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

: 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 NH 4+ . The glucuronidase (GUS) activities of BcAMT1;5 pro ::GUS showed a similar change in response to different N conditions. Overexpression of BcAMT1;5 accelerated the growth of transgenic seedlings, increased NH 4+ net inﬂuxes, and enhanced the content and accumulation of NH 4+ and NO 3 − 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.


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 (NO 3 − ), and nitrous oxide.Ammonium (NH 4 + ) and NO 3 − are major sources of inorganic N. Most plants preferentially use NO 3 − as the N source, and rising carbon dioxide (CO 2 ) in the atmosphere can inhibit its reduction; however, it has no obvious influence on NH 4 + assimilation [2].Because NH 4 + can be directly absorbed via the glutamine synthetase (GS) and NADH-dependent glutamate synthase (GOGAT) pathways, NH 4 + 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 NH 4 + can be toxic to plants [5].Consequently, the absorption and transport of NH 4 + must be regulated in plants.
Ammonium transporters (AMTs) on the plasma membranes of root cells transport NH 4 + into plants [6], which include the superfamily of AMT, methylamine permease (MEP), and rhesus factor protein (Rh), mainly mediate the acquisition and transport of NH 4 + to supply NH 4 + 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 NH 4 + 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 NH 4 + from soil; AtAMT1;2 mediates NH 4 + uptake by root cells via the apoplasmic transport route [8]; and AtAMT1;4 functions in transporting NH 4 + 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 NH 4 + into N-deficient roots and promoting NH 4 + absorption [11].In rice, the expression of OsAMT1;1-OsAMT1;3 is cooperatively regulated by the uptake of low NH 4 + ; 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 NH 4 + -resupply and AMT1;5 is decreased by NH 4 + resupply.The AMT1;5 gene is upregulated at an early stage, especially in young fruits, suggesting that it may function providing NH 4 + into young fruits during fruit development [13].In flowering Chinese cabbage, BcAMT1;2 is up-regulated by NH 4 + and NO 3 − , especially a mixture of NH 4 + and NO 3 − , and mediates the interaction between NH 4 + and NO 3 − [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 NH 4 + 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 NH 4 + , AtAMT1;5 is responsible for approximately the uptake of 10% NH 4 + [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.

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 NaNO 3 , 2.0 mmol L −1 KCl, 1.0 mmol L −1 KH 2 PO 4 , 1.0 mmol L −1 MgSO 4 , 0.5 mmol L −1 CaCl 2 , 0.1 mmol L −1 Fe-EDTA, 50 µmol L −1 H 3 BO 3 , 12 µmol L −1 MnSO 4 , 1 µmol L −1 CuSO 4 , 1 µmol L −1 ZnCl 2 , and 0.2 µmol L −1 Na 2 MoO 4 ).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 cultiva-tion for 10-15 d, some seedlings were still provided by normal N (4 mmol L −1 NaNO 3 ), 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 NaNO 3 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 NH 4 + (i.e., 0.5, 2, 10 mmol L −1 ) and different ratios of NH 4 + and NO 3 − (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).

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.

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.

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].

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 (∆mep1, ∆mep2, ∆mep3, and ura3) [20].The yeast mutant cell was unable to grow on medium containing lower than 5 mmol L −1 NH 4 + 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.

Construction of pCAMBIA1391-BcAMT1;5 pro ::GUS for Arabidopsis Transformation and Glucorinidase (GUS) Assays
The sequence of the BcAMT1;5 promoter (BcAMT1;5 pro ) was amplified by PCR from the DNA of flowering Chinese cabbage using AMT1;5 pro primers (Table S1), and ligated into the pCAMBIA1391 vector (Dingguo Biotechnology), yielding the pCAMBIA1391-BcAMT1;5 pro ::GUS construct.The empty vector and BcAMT1;5 pro ::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;5 pro ::GUS were analysed when seedlings were transferred to N-free Murashige and Skoog Medium (MS; PhytoTech, Lenexa, KS, USA), 0.25 mmol L −1 NH 4 + , 0.25 mmol L −1 NO 3 − , and a mixture of 0.0625 mmol L −1 NH 4 + and 0.1875 mmol L −1 NO 3 − for 2 h.The images were observed and photograph using a digital stereo microscope (SMZ171; Shanghai Optical Instrument Factory, Shanghai, China).

Construction of BcAMT1;5-Overexpressing Lines in Arabidopsis
Arabidopsis (Col-0) was transformed with GV1301 harboring pCAMBIA3301-35S pro :: BcAMT1;5::NOS term 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.

Plant Culture for Growth Test, NH 4 + 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 NO 3 − as the N source) for 4 d and transferred to vertical plates containing 0.25 mmol L −1 NH 4 + half-strength MS for 10 d.Seedlings were harvested to measure the biomass, primary root length, and NH 4 + content.The NH 4 + content was measured, as described by Ivančič and Degobbis [22].The NH 4 + and NO 3 − 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 NH 4 + 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 halfstrength MS agar medium (containing 0.0625 mmol L −1 NH 4 + + 0.1875 mmol L −1 NO 3 − ) for 7 d.The content and accumulation of NH 4 + and NO 3 − were determined, as described by Ivančič and Degobbis [22].Shoots and roots were harvested for qPCR.

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.

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.

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.

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 Cterminus 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.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  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.

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).
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.

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.

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 To detect NH 4 + 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 NH 4 + 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 NH 4 + as the sole N source, suggesting that BcAMT1;5 is a functional AMT.
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.

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.

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

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.
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.

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

Expression Profiles for BcAMT1;5 Gene in Different N Regimes
After preculture for 12 d in nutrient solutions with 4 mmol L −1 NO 3 − , 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).
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.

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 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 NH 4 + for 2 h, while it was evidently decreased at the levels of 2 and 10 mmol L −1 NH 4 + , being 67.60% and 77.83% of the control (Figure 6A).When adding different concentrations of NH 4 + 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 NH However, at normal N levels, adding the ratios of NH 4 + to NO 3 − at 5:95 and 25:75, upregulated 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, NH 4 + concentration, and the ratios of NH 4 + and NO 3 − nutrition.
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.

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

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.

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).

The GUS Activity of BcAMT1;5 pro ::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;5 pro ) (Table S3) and constructed the fusion vector of BcAMT1;5 pro and pCAMBIA1391 empty vector, in which the GUS gene is not driven by any promoter.Histochemical staining of pCAMBIA1391-BcAMT1;5 pro ::GUS transformants was performed in response to different N treatments, being N-free, NH 4 + , NO 3 − , and a mixture of NH 4 + and NO 3 − .The GUS activity of pCAMBIA1391 empty vector did not change under N forms, while those of BcAMT1;5 pro showed significant changes in response to different N treatments.Its GUS activities were enhanced in response to N-free and NO 3 − treatments and decreased in response to NH 4 + or the mixture of NH 4 + and NO 3 − , especially NH 4 + .The GUS staining was chiefly observed in the vascular tissues of seedlings (Figure 8).

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).

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 NO 3 − for 4 d and grown for 10 d on vertical agar plates containing 0.25 mmol L −1 NH 4 + .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 NH 4 + .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 NH 4 + content (Figure 9D).Based on the result of NH 4 + influx, ox-6 significantly increased the net NH 4 + ion influx by 1.48-fold (Figure 9E).

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, NH 4 + resistance of overexpressing BcAMT1;5 Arabidopsis was transferred to 20 mmol L −1 MeA, an analog of NH 4 + .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 NH 4 + toxicity (i.e., shorting roots and yellowish leaves).This indicates that the BcAMT1;5overexpressed lines enhanced the absorption of NH 4 + .
Horticulturae 2023, 9, x FOR PEER REVIEW 11 of 16 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 wildtype), 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 3.57-4.58times those of the wild-type (Figure 11E).), 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 NH 4 + and NO 3 − uptake and transportation were evaluated under a mixture nutrition of NH 4 + and NO 3 − , including ion flux rates, N content, and N accumulation in Arabidopsis seedlings.As shown in Figure 11A, the ox-6 line had larger net NH 4 + influxes than that of wild-type (i.e., 1.27-fold of wild-type), while there was little difference in net NO 3 − influxes between the wild-type and ox-6 line.As for N concentration, the NH 4 + and NO 3 − 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 NH 4 + and NO 3 − 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 NH 4 + and NO 3 − .In the roots of overexpressing BcAMT1;5 line, the expression level of AtGLN1;1, encoding GS, was 1.52fold 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 3.57-4.58times those of the wild-type (Figure 11E).

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 ( 201 DFAGSGVVHMVGGIAGLWGALIEGPR 226 ), 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,

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 ( 201 DFAGSGVVHMVGGIAGLWGALIEGPR 226 ), 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 NH 4 + 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 NH 4 + 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 NH 4 + 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 BcAMT1;5-overexpressing line.Increased N assimilation gene expression levels or activities may accelerate the assimilation of NH 4 + to glutamine, thus avoiding NH 4 + 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 NH 4 + accumulation in roots [41].In Arabidopsis, excessive NH 4 + assimilation by GS can cause NH 4 + 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 NH 4 + toxicity.These results indicated that different members of the AMTs subfamily may play roles in NH 4 + uptake and assimilation through different mechanisms.Different AMT1 members are required for NH 4 + 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.

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 NH 4 + 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 NH 4 + .The GUS activities of BcAMT1;5 pro ::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 NH 4 + influx, changed N-related assimilation genes expression, and increased the content and accumulation of NH 4 + and NO 3 − 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.

Figure 1 .
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 TableS2.(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

Figure 1 .
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 TableS2.(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 2 .
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 3 .
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 2 .
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 .
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 3 .
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 .
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 4 .
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 .
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 .
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.
4 + inhibited the expression of BcAMT1;5 in flowering Chinese cabbage and increased it with NH 4 + concentration.Similarly, supplying different ratios of NH 4 + and NO 3 − 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 NH 4 + and the mixture of NH 4 + and NO 3 − decreased BcAMT1;5 expression compared with that of sole NO 3 − treatment (Figure 6C).

Figure 6 .
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 Nstarvation 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 .
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 NH 4 + for 2 h after N-deficiency for 48 h.(A) The normal N level (4 mmol L −1 NO 3 − ) (B).BcAMT1;5 expression in response to different ratios of NO 3 − and NH 4 + 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.
This indicates that the transcript of BcAMT1;5 in flowering Chinese cabbage has a circadian rhythm.

Figure 7 .
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.

Horticulturae 2023, 9 ,
x FOR PEER REVIEW 9 of 167).This indicates that the transcript of BcAMT1;5 in flowering Chinese cabbage has a circadian rhythm.

Figure 7 .
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 9 .
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 .
Figure 9. Overexpression of BcAMT1;5 in Arabidopsis improves plant growth at 0.25 mmol L −1 NH 4 + 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 NO 3 − and were transferred to 0.25 mmol L −1 NH 4 + 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) NH 4 + content of whole plants of wild-type and the lines of overexpressing BcAMT1;5.(E) NH 4 + 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 NH 4 + fluxes; histogram: diagram of NH 4 + 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 10 .
Figure 10.The growth phenotype and fresh weight of the whole plants of overexpressing BcAMT1;5 on NH 4 + 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 NO 3 − 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.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 NH 4 + and NO 3 − (0.0625 mmol L −1 NH 4 + + 0.1875 mmol L −1 NO 3 − ), and the fresh weight of shoots and roots was 1.24-and 1.34-fold that of the wild-type, respectively (FigureS3A).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 (FigureS3B).The effects of overexpressing BcAMT1;5 lines on NH 4 + and NO 3 − uptake and transportation were evaluated under a mixture nutrition of NH 4 + and NO 3 − , including ion flux rates, N content, and N accumulation in Arabidopsis seedlings.As shown in Figure11A, the ox-6 line had larger net NH 4 + influxes than that of wild-type (i.e., 1.27-fold of wild-type), while there was little difference in net NO 3 − influxes between the wild-type and ox-6 line.As for N concentration, the NH 4 + and NO 3 − 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 NH 4 + and NO 3 − was significantly enhanced in the ox-6 line compared with those in the wild type (Figure11C).Furthermore, the expression levels of related N-assimilation genes were investigated under a mixture of NH 4 + and NO 3 − .In the roots of overexpressing BcAMT1;5 line, the expression level of AtGLN1;1, encoding GS, was 1.52fold 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 (Figure11D).In leaves, the transcript levels of AtGLN1;1, AtGLN2, and AtGDH2, which encoded glutamate dehydrogenase (GDH), were significantly increased by 3.57-4.58times those of the wild-type (Figure11E).

Figure 11 .
Figure 11.Overexpressing BcAMT1;5 in Arabidopsis improves plant growth at the mixture nutrition of 0.0625 mmol L −1 NH 4 + + 0.1875 mmol L −1 NO 3 − as N source.(A) NH 4 + and NO 3 − ion fluxes.(B) NH 4 + and NO 3 − content.(C) NH 4 + and NO 3 − 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.

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.