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Article

BcAMT1;5 Mediates Nitrogen Uptake and Assimilation in Flowering Chinese Cabbage and Improves Plant Growth When Overexpressed in Arabidopsis

by
Yunna Zhu
1,2,†,
Lihua Zhong
2,3,†,
Xinmin Huang
2,4,
Wei Su
2,
Houcheng Liu
2,
Guangwen Sun
2,
Shiwei Song
2,* and
Riyuan Chen
2,*
1
Guangdong Provincial Key Laboratory of Utilization and Conservation of Food and Medicinal Resources in Northern Region, Henry Fok College of Biology and Agriculture, Shaoguan University, Shaoguan 512005, China
2
College of Horticulture, South China Agricultural University, Guangzhou 510642, China
3
College of Agriculture and Food Engineering, Baise University, Baise 533000, China
4
College of Biology and Food Engineering, Guangdong University of Petrochemical Technology, Maoming 525000, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2023, 9(1), 43; https://doi.org/10.3390/horticulturae9010043
Submission received: 22 November 2022 / Revised: 26 December 2022 / Accepted: 26 December 2022 / Published: 1 January 2023
(This article belongs to the Special Issue New Advance in Germplasm Resources, Biotechnology and Genetic Breeding of Vegetable Crops)
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Abstract

Nitrogen (N) is a major limiting factor for plant growth and vegetable production. Understanding the regulatory mechanisms of N uptake, transport, and assimilation is key to improving N use efficiency in plants. Ammonium transporters (AMTs) play an important role in plant N metabolism. In this study, we isolated an important AMT1 subfamily member (BcAMT1;5) with a highly conserved signatural AMT1 subfamily motif from flowering Chinese cabbage. Based on functional complementation in yeast mutant 31019b and overexpression of BcAMT1;5 in Arabidopsis, BcAMT1;5 is a functional AMT. Tissue expression analysis showed that BcAMT1;5 was mainly expressed in roots and showed multiple N regime transcript patterns to respond to varying nutritional conditions. This was up-regulated by N-deficiency and down-regulated by supplying NH4+. The glucuronidase (GUS) activities of BcAMT1;5pro::GUS showed a similar change in response to different N conditions. Overexpression of BcAMT1;5 accelerated the growth of transgenic seedlings, increased NH4+ net influxes, and enhanced the content and accumulation of NH4+ and NO3− at low N concentrations. Additionally, it increased the transcript levels of N assimilation-related genes in shoots. These results indicate that BcAMT1;5 may participate in N uptake and assimilation under various N conditions in flowering Chinese cabbage, but it was differed obviously from other AMT1s.

1. Introduction

Nitrogen (N) is pivotal to vegetable yield and quality, and the N fertilizer application is a crucial aspect of modern vegetable management practices and one of the determinants of increasing vegetable yield, thereby keeping pace with the increase in the human population [1]. However, most of the N fertilizers (>60%) are not absorbed by plants but are lost to the environment in the form of ammonia, nitrate (NO3−), and nitrous oxide. Ammonium (NH4+) and NO3− are major sources of inorganic N. Most plants preferentially use NO3− as the N source, and rising carbon dioxide (CO2) in the atmosphere can inhibit its reduction; however, it has no obvious influence on NH4+ assimilation [2]. Because NH4+ can be directly absorbed via the glutamine synthetase (GS) and NADH-dependent glutamate synthase (GOGAT) pathways, NH4+ plays a key role in plant symbiosis [3], especially when plants suffer to N-deficiency because they require less energy for assimilation [4]. However, excess NH4+ can be toxic to plants [5]. Consequently, the absorption and transport of NH4+ must be regulated in plants.
Ammonium transporters (AMTs) on the plasma membranes of root cells transport NH4+ into plants [6], which include the superfamily of AMT, methylamine permease (MEP), and rhesus factor protein (Rh), mainly mediate the acquisition and transport of NH4+ to supply NH4+ nutrients for plants or microorganisms. The MEP and Rh proteins are mainly found in fungi, bacteria, and animals, whereas AMT is found in plants and animals [7]. There are two subfamilies of AMT in plants: AMT1 and AMT2 [6,7]. Most AMT1 subfamily genes, as high-affinity members, respond to N-starvation. In Arabidopsis, the AMT1 subfamily includes AtAMT1;1–AtAMT1;5, AtAMT1;1–AtAMT1;3, which contribute to approximately 90% of the total high-affinity NH4+ uptake capacity in roots, and AtAMT1;5 contributes to the rest [8]. The AtAMT1;1, AtAMT1;3, and AtAMT1;5 proteins mainly participate in absorbing NH4+ from soil; AtAMT1;2 mediates NH4+ uptake by root cells via the apoplasmic transport route [8]; and AtAMT1;4 functions in transporting NH4+ into pollen [9]. This indicates that all AtAMT proteins have different roles in absorption and transport, displaying distinct ammonium transport affinities and capacities [10].
In maize, ZmAMT1;1a and ZmAMT1;3 transcript is induced by resupplying NH4+ into N-deficient roots and promoting NH4+ absorption [11]. In rice, the expression of OsAMT1;1–OsAMT1;3 is cooperatively regulated by the uptake of low NH4+; however, they undergo different regulatory mechanisms [12]. In apples, both AMT1;2 and AMT1;5 are induced by N-deficiency, while AMT1;2 is up-regulated by NH4+-resupply and AMT1;5 is decreased by NH4+ resupply. The AMT1;5 gene is upregulated at an early stage, especially in young fruits, suggesting that it may function providing NH4+into young fruits during fruit development [13]. In flowering Chinese cabbage, BcAMT1;2 is up-regulated by NH4+ and NO3−, especially a mixture of NH4+ and NO3−, and mediates the interaction between NH4+ and NO3− [14]. However, the same homologous genes from different plants or genes from the same plant exhibit different expression characteristics. For example, AtAMT1;4 participates in NH4+ transport in pollen with a higher mRNA abundance in Arabidopsis pollen [9], whereas BcAMT1;4 mainly participates in leaf ammonium transport [15]. The AMT1 proteins appear to have different mechanisms depending on their physiological roles [16].
A previous study has reported that AtAMT1;1–AtAMT1;3 mediate mainly high-affinity NH4+, AtAMT1;5 is responsible for approximately the uptake of 10% NH4+ [8]. So, most studies on AMTs are concerned with AMT1;1–AMT1;3, and very little information concerning the function of AMT1;5 exists. Thus, in the present study, an AMT1-type gene, BcAMT1;5, will be isolated from flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee), and its expression characteristics and functions will be analyzed.

2. Materials and Methods

2.1. Plant Materials and Culture Conditions

The experiment was conducted in a greenhouse at the South China Agricultural University. Flowering Chinese cabbage (Youlv 501, cultivated by Guangzhou Academy of Agriculture Sciences, Guangdong Province, China) seeds were sterilized and sown in plug trays filled with perlite and then further grown in modified Hoagland solution (4.0 mmol L−1 NaNO3, 2.0 mmol L−1 KCl, 1.0 mmol L−1 KH2PO4, 1.0 mmol L−1 MgSO4, 0.5 mmol L−1 CaCl2, 0.1 mmol L−1 Fe-EDTA, 50 μmol L−1 H3BO3, 12 μmol L−1 MnSO4, 1 μmol L−1 CuSO4, 1 μmol L−1 ZnCl2, and 0.2 μmol L−1 Na2MoO4). Uniform seedlings at three-leaf heart stage were selected and transplanted to several plastic boxes with modified Hoagland solution. The pH values of the nutrient solution were adjusted to 5.8–6.0 every two days, and the nutrient solution was changed every four days. The air circulation of nutrient solution was maintained by an air pump, with ventilation of 15 min per hour. After transplanting for 48 d, roots, functional leaves, young leaves, stems, petioles, and flowers of flowering Chinese cabbage were collected at the flowering stage. After cultivation for 10–15 d, some seedlings were still provided by normal N (4 mmol L−1 NaNO3), the remaining seedlings were washed in deionized water and transferred to an N-free modified Hoagland solution containing 4 mmol L−1 NaCl instead of 4 mmol L−1 NaNO3 for 48 h. During this period, roots and shoots were harvested at 0, 0.5, 1, 2, 4, 24, and 48 h. For the treatments of different N regimes, seedlings that were N-deficient for 48 h and supplied with normal N were transferred to different concentrations of NH4+ (i.e., 0.5, 2, 10 mmol L−1) and different ratios of NH4+ and NO3− (i.e., 0:100, 5:95, 25:75, 50:50, and 100:0) for 2 h, and the samples were harvested. In the circadian rhythm tests, seedlings were grown in a greenhouse (25 to 30 °C, natural light) and then transferred to a growth incubator (T: 25 °C; relative humidity: 70%; light intensity: 150 μmol m–2 s–1; light period: 12/12 h light/dark period, light from 7:00 to 18:59, and darkness from 19:00 to 6:59). After 3 d, leaf and root samples were taken every 3 h from 3:00 to 24:00. All the samples were randomly selected from four plants per biological replicate. After liquid nitrogen refrigeration, the samples were stored at −80 °C for quantitative real-time PCR (qPCR).

2.2. Cloning of BcAMT1;5 and Bioinformatics Analysis

Based on AMT1;5 sequence of Brassica rapa (retrieved from GenBank under accession no. XM_009137637.1) specific primers were designed (Table S1). According to the protocols of the Plant RNAiso Plus Kit and the PrimeScript™ 1st Strand cDNA Synthesis Kit (TaKaRa, Kyoto, Japan), the total RNA was extracted from the leaves and roots of flowering Chinese cabbage and was reverse transcribed to cDNA. The open reading frame (ORF) sequence of BcAMT1;5 was amplified by PCR using cDNA from flowering Chinese cabbage as a template, and the products were ligated into the pCAMBIA3301 vector (Dingguo Biotechnology, Beijing, China) and sequenced.
Using online tools, the following characteristics of BcAMT1;5 sequence were predicted: physicochemical properties (ProtParam, http://web.expasy.org/protparam/, accessed on 15 October, 2022), transmembrane motifs (Protter, http://wlab.ethz.ch/protter/, accessed on 15 October, 2022), PROSITE motifs (ScanProsite, http://prosite.expasy.org/scanprosite/, accessed on 15 October, 2022), and subcellular localization (http://www.softberry.com, accessed on 15 October, 2022). Multiple sequence alignment of 29 AMT proteins from flowering Chinese cabbage (Brassica campestris), Arabidopsis thaliana, Brassica rapa, Oryza sativa, and Populus trichocarpa were conducted. Phylogenetic tree was conducted using the neighbor-joining method in MEGA 6.0 [17]. WEBLOGO (http://weblogo.berkeley.edu/logo.cgi/, accessed on 15 October, 2022) was used to generate conserve domains defining of the AMT subfamilies [18].

2.3. Sub-Cellular Location

The AMT1;5 primers (Table S1) were used to amplify the coding sequence of BcAMT1;5. At the Xba I and Sma I sites, the BcAMT1;5 amplicon was ligated into pBI121-GFP vector harboring the CaMV 35S promoter and GFP. The sub-cellular location was conducted as described by Zhong et al. [15]. After two days of cocultivation, the fluorescence levels of the fusion proteins were observed by using a positive fluorescence microscope (Axio Imager D2; Zeiss, Dresden, Germany) at 480 nm excitation and 525 nm emission wavelengths.

2.4. qPCR

According to the method reported by Zhong et al. [15], the qPCR was performed in a LightCycler 480 Real-Time PCR System (Roche, Basel, Switzerland) using SYBR Premix Ex Taq (TaKaRa) and the primer pairs listed in Table S1. Three biological replicates were analyzed for each treatment. Genes encoding glyceraldehyde-3-phosphate dehydrogenase and β-actin were used as internal controls. The relative gene expression was calculated, as previously described by Livak and Schmittgen [19].

2.5. Functional Complementation in Yeast Mutant 31019b

In the pYES2 vector (Waryong Biotechnology, Beijing, China), the BcAMT1;5 ORF was cloned into the EcoRI and XbaI sites to construct yeast expression vector, and then the recombinant and empty pYES2 plasmids were transformed into the yeast mutant strain 31019b (Δmep1, Δmep2, Δmep3, and ura3) [20]. The yeast mutant cell was unable to grow on medium containing lower than 5 mmol L−1 NH4+ as the only N source [8,20]. As described the previous studies [8,20], the functional complementation of BcAMT1;5 was operated in the yeast mutant strain 31019b. A three-day incubation of yeast cells at 30 °C was evaluated and photographed.

2.6. Construction of pCAMBIA1391-BcAMT1;5pro::GUS for Arabidopsis Transformation and Glucorinidase (GUS) Assays

The sequence of the BcAMT1;5 promoter (BcAMT1;5pro) was amplified by PCR from the DNA of flowering Chinese cabbage using AMT1;5pro primers (Table S1), and ligated into the pCAMBIA1391 vector (Dingguo Biotechnology), yielding the pCAMBIA1391-BcAMT1;5pro::GUS construct. The empty vector and BcAMT1;5pro::GUS transgenic plants were generated in a wild-type (Col-0) background through A. tumefaciens-mediated transformation, and second-generation seeds were collected to analyze the promoter activities. As described by Zhu et al. [14], the promoter activities of empty vector and BcAMT1;5pro::GUS were analysed when seedlings were transferred to N-free Murashige and Skoog Medium (MS; PhytoTech, Lenexa, KS, USA), 0.25 mmol L−1 NH4+, 0.25 mmol L−1 NO3−, and a mixture of 0.0625 mmol L−1 NH4+ and 0.1875 mmol L−1 NO3− for 2 h. The images were observed and photograph using a digital stereo microscope (SMZ171; Shanghai Optical Instrument Factory, Shanghai, China).

2.7. Construction of BcAMT1;5-Overexpressing Lines in Arabidopsis

Arabidopsis (Col-0) was transformed with GV1301 harboring pCAMBIA3301-35Spro::BcAMT1;5::NOSterm by flower bud dipping [21], and several transformants were selected by phosphinothricin-resistance, PCR test using bar primers, and qPCR test of leaves using specific BcAMT1;5 primers (Table S1). Fourth-generation seeds were collected to obtain independent homozygous BcAMT1;5-transformed lines. Three lines were used in the experiment of growth phenotyping, according to the results of growth phenotyping, and one line was selected to further analyze ion fluxes, N content, N accumulation, and gene expression.

2.8. Plant Culture for Growth Test, NH4+ Uptake, Ion Fluxes and Gene Expression

For the growth test, Arabidopsis seeds were germinated on half-strength MS agar medium (containing 4 mmol L−1 NO3− as the N source) for 4 d and transferred to vertical plates containing 0.25 mmol L−1 NH4+ half-strength MS for 10 d. Seedlings were harvested to measure the biomass, primary root length, and NH4+ content. The NH4+ content was measured, as described by Ivančič and Degobbis [22]. The NH4+ and NO3− ion fluxes were measured by scanning ion-selective electrode technique (MA01002 system; Younger USA Science and Technology Limited Liability, Amherst, MA, USA) according to Zhu et al. [14]. This work was performed at Xuyue Science and Technology Limited Company (Beijing, China). Six similar seedlings from each treatment were selected for ion flux analyses. For the NH4+ toxic analog, methylammonium (MeA), the consistent seedlings were transferred to vertical plates containing 20 mmol L−1 MeA for 10 d. For the responses to a mixture of N nutrition, Arabidopsis seeds were precultured for 7 d and then transformed into half-strength MS agar medium (containing 0.0625 mmol L−1 NH4+ + 0.1875 mmol L−1 NO3−) for 7 d. The content and accumulation of NH4+ and NO3− were determined, as described by Ivančič and Degobbis [22]. Shoots and roots were harvested for qPCR.

2.9. Statistical Analysis

The experiment data were analyzed by Microsoft excel (Microsoft Corporation, Washington, DC, USA) and SPSS 21 (SPSS Incorporation, Chicago, IL, USA). The figures were drawn by using SigmaPlot 11.1 (Jandel Scientific software, San Rafael, CA, USA), and Duncan’s test by SPSS 21 was used to compare the differences among treatments, with p < 0.05 or p < 0.01 as the significance threshold.

3. Results

3.1. Molecular Identification of AMT1;5 Homolog from Flowering Chinese Cabbage

To explore the biological characteristics of AMT1;5 in flowering Chinese cabbage, the Arabidopsis AtAMT1; 5 sequence [8] was used as a reference to search for homologs in the Brassica rapa genomic database. We obtained a homologous sequence using cDNA from flowering Chinese cabbage, designated BcAMT1;5 (GenBank accession number: MF966940), the ORF of which was 1503 bp in length, encoding a protein of 500 amino acids, with a predicted molecular weight of 53.17 kDa and an isoelectric point of 6.24.
Phylogenetic tree was constructed according to the amino acid sequences of BcAMT1;5 from flowering Chinese cabbage and the AMT1 and AMT2 subfamily members from Arabidopsis thaliana, Brassica rapa, Oryza sativa, and Populus trichocarpa. This shows that BcAMT1;5 belongs to the AMT1 cluster and is highly homologous to AMT1;5 from Arabidopsis and B. rapa, which share 91.00% and 99.60% sequence identity, respectively (Figure 1A, B). The BcAMT1;5-coding protein is predicted to be an integral membrane protein containing 10 transmembrane domains (TMs), with both the N-terminus and C-terminus inside the cytoplasm, with a signature motif of the AMT1 subfamily in the 6th TMs (red box in Figure 1B). Overall, these results indicate that BcAMT1;5 is a member of the AMT1 subfamily, which has been identified in flowering Chinese cabbage.

3.2. Subcellular Localization of BcAMT1;5 and its Functional Complementation Analysis in Yeast Mutant Cells

Using the petiole epidermis of the instantaneously overexpressing pBI121-BcAMT1;5-GFP in onion, we observed that the green fluorescent protein (GFP) signal was mainly localized in the plasma membrane while the ones of pBI121-GFP was localized in the plasma membrane, cell nucleus, and cytoplasm (Figure 2). The intensity of fluorescence signal showed that the ones of pBI121-GFP was stronger than that of pBI121-BcAMT1;5-GFP either in GFP field or merged fields of GFP and bright (Figure S1).
To detect NH4+ transportation function of BcAMT1;5, the ORF of BcAMT1;5 was cloned into the pYES2 yeast vector and then transformed into the yeast mutant strain 31019b. Recombinant yeast strains harboring the empty vector (pYES2) hardly grew and those harboring pYES2-BcAMT1;5 grew normally on medium containing 2 mmol L−1 NH4+ as the sole N source. Both pYES2-BcAMT1;5 and pYES2 yeast strains grew normally on medium containing 2 mmol L−1 arginine (Figure 3). This indicated that BcAMT1;5 complemented the defect in the yeast mutant strain 31019b, which cannot grow on medium with less than 5 mmol L−1 NH4+ as the sole N source, suggesting that BcAMT1;5 is a functional AMT.

3.3. Expression Profiles for BcAMT1;5 Gene in Different Tissues of Flowering Chinese Cabbage

To further characterize BcAMT1;5 in flowering Chinese cabbage, its expression patterns were investigated in the roots, leaves, stems, petioles, and flowers. The BcAMT1;5 gene was expressed most highly in the roots and flowers, and either low-level transcripts or no transcripts were detected in the leaves and stems (Figure 4). Thus, the expression patterns of BcAMT1;5 were analyzed only in the roots in the following experiments.

3.4. Expression Profiles for BcAMT1;5 Gene in Different N Regimes

After preculture for 12 d in nutrient solutions with 4 mmol L−1 NO3−, flowering Chinese cabbage seedlings were transferred to N-free nutrient solutions. The N-starvation treatment increased the transcription level of BcAMT1;5 in the roots, with an increase in the time of N-starvation treatment, with the highest level occurring 48 h after the N-starvation treatment, and this was almost 5.69 times higher than that at 0 h (Figure 5).
To determine the molecular response of BcAMT1;5 to plant N status, its expression levels were investigated using qPCR. After N-starvation for 48 h, BcAMT1;5 expression in roots was slightly increased at the level of 0.5 mmol L−1 NH4+ for 2 h, while it was evidently decreased at the levels of 2 and 10 mmol L−1 NH4+, being 67.60% and 77.83% of the control (Figure 6A). When adding different concentrations of NH4+ at normal N levels, the transcript levels of BcAMT1;5 were sharply inhibited, with levels of 13.97–65.67% of control grown with normal N supply (Figure 6B). Therefore, the addition of NH4+ inhibited the expression of BcAMT1;5 in flowering Chinese cabbage and increased it with NH4+ concentration. Similarly, supplying different ratios of NH4+ and NO3− nutrition clearly affected the transcription levels of BcAMT1;5 in flowering Chinese cabbage (Figure 6C,D). After N-starvation for 48 h, the addition of NH4+ and the mixture of NH4+ and NO3− decreased BcAMT1;5 expression compared with that of sole NO3− treatment (Figure 6C). However, at normal N levels, adding the ratios of NH4+ to NO3− at 5:95 and 25:75, up-regulated BcAMT1;5 transcription, and adding the ratios of 50:50 and 100:0 significantly down-regulated its expression level at the level of 0.05 (Figure 6D). These results indicated that BcAMT1;5 transcription was obviously affected by the external environment N constitution, i.e., N-deficiency, NH4+ concentration, and the ratios of NH4+ and NO3− nutrition.

3.5. Expression of BcAMT1;5 Gene in Light Regimes

The transcript of BcAMT1;5 exhibited diurnal rhythms, showing that BcAMT1;5 had greater expression during the light period than during the dark period, with the lowest expression at 00:00 and the highest at 12:00 (about 3.71–fold that observed at 00:00) (Figure 7). This indicates that the transcript of BcAMT1;5 in flowering Chinese cabbage has a circadian rhythm.

3.6. The GUS Activity of BcAMT1;5pro::GUS in Response to Different N Conditions

To further understand the expression levels of BcAMT1;5 under different N forms, we isolated the promoter of BcAMT1;5 (BcAMT1;5pro) (Table S3) and constructed the fusion vector of BcAMT1;5pro and pCAMBIA1391 empty vector, in which the GUS gene is not driven by any promoter. Histochemical staining of pCAMBIA1391-BcAMT1;5pro::GUS transformants was performed in response to different N treatments, being N-free, NH4+, NO3−, and a mixture of NH4+ and NO3−. The GUS activity of pCAMBIA1391 empty vector did not change under N forms, while those of BcAMT1;5pro showed significant changes in response to different N treatments. Its GUS activities were enhanced in response to N-free and NO3− treatments and decreased in response to NH4+ or the mixture of NH4+ and NO3−, especially NH4+. The GUS staining was chiefly observed in the vascular tissues of seedlings (Figure 8).

3.7. Heterologous Expression of BcAMT1;5 in Arabidopsis

To understand the potential role of BcAMT1;5 in plants, BcAMT1;5 was overexpressed in the Arabidopsis wild-type line (Col-0), several homozygous lines were constructed, and the overexpression of BcAMT1;5 was shown in Figure S2. The seedlings were precultured at 4 mmol L−1 NO3− for 4 d and grown for 10 d on vertical agar plates containing 0.25 mmol L−1 NH4+. A phenotypic analysis of transgenic lines showed that overexpression of BcAMT1;5 in Arabidopsis obviously promoted the growth of seedlings at 0.25 mmol L−1 NH4+. The fresh weight of shoots and roots was significantly increased in most overexpression lines (Figure 9B), and the length of the primary root was increased (Figure 9C). Compared with the wild-type, transgenic lines had significantly increased NH4+ content (Figure 9D). Based on the result of NH4+ influx, ox-6 significantly increased the net NH4+ ion influx by 1.48-fold (Figure 9E).
To further evaluate the function of BcAMT1;5, NH4+ resistance of overexpressing BcAMT1;5 Arabidopsis was transferred to 20 mmol L−1 MeA, an analog of NH4+. As shown in Figure 10A, the growth of seedlings was weaker in each line overexpressing BcAMT1;5 than in the wild type, with the shortened primary root length and the yellower shoot. Compared with the fresh weight of the wild type, the fresh weight of the transgenic lines was sharply reduced, being 38.46–59.36% (Figure 10B). Therefore, overexpression of BcAMT1;5 in Arabidopsis significantly inhibited the growth of seedlings, showing symptoms of NH4+ toxicity (i.e., shorting roots and yellowish leaves). This indicates that the BcAMT1;5-overexpressed lines enhanced the absorption of NH4+.
To further investigate the role of overexpressing BcAMT1;5, the ox-6 line was selected as the research object for the subsequent test. Compared with the wild-type, overexpression of BcAMT1;5 significantly promoted the growth of Arabidopsis seedlings under a mixture nutrition of NH4+ and NO3− (0.0625 mmol L−1 NH4+ + 0.1875 mmol L−1 NO3−), and the fresh weight of shoots and roots was 1.24- and 1.34-fold that of the wild-type, respectively (Figure S3A). Furthermore, root morphology indices of the ox-6 line were significantly increased by 1.23- to 1.82-fold of the wild-type, including the length of primary root, the number of lateral roots, and the density of lateral roots (Figure S3B).
The effects of overexpressing BcAMT1;5 lines on NH4+ and NO3− uptake and transportation were evaluated under a mixture nutrition of NH4+ and NO3−, including ion flux rates, N content, and N accumulation in Arabidopsis seedlings. As shown in Figure 11A, the ox-6 line had larger net NH4+ influxes than that of wild-type (i.e., 1.27-fold of wild-type), while there was little difference in net NO3− influxes between the wild-type and ox-6 line. As for N concentration, the NH4 + and NO3− contents were evidently increased in the ox-6 line. Because of the increase in fresh weight and N content in overexpressing BcAMT1;5, the accumulation of NH4+ and NO3− was significantly enhanced in the ox-6 line compared with those in the wild type (Figure 11C). Furthermore, the expression levels of related N-assimilation genes were investigated under a mixture of NH4+ and NO3−. In the roots of overexpressing BcAMT1;5 line, the expression level of AtGLN1;1, encoding GS, was 1.52-fold higher than that of the wild-type, whereas the expression levels of AtGLN1;2, AtGLN2, and AtGLT1, which encoded GOGAT, were significantly decreased, being 0.04–0.49% of the wild-type (Figure 11D). In leaves, the transcript levels of AtGLN1;1, AtGLN2, and AtGDH2, which encoded glutamate dehydrogenase (GDH), were significantly increased by 3.57–4.58 times those of the wild-type (Figure 11E).

4. Discussion

In the present study, we identified one AMT gene, BcAMT1;5, from B. campestris, which is a member of the AMT1 subfamily. It contains 10 TMs and an AMT1 subfamily signature motif (201DFAGSGVVHMVGGIAGLWGALIEGPR226), which is located in the sixth transmembrane helix, and is conserved among AMT members of Arabidopsis, B. rapa, and B. campestris. This conserved motif can be used to identify other AMT subfamily members in plant species [8]. The GFP signal of pBI121-BcAMT1;5-GFP protein was mainly located in the plasma membrane. Overexpression of pYES2-BcAMT1;5 recovered the normal growth of the yeast mutant strain 31019b with 2 mmol L−1 NH4+ as the sole N source, being similar to AMT members in other plant species [8,15,23,24]. Therefore, BcAMT1;5 in flowering Chinese cabbage encodes a functional ammonium transporter.
In previous studies, multiple AMT1s genes in Arabidopsis [8] and Malus robusta [25] showed different spatial expression and NH4+ uptake capacity to respond differentially to varying environments and nutritional conditions. Similarly, the results showed that BcAMT1;5 was mainly expressed in the roots and flowers of flowering Chinese cabbage, but was not expressed or expressed less in the leaves and stems. The first study of AMT1 genes in Arabidopsis showed that these genes are transcribed in all major organs, but are highly expressed in roots [26]. However, the opposite results were observed in M. robusta, the transcription of MrAMT1;5 is mainly observed in leaves and stems, and it is less expressed in roots [25]. The AMT1;5 gene in apples showed higher expression levels in mature leaves and young fruits [13]. The expression features of AMTs in different plant species demonstrate intricate tissue specificities, suggesting that they may have distinct physiological functions in NH4+ uptake and utilization [13].
Previous studies have shown that the expression patterns of AMT1 genes in response to different N regimes are common in plants [8,13,14,23,24,25]. In this study, BcAMT1;5 transcription was visibly up-regulated by N-starvation, was inhibited by supplying NH4+; additionally, adding different ratios of NH4+ and NO3− distinctly changed the transcript level of BcAMT1;5, regardless of N-deficiency or normal N levels. Previous studies have reported that NO3− may regulate AMT gene expression to affect cytosolic NH4+ homeostasis or participate in more complex feedback responses by affecting plant metabolism [27,28]. Our previous study reported that BcAMT1;2 may be involved in the interaction between NH4+ and NO3− [14]. In addition, we measured the GUS activities of BcAMT1;5pro::GUS in response to different N regimes, which was expressed mainly in the vascular tissues of Arabidopsis seedlings, up-regulated by N-deficiency and the addition of NO3−, and down-regulated by adding NH4+ or a mixture of NO3− and NH4+. These results indicate that the expression of BcAMT1;5 in flowering Chinese cabbage was affected by external N status.
It is well known that circadian rhythm is involved in regulating NH4+ uptake in plants, and many studies have shown that AMT transcription is correlated with diurnal variation [23,29,30]. In agreement with previous results, the expression of BcAMT1;5 in the roots of flowering Chinese cabbage was strongly affected by diurnal variation, with the highest transcript being found at 12:00 and the lowest ones being found at 21:00–24:00. This indicates that NH4+ uptake and assimilation by regulated AMT genes might be controlled by carbohydrate availability [15,23,29,31,32].
To further understand the biological function of BcAMT1;5, we constructed Arabidopsis lines overexpressing BcAMT1;5. Compared with that of the wild-type, the overexpressing BcAMT1;5 lines promoted plant growth with higher NH4+ content and ion influxes at the low NH4+ level, which is consistent with the overexpression of AtAMT1;5 in Arabidopsis lines [8] and BcAMT1;2 in B. campestris lines [14]. Compared with the wild type, overexpressing BcAMT1;5 sharply inhibited seedling growth at 20 mmol L−1 of MeA. These results again show that BcAMT1;5, isolated from flowering Chinese cabbage, encodes a functional AMT protein. In agreement with a previous study [8], AtAMT1;5 can access external NH4+ by uptake across the plasma membrane of rhizodermis cells and is responsible for the residual high-affinity NH4+ uptake by AtAMT1;1–AtAMT1;3. The OsAMTs protein can promote rice root branching and root system buildup at low NH4+ concentrations [33]. In the mixture of NH4+ and NO3, overexpressing BcAMT1;5 significantly accelerated the growth of Arabidopsis seedlings, with a higher fresh weight and lateral root number, and a longer primary root length, in contrast to that of the wild type. Additionally, net NH4+ influxes were obviously increased, but net NO3− influxes were not obviously affected, and the content and accumulation of NH4+ and NO3− were higher than those of the wild-type, indicating that the overexpression of BcAMT1;5 clearly increased the N influx into roots. Our previous study has reported that BcAMT1.2-overexpressing lines increase the net influx of NH4+ and the net effluxes of NO3−, with the increase in NH4+ content and the decrease in NO3− content [14]. Although both BcAMT1;2 and BcAMT1;5 are involved in NH4+ uptake, they may have different effects on NO3− uptake by flowering Chinese cabbage.
Overexpression of AMT genes may change cellular NH4+ pools or accumulate excess NH4+, which can be toxic to plant cells [34]. If N assimilation can keep up with the increase in NH4+ uptake, the detrimental effects of NH4+ can be avoided [35]. It is well known that NH4+ needs to be assimilated by the GS/GOGAT pathway, the GDH pathway is complementary to NH4+ assimilation, and NH4+ and 2-oxoglutarate can synthesize glutamate catalyzed by GDH [36]. In Arabidopsis, AtGLN, AtGDH, and AtGLT encode GS, GDH, and GOGAT, respectively [36,37]. In rice, GS activity is essential for plant growth because it supplies amino acids and reduces NH4+ toxicity [38]. A previous study reported that the transcription of N assimilation-related genes is regulated by the NH4+ status in plants [39]. In this study, the results showed that the transcription of AtGLN1;1 in roots of the BcAMT1;5-overexpressing line was significantly up-regulated in contrast to the wild-type, while the expression levels of AtGLN1;1, AtGLN2, and AtGDH2 were strongly enhanced in shoots of the BcAMT1;5 overexpressing line. However, we observed that the ones of AtGLN1;2, AtGLN2, and AtGLT1 were evidently down-regulated in the roots of the BcAMT1;5-overexpressing line. Increased N assimilation gene expression levels or activities may accelerate the assimilation of NH4+ to glutamine, thus avoiding NH4+ toxicity [28]. Several previous studies have reported that overexpressing OsAMT1;1, OsAMT1;2 in rice and BcAMT1;2 in Arabidopsis enhance the expression levels or activities of GS [14,39,40]. Glutamine, a core metabolite of N assimilation, plays a dual role in regulating ZmAMT1s transcript in maize, with positive induction under low glutamine levels and negative feedback regulation under high glutamine levels, with the aim of preventing excessive NH4+ accumulation in roots [41]. In Arabidopsis, excessive NH4+ assimilation by GS can cause NH4+ toxicity [42]. Therefore, we speculated that the decrease in AtGLN1;2, AtGLN2, and AtGLT1 transcripts in the roots of BcAMT1;5-overexpressing lines could help avoid NH4+ toxicity. These results indicated that different members of the AMTs subfamily may play roles in NH4+ uptake and assimilation through different mechanisms. Different AMT1 members are required for NH4+ uptake and assimilation [12]. According to the analysis of cis-acting regulatory elements, the sequence of BcAMT1;5 promoter contained several binding elements about metabolisms (i.e., TATABOX5 for N metabolism, DOFCOREZM for carbon metabolism) and light regulatory elements (i.e., I box, INRNTPSADB). These cis-acting elements might play important roles in the function of BcAMT1;5 regulating N absorption and assimilation.

5. Conclusions

In this study, we identified and characterized BcAMT1;5 from flowering Chinese cabbage. BcAMT1;5 protein is located in plant membrane, having a highly conserved signatural motif of the AMT1 subfamily, and it can recover NH4+ transportation of 31019b yeast mutant. The expression patterns indicated that BcAMT1;5 was mainly expressed in the roots of flowering Chinese cabbage and showed distinct responses to different N conditions, which were up-regulated by N-deficiency and down-regulated by supplying NH4+. The GUS activities of BcAMT1;5pro::GUS overexpressing lines showed similar changes in response to different N conditions. Furthermore, at low N concentrations, overexpression of BcAMT1;5 accelerated seedling growth, increased NH4+ influx, changed N-related assimilation genes expression, and increased the content and accumulation of NH4+ and NO3− in contrast to that in the wild-type. This suggests that transcriptional regulation of BcAMT1;5 in flowering Chinese cabbage may participate in N uptake and assimilation under various N conditions, however, it was differed obviously from other AMT1s. Therefore, further studies need to be conducted in the future to investigate the molecular mechanisms that link AMT members and the uptake and assimilation of N in response to different N supplies.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae9010043/s1, Table S1: Primer sequences used in this study; Table S2: The sequence information for each AMT protein used for the phylogenetic analysis; Figure S1: The fluorescence signal intensity of pBI121-GFP and pBI121-BcAMT1;5-GFP in onion epidermal cells; Figure S2: Identification of qPCR in different BcAMT1;5-overexpressing lines; Figure S3: Overexpression of BcAMT1;5 in Arabidopsis improves plant growth with a mixture of 0.0625 mmol L−1 NH4+ and 0.1875 mmol L–1 NO3– as the N source; Table S3: The prediction analysis of regulatory elements for the promoter of BcAMT1;5.

Author Contributions

Conceptualization, S.S. and R.C.; methodology, Y.Z. and L.Z.; data analysis, X.H. and Y.Z.; writing—original draft preparation, Y.Z. and L.Z.; writing—review and editing, Y.Z. and L.Z.; revision, W.S., G.S., and H.L.; funding acquisition, R.C., S.S., and Y.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (31972481 and 32072656), the Natural Science Foundation of Guangdong Province, China (2019A1515011680), the Guangdong Provincial Special Fund for Modern Agriculture Industry Technology Innovation Teams (2022KJ131), the Characteristic Innovation Project of Guangdong Provincial Department of Education (2019KTSCX164), and the Science and Technology Research Project of Shaoguan, Guangdong Province (200810224537535).

Data Availability Statement

Not applicable.

Acknowledgments

We thank Guibing Hu from South China Agricultural University for kindly providing the pBI121-GFP vector, as well as Bruno André from the Université Libre de Bruxelles for kindly providing the yeast mutant strain 31019b.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic tree of ammonium transporter (AMT) homologs and the alignment of AMT1;5 amino acid sequences from Brassica campestris, B. rapa, and Arabidopsis. (A) The phylogenetic tree of AMT family proteins was constructed using MEGA 6.0. The numbers at the nodes are bootstrap values. At, Arabidopsis thaliana; Bc, Brassica campestris; Br, Brassica rapa; Os, Oryza sativa; Ptr, Populus trichocarpa. BcAMT1;5 from B. campestris is marked by a black triangle. The sequence information for each AMT protein is provided in Table S2. (B) Sequence alignment of BcAMT1;5, BrAMT1;5, and AtAMT1;5 performed by using Clustalw in MEGA 6.0. Single-letter designations and white letters on black present different amino acids residues and identical residues at the aligned positions of two or three sequences, respectively. Transmembrane domains (TMs) of AMTs were predicted by Protter (http://wlab.ethz.ch/protter/). Gray boxes above the sequences show 10 potential TMs positions, the dotted line and solid line show putative inside- and outside-cytoplasm domains, respectively. The sequences marked in the red box represent the signature motif identified by Couturier et al. [23] for the AMT1 subfamily.
Figure 1. Phylogenetic tree of ammonium transporter (AMT) homologs and the alignment of AMT1;5 amino acid sequences from Brassica campestris, B. rapa, and Arabidopsis. (A) The phylogenetic tree of AMT family proteins was constructed using MEGA 6.0. The numbers at the nodes are bootstrap values. At, Arabidopsis thaliana; Bc, Brassica campestris; Br, Brassica rapa; Os, Oryza sativa; Ptr, Populus trichocarpa. BcAMT1;5 from B. campestris is marked by a black triangle. The sequence information for each AMT protein is provided in Table S2. (B) Sequence alignment of BcAMT1;5, BrAMT1;5, and AtAMT1;5 performed by using Clustalw in MEGA 6.0. Single-letter designations and white letters on black present different amino acids residues and identical residues at the aligned positions of two or three sequences, respectively. Transmembrane domains (TMs) of AMTs were predicted by Protter (http://wlab.ethz.ch/protter/). Gray boxes above the sequences show 10 potential TMs positions, the dotted line and solid line show putative inside- and outside-cytoplasm domains, respectively. The sequences marked in the red box represent the signature motif identified by Couturier et al. [23] for the AMT1 subfamily.
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Figure 2. Subcellular localization of pBI121-GFP, pBI121-BcAMT1;5-GFP fusion proteins in onion epidermal cells. GFP: the green fluorescence images observed by fluorescence microscopy; bright field: the images observed by bright field microscopy; merged: the overlay of the two images of GFP and bright field. The scale bar is 50 μm.
Figure 2. Subcellular localization of pBI121-GFP, pBI121-BcAMT1;5-GFP fusion proteins in onion epidermal cells. GFP: the green fluorescence images observed by fluorescence microscopy; bright field: the images observed by bright field microscopy; merged: the overlay of the two images of GFP and bright field. The scale bar is 50 μm.
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Figure 3. Functional complementation of BcAMT1;5 in yeast mutant 31019b cells. The optical density of yeast cell suspensions was adapted to 1.0 at 600 nm (dilution 1). After serial dilution by factors of 10, 3 μL of the yeast cell suspension each dilution was spotted on the yeast nitrogen base medium. A three-day incubation of yeast cells at 30 °C was evaluated and photographed.
Figure 3. Functional complementation of BcAMT1;5 in yeast mutant 31019b cells. The optical density of yeast cell suspensions was adapted to 1.0 at 600 nm (dilution 1). After serial dilution by factors of 10, 3 μL of the yeast cell suspension each dilution was spotted on the yeast nitrogen base medium. A three-day incubation of yeast cells at 30 °C was evaluated and photographed.
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Figure 4. BcAMT1;5 expression patterns in different organs of flowering Chinese cabbage. Each value shows the mean ± SE (n = 3). Different lowercase letters show significant differences at p < 0.05.
Figure 4. BcAMT1;5 expression patterns in different organs of flowering Chinese cabbage. Each value shows the mean ± SE (n = 3). Different lowercase letters show significant differences at p < 0.05.
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Figure 5. BcAMT1;5 expression in response to N-starvation for 0, 0.5, 1, 2, 4, 24, and 48 h. Each value shows the mean ± SE (n = 3). Different lowercase letters show significant differences at p < 0.05.
Figure 5. BcAMT1;5 expression in response to N-starvation for 0, 0.5, 1, 2, 4, 24, and 48 h. Each value shows the mean ± SE (n = 3). Different lowercase letters show significant differences at p < 0.05.
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Figure 6. BcAMT1;5 expression patterns in root of flowering Chinese cabbage in response to different nitrogen (N) regimes. BcAMT1;5 expression in response to 0, 0.5, 2, and 10 mmol L−1 NH4+ for 2 h after N-deficiency for 48 h. (A) The normal N level (4 mmol L−1 NO3−) (B). BcAMT1;5 expression in response to different ratios of NO3− and NH4+ nutrition (the total N = 4 mmol L−1) for 2 h after N-starvation for 48 h (C) and at the normal N level (D). Values and error bars show the mean ± SE (n = 3). Different lowercase letters indicate significant differences at p < 0.05.
Figure 6. BcAMT1;5 expression patterns in root of flowering Chinese cabbage in response to different nitrogen (N) regimes. BcAMT1;5 expression in response to 0, 0.5, 2, and 10 mmol L−1 NH4+ for 2 h after N-deficiency for 48 h. (A) The normal N level (4 mmol L−1 NO3−) (B). BcAMT1;5 expression in response to different ratios of NO3− and NH4+ nutrition (the total N = 4 mmol L−1) for 2 h after N-starvation for 48 h (C) and at the normal N level (D). Values and error bars show the mean ± SE (n = 3). Different lowercase letters indicate significant differences at p < 0.05.
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Figure 7. BcAMT1;5 expression in response to circadian rhythm and light intensity in flowering Chinese cabbage roots. Seedlings were grown in normal nutrient solution, 150 μmol m−2 s−1 light intensity and a 12 h/12 h light/dark period (light from 7:00 to 18:59; darkness from 19:00 to 6:59). Roots were collected at 3 h intervals. Black boxes represent the dark period, the white boxes represent the light period. Values and error bars indicate means ± SE (n = 3). Different lowercase letters show significant differences at p < 0.05.
Figure 7. BcAMT1;5 expression in response to circadian rhythm and light intensity in flowering Chinese cabbage roots. Seedlings were grown in normal nutrient solution, 150 μmol m−2 s−1 light intensity and a 12 h/12 h light/dark period (light from 7:00 to 18:59; darkness from 19:00 to 6:59). Roots were collected at 3 h intervals. Black boxes represent the dark period, the white boxes represent the light period. Values and error bars indicate means ± SE (n = 3). Different lowercase letters show significant differences at p < 0.05.
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Figure 8. Histochemical staining for glucuronidase (GUS) activities in Arabidopsis seedlings transformed with pCAMBIA1391 and pCAMBIA1391-BcAMT1;5pro::GUS. Transgenic seedlings were subjected to N-free treatment for 4 d and supplied with 0.25 mmol L−1 NH4+, 0.25 mmol L−1 NO3−, a mixture of 0.1875 mmol L−1 NO3−, and 0.0625 mmol L−1 NH4+ for 2 h after N-free treatment. +A, +N, and +NA indicate NH4+, NO3−, as well as the mixture of NO3− and NH4+, respectively.
Figure 8. Histochemical staining for glucuronidase (GUS) activities in Arabidopsis seedlings transformed with pCAMBIA1391 and pCAMBIA1391-BcAMT1;5pro::GUS. Transgenic seedlings were subjected to N-free treatment for 4 d and supplied with 0.25 mmol L−1 NH4+, 0.25 mmol L−1 NO3−, a mixture of 0.1875 mmol L−1 NO3−, and 0.0625 mmol L−1 NH4+ for 2 h after N-free treatment. +A, +N, and +NA indicate NH4+, NO3−, as well as the mixture of NO3− and NH4+, respectively.
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Figure 9. Overexpression of BcAMT1;5 in Arabidopsis improves plant growth at 0.25 mmol L−1 NH4+ as an N source. (A) The growth phenotype of wild-type and the fourth-generation transgenic lines. Seedlings were precultured vertically on solid medium at 4 mmol L−1 NO3− and were transferred to 0.25 mmol L−1 NH4+ for 10 d. (B) Fresh weight of the wild-type and the lines of overexpressing vector and BcAMT1;5. (C) The length of the primary root of wild-type and the lines of overexpressing BcAMT1;5. (D) NH4+ content of whole plants of wild-type and the lines of overexpressing BcAMT1;5. (E) NH4+ ion flux of wild-type and the line 6 of overexpressing BcAMT1;5. WT: wild-type, ox-2, ox-3, and ox-6 present the line 2, 3, and 6 of overexpressing BcAMT1;5, respectively. Line chart: time series plot of NH4+ fluxes; histogram: diagram of NH4+ fluxes. Data represent mean ± SE (n = 10 in (A–C); n = 3 in (D); n = 6 in (E)), different letters above the bars indicate significant differences at p < 0.05, and ** in the histogram shows significant differences at p < 0.01.
Figure 9. Overexpression of BcAMT1;5 in Arabidopsis improves plant growth at 0.25 mmol L−1 NH4+ as an N source. (A) The growth phenotype of wild-type and the fourth-generation transgenic lines. Seedlings were precultured vertically on solid medium at 4 mmol L−1 NO3− and were transferred to 0.25 mmol L−1 NH4+ for 10 d. (B) Fresh weight of the wild-type and the lines of overexpressing vector and BcAMT1;5. (C) The length of the primary root of wild-type and the lines of overexpressing BcAMT1;5. (D) NH4+ content of whole plants of wild-type and the lines of overexpressing BcAMT1;5. (E) NH4+ ion flux of wild-type and the line 6 of overexpressing BcAMT1;5. WT: wild-type, ox-2, ox-3, and ox-6 present the line 2, 3, and 6 of overexpressing BcAMT1;5, respectively. Line chart: time series plot of NH4+ fluxes; histogram: diagram of NH4+ fluxes. Data represent mean ± SE (n = 10 in (A–C); n = 3 in (D); n = 6 in (E)), different letters above the bars indicate significant differences at p < 0.05, and ** in the histogram shows significant differences at p < 0.01.
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Figure 10. The growth phenotype and fresh weight of the whole plants of overexpressing BcAMT1;5 on NH4+ toxic analog methylammonium (MeA). (A) Growth of wild-type and transgenic lines (line 2, 3, 6) on agarose containing 20 mmol L−1 MeA for 10 d after being precultured 4 mmol L−1 NO3− for 4 d. (B) Fresh weight of the same plants as shown in (A). WT: wild-type, ox-2, ox-3, and ox-6 present the line 2, 3, and 6 of overexpressing BcAMT1;5, respectively. Bars indicate mean ± SE (n = 10), and different letters above the bars indicate significant differences at p < 0.05.
Figure 10. The growth phenotype and fresh weight of the whole plants of overexpressing BcAMT1;5 on NH4+ toxic analog methylammonium (MeA). (A) Growth of wild-type and transgenic lines (line 2, 3, 6) on agarose containing 20 mmol L−1 MeA for 10 d after being precultured 4 mmol L−1 NO3− for 4 d. (B) Fresh weight of the same plants as shown in (A). WT: wild-type, ox-2, ox-3, and ox-6 present the line 2, 3, and 6 of overexpressing BcAMT1;5, respectively. Bars indicate mean ± SE (n = 10), and different letters above the bars indicate significant differences at p < 0.05.
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Figure 11. Overexpressing BcAMT1;5 in Arabidopsis improves plant growth at the mixture nutrition of 0.0625 mmol L−1 NH4+ + 0.1875 mmol L−1 NO3− as N source. (A) NH4+ and NO3− ion fluxes. (B) NH4+ and NO3− content. (C) NH4+ and NO3− accumulation. The expression levels of N assimilation genes in roots (D) and in shoots (E). WT and ox-6 present wild-type and the line 6 of overexpressing BcAMT1;5, respectively. Data represent mean ± SE (n = 16 in A; n = 3 in (A–E)), ns represents no significant difference, * and ** in the histogram shows significant differences at p < 0.05 and p < 0.01, respectively.
Figure 11. Overexpressing BcAMT1;5 in Arabidopsis improves plant growth at the mixture nutrition of 0.0625 mmol L−1 NH4+ + 0.1875 mmol L−1 NO3− as N source. (A) NH4+ and NO3− ion fluxes. (B) NH4+ and NO3− content. (C) NH4+ and NO3− accumulation. The expression levels of N assimilation genes in roots (D) and in shoots (E). WT and ox-6 present wild-type and the line 6 of overexpressing BcAMT1;5, respectively. Data represent mean ± SE (n = 16 in A; n = 3 in (A–E)), ns represents no significant difference, * and ** in the histogram shows significant differences at p < 0.05 and p < 0.01, respectively.
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MDPI and ACS Style

Zhu, Y.; Zhong, L.; Huang, X.; Su, W.; Liu, H.; Sun, G.; Song, S.; Chen, R. BcAMT1;5 Mediates Nitrogen Uptake and Assimilation in Flowering Chinese Cabbage and Improves Plant Growth When Overexpressed in Arabidopsis. Horticulturae 2023, 9, 43. https://doi.org/10.3390/horticulturae9010043

AMA Style

Zhu Y, Zhong L, Huang X, Su W, Liu H, Sun G, Song S, Chen R. BcAMT1;5 Mediates Nitrogen Uptake and Assimilation in Flowering Chinese Cabbage and Improves Plant Growth When Overexpressed in Arabidopsis. Horticulturae. 2023; 9(1):43. https://doi.org/10.3390/horticulturae9010043

Chicago/Turabian Style

Zhu, Yunna, Lihua Zhong, Xinmin Huang, Wei Su, Houcheng Liu, Guangwen Sun, Shiwei Song, and Riyuan Chen. 2023. "BcAMT1;5 Mediates Nitrogen Uptake and Assimilation in Flowering Chinese Cabbage and Improves Plant Growth When Overexpressed in Arabidopsis" Horticulturae 9, no. 1: 43. https://doi.org/10.3390/horticulturae9010043

APA Style

Zhu, Y., Zhong, L., Huang, X., Su, W., Liu, H., Sun, G., Song, S., & Chen, R. (2023). BcAMT1;5 Mediates Nitrogen Uptake and Assimilation in Flowering Chinese Cabbage and Improves Plant Growth When Overexpressed in Arabidopsis. Horticulturae, 9(1), 43. https://doi.org/10.3390/horticulturae9010043

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