NO₃^– concentration (Figure 2D). This is consistent with previous studies (Fan et al., 2016; Qu et al., 2016). Consequently, we conclude that N status and form influence AMT and NRT transcripts and that these genes are involved in the regulation of NH₄⁺ and NO₃^– fluxes, respectively.

NO₃^– Accelerates Net NH₄⁺ Influxes in B. campestris

Previous studies have reported that NH₄⁺ and NO₃^– might interact with each other under coexistence (Hachiya et al., 2012). Net N fluxes include total N influxes and total N effluxes. When net N influxes increased, total N influxes were enhanced, and/or total N effluxes were reduced (Hachiya and Sakakibara, 2017). In this study, net NH₄⁺ influxes, with and without containing NO₃^–, increased sharply, then decreased rapidly, and slowly relaxed to a stable level with the addition of NH₄⁺ (Figure 3A). Drastic initial changes in NH₄⁺ fluxes may be caused by depolarization and polarization which are affected by electrical membrane potential after adding more NH₄⁺ (Wang et al., 1994).

In addition, at a high external concentration of NH₄⁺, plants may activate the NH₄⁺ efflux system to cope with high NH₄⁺ influx (Britto and Kronzucker, 2001; Babourina et al., 2007; Hachiya and Sakakibara, 2017). However, to date there have been no reports of any gene that encodes protein that is specifically involved in the NH₄⁺ efflux system (Babourina et al., 2007), NH₄⁺ effluxes may be mediated via aquaporin channels or non-selective K⁺ channels (Hachiya and Sakakibara, 2017). Babourina et al. (2007) reported that K⁺ net fluxes are not correlated with net NH₄⁺ fluxes. Moreover, before adding NH₄⁺, net NH₄⁺ influxes in bathing solution containing NO₃^– were higher than in those lacking NO₃^–. A similar tendency was observed after adding NH₄⁺ (Figure 3A). This indicated that the presence of NO₃^– might have a positive effect on net NH₄⁺ uptake, which is consistent with previous studies performed on other species (Kronzucker et al., 1999; Babourina et al., 2007; Luo et al., 2013); however, it is contrary to the results reported by Arkon et al., 2012. Using isotope labeling, Kronzucker et al. (1999) reported that a larger proportion of ¹³NH₄⁺ signal is allocated to the xylem in the presence of both NH₄⁺ and NO₃^– than that with sole NH₄⁺. NO₃^– may influence the expression of AMTs involved in cytosolic NH₄⁺ homeostasis or be involved in a more complex feedback response via plant metabolism (Babourina et al., 2007; Hachiya and Sakakibara, 2017). In Arabidopsis, NO₃^– mediates NH₄⁺ uptake and assimilation by NRT1.1 (Jian et al., 2018).

Compared with the transcript levels in N-deficiency, BcAMT1.1, BcAMT1.3, and BcAMT1.5 transcript levels were repressed by NH₄⁺ to the growth medium, whereas levels were unaffected or increased slightly in response to NO₃^– (Figures 4A, B). Nevertheless, BcAMT1.2 expression was significantly reduced by N-deficiency (Supplementary Figures S6B, C), and enhanced by the addition of NH₄⁺, particularly in the presence of NO₃^– (Figures 4A, B). The increased AMT activity may lead to a higher rate of NH₄⁺ uptake into internal compartments (vacuole or plastids) or further transport to the xylem. Both events would lead to a lower NH₄⁺ concentration in the cytoplasm of root cells (Babourina et al., 2007). In Arabidopsis, AtAMT1.1, AtAMT1.3, and AtAMT1.5 are located in rhizodermal cells, and AtAMT1.2 is located in root endodermal and cortical cells (Yuan et al., 2007). Specific localization in the root zone of AMTs determines the pathways of NH₄⁺ uptake, transport and allocation to shoots (Duan et al., 2018). When external NH₄⁺ is high, apoplastic transport mediated by AtAMT1.2 prevails at the root endodermis (Yuan et al., 2007; Duan et al., 2018). AtAMT1.2 exclusively regulates NH₄⁺ flux into the vasculature (Yuan et al., 2007; Straub et al., 2017) and favors N allocation to the shoot (Duan et al., 2018). BcAMT1.2_pro::GUS activity, which was expressed mainly in the vascular tissues in Arabidopsis, was enhanced by adding NH₄⁺ or NO₃^– compared with that in N-deficiency (Figures 6A–F). Therefore, we speculated that BcAMT1.2 may participate in the interaction of NH₄⁺ and NO₃^–.

NH₄⁺ Decreases Net NO₃^– Influxes in B. campestris

NH₄⁺ had an influence on NO₃^– fluxes. Before and after adding NO₃^–, net NO₃^– influxes of bathing solution containing NH₄⁺ were lower than those without NO₃^–, whereas net NO₃^– effluxes of bathing solution with NH₄⁺ were lower than those without NH₄⁺ (Figure 3B). This indicated that NH₄⁺ might decrease net NO₃^– influxes, which is consistent with the discoveries in other plants (Kronzucker et al., 1999; Arkon et al., 2012; Luo et al., 2013). BcNRT1.1 and BcNRT2.1, a dual-affinity transport system and high affinity transport system, respectively, were downregulated by NH₄⁺ (Figures 4A, B). Furthermore, the expression of BcNRT1.8, which regulates the xylem loading of NO₃^–, was decreased by NH₄⁺, whereas that of BcNAXT1, which regulates NO₃^– efflux system, was increased (Figures 4A, B). The addition of NH₄⁺ not only decreased NO₃^– absorption, but also NO₃^– xylem loading, and consequently NO₃^– influxes were decreased or NO₃^– effluxes were increased. Previous studies have reported that the acidification of the rhizosphere caused by NAXT1 inhibits NO₃^– absorption (Hachiya and Sakakibara, 2017). Furthermore, the overexpression of OsNRT2.3b enhances NO₃^– uptake in response to sole NO₃^– treatment, whereas OsNRT2.3b expression is inhibited in response to treatment with mixtures of NH₄⁺ and NO₃^– (Fan et al., 2016). Therefore, NH₄⁺ may affect the absorption of NO₃^– by regulating NRT transcripts in the coexistence of NH₄⁺ and NO₃^–.

BcAMT1.2 Mediated the Interaction of NH₄⁺ and NO₃^– Coexistence

One AMT1-type homologous gene, namely BcAMT1.2, was isolated from B. campestris (Figures 5A, B). The protein encoded by BcAMT1.2, which is located in the plasma membrane, may be a functional AMT (Supplementary Figure S4). In a low concentration of NH₄⁺, overexpressing BcAMT1.2 lines accelerated the growth of Arabidopsis which increased NH₄⁺ content compared with the wildtype (Figures 7B–E). This is consistent with overexpressing AtAMT1.2 in Arabidopsis mutant lines (Yuan et al., 2007). In the NH₄⁺ and NO₃^– mixture, net NH₄⁺ influxes of BcAMT1.2-ox lines were obviously increased (Figure 8A), and net NO₃^– influxes were decreased and changed from net influxes to net effluxes (Figure 8B), NO₃^– content of BcAMT1.2-ox lines was lower than that of the wildtype (Figure 8D), indicating that the constitutive expression of BcAMT1.2 clearly reduced the NO₃^– influx into roots. Although net NH₄⁺ influxes of BcAMT1.2-ox line were increased (Figures 8A, C), NH₄⁺ content was not increased, in contrast to the wildtype (Figure 8D).

The expression of N assimilation genes is regulated by NH₄⁺ in plants (Ranathunge et al., 2014). Previous studies have shown that GS and GOGAT can remove NH₄⁺ from the cytoplasm to relieve its toxicity (Babourina et al., 2007; Hachiya and Sakakibara, 2017). In Arabidopsis, GLN1 and GLN2 encode GS isoenzymes, located in the cytosol (GS1) and chloroplast (GS2), respectively (Lothier et al., 2011; Guan et al., 2016). GLN1.2 in Arabidopsis is essential for NH₄⁺ detoxification and N assimilation under ample nitrate supply (Lothier et al., 2011). GOGAT, encoded by GLT1, is responsible for NH₄⁺ assimilation in non-photorespiratory organs with GDH (Liu and von Wirén, 2017). We observed that the expression levels of N assimilation genes (AtGLN1.2, AtGLN2, and AtGLT1) were significantly increased (Figures 8E, F), implying that an increase in BcAMT1.2 mRNA abundance could also directly or indirectly affect NH₄⁺ assimilation. SaAMT1.2 expression levels have been observed to be positively correlated with GS-specific activity in sandalwood (Santalum album) (Yusuf and Deepa, 2017). Overexpressing OsAMT1.1 in rice increases the amounts of amino acids, photosynthetic pigments, and sugars with higher NH₄⁺ levels to improve nitrogen use efficiency, plant growth, and grain yield (Ranathunge et al., 2014). Therefore, overexpressing BcAMT1.2 may affect the homeostasis between nitrogen and carbon to regulate plant growth.

However, the mechanisms underlying this phenomenon remain unknown; thus, warrant further investigation in the future. Co-expression experiments in oocytes have revealed that a complex of AMT1.2 with CBL1 and the active CIPK23 kinase is required for AMT1.2 regulation, whereas a noncatalytic CIPK23 is not sufficient to inactivate NH₄⁺ transportation. CIPK23 and CBL1 appear to occupy a key position in cellular NH₄⁺ and NO₃^– homeostasis (Straub et al., 2017). Interestingly, the phosphorylation of NRT1.1 is also regulated by CIPK23 (Ho and Tsay, 2010).

In addition, Giehl et al. (2017) reported that AtAMT2.1 contributes to NH₄⁺ uptake in the millimolar range, and mediates a high accumulation of NH₄⁺ in xylem sap, which contributes to long-distance translocation from root to shoot. Further studies are required to clarify whether BcAMT2s play a similar role in the interaction between NH₄⁺ and NO₃^– in B. campestris. It may be related to NO₃^– signaling, uptake, and reduction during the interaction of NH₄⁺ and NO₃^– (Hachiya et al., 2012). How AMT1.2 affects the interaction between NH₄⁺ and NO₃^– to exert its effects, and whether other proteins and signaling cascades are involved, are interesting questions that await future research.

Data Availability Statement

All datasets for this study are included in the article/Supplementary Material.

Author Contributions

SS and RC conceived and designed the research. YZ and XH carried out the experiments. WS analyzed the data. YH, GS, and HL reviewed and edited the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (31972481, 31401855) and the China Agriculture Research System (CARS-25-C-04).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We are grateful to Dr. Bruno André (Université Libre de Bruxelles, Belgium) for providing the yeast mutant strain 31019b.

Abbreviations:

AMT, ammonium transporter; CBL, calcineurin B-like protein; CIPK, CBL-interacting serine/threonine-protein kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GDH, glutamate dehydrogenase; GOGAT, glutamate dehydrogenase and NADH-dependent glutamate synthase; GS, glutamine synthetase; GUS, β-glucuronidase; HATS, high-affinity transport system; KD, kilo-dalton; MES, 2-(N-morpholino) ethanesulfonic acid hydrate buffer; LATS, low-affinity transport system; N, nitrogen; NH₄⁺, ammonium; NO₃^–, nitrate; NRT, nitrate transporter; ORF, open reading frame; qPCR, quantitative real-time polymerase chain reaction; SIET, scanning ion-selective electrode technique; TM, transport membrane.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.01776/full#supplementary-material

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