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Article

Nitrogen Absorption Pattern Detection and Expression Analysis of Nitrate Transporters in Flowering Chinese Cabbage

by
Shuaiwei Zhang
,
Yuepeng Zhang
,
Yudan Wang
,
Yanwei Hao
,
Wei Su
,
Guangwen Sun
,
Houcheng Liu
,
Riyuan Chen
* and
Shiwei Song
*
College of Horticulture, South China Agricultural University, Guangzhou 510642, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2022, 8(3), 188; https://doi.org/10.3390/horticulturae8030188
Submission received: 22 January 2022 / Revised: 10 February 2022 / Accepted: 17 February 2022 / Published: 22 February 2022
(This article belongs to the Section Plant Nutrition)
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Abstract

:
Nitrate transporters (NRTs) play an important role in nitrate absorption and internal distribution in plant roots and other parts. Experiments were carried out to explore the sequences and expression characteristics of NRT genes, and their correlation with the N uptake in flowering Chinese cabbage. We have isolated three important BcNRTs (BcNRT1.1, BcNRT1.2, and BcNRT2.1) from flowering Chinese cabbage. Spatio-temporal expression analysis found that BcNRT1.1 and BcNRT2.1 were mainly expressed in roots, while BcNRT1.2 was more expressed in roots than in leaves during vegetative growth and was mainly expressed in leaves during reproductive growth. The NO3− uptake rate of the entire growth period was significantly correlated with BcNRT1.1 and BcNRT1.2 expression in roots. In addition, the total N content was increased with the increase in NO3− concentration in flowering Chinese cabbage. The NH4+ uptake was slightly induced by NH4+, but the total N content had no significant difference under the NH4+ concentration of 1–8 mmol/L. We also found that lower concentrations of NH4+ promoted the expression of BcNRT1.1 and BcNRT1.2 while inhibiting the expression of BcNRT2.1 in the roots of flowering Chinese cabbage. The amount of total N uptake in the treatment with 25/75 of NH4+/NO3− was significantly higher than that of the other two treatments (0/100 and 50/50). In the mixture of NH4+ and NO3−, total N uptake was significantly correlated with the BcNRT1.2 expression. We concluded that mixed nutrition with an NH4+/NO3− of 25/75 could significantly increase total nitrogen uptake in flowering Chinese cabbage, in which two members of the NRT1 subfamily (BcNRT1.1 and BcNRT1.2) might play a major regulatory role in it. This study is a beneficial attempt to dig deeper into the NRT genes resources and lays the foundation for the ultimate use of genetic improvement methods to increase the NUE with less nitrogen fertilizer in flowering Chinese cabbage.

1. Introduction

Nitrate is one of the most important nitrogen (N) sources for plants due to its function as both a nutritional component and a signaling molecule [1]. For the growth environment of most vegetables, the content of nitrate nitrogen (NO3−) in the soil is higher than that of ammonium nitrogen (NH4+), and the N sources absorbed and utilized are mainly NO3−. Nitrate quickly stimulates comprehensive nutrient transportation and assimilation, and the C/N metabolism and regulation pathways enable plant growth plasticity to respond to fluctuating environments [2]. NO3− is absorbed by roots through NO3− transporters and then transported to the whole plant, or it combines with carbon to produce amino acids before redistribution [3]. Similar to most ions, the absorption of NO3− by plants is an active absorption process, which generally absorbs nitrate ions in the soil through nitrate transporters that span the epidermis and cortical plasma membrane of plant roots [4]. Effective acquisition and distribution of NO3− are very important for plants to successfully compete for the limited and unevenly distributed nitrogen in the soil and maintain growth [5].
Nitrate transporter (NRT) genes are divided into two subgroups, NRT1 and NRT2 [6]. Previous research on the expression and function of these two transporter families has shown that NRT1 is a low-affinity transporter system (LATS), while NRT2 is a high-affinity transporter system (HATS) [7]. When the external NO3− concentration is greater than 1 mM, the low-affinity nitrate transport system works, but when the NO3− concentration is less than 1 mM, plants use the high-affinity nitrate transport system to absorb NO3− [8]. Regarding the biological functions, have been studied the most in model plants, such as Arabidopsis and rice. Different NRTs have different functions, such as four NRT2 (NRT2.1, NRT2.2, NRT2.4, and NRT2.5) and two NPF (NRT1.1 and NRT1.2), which have been identified as important nitrate transporters for root uptake of nitrate [9,10]. The identification of the first nitrate transporter, AtNRT1.1, in Arabidopsis represents an important milestone on the road to understanding the molecular basis of nitrate use in plants [11]. The initial study found that AtNRT1.1 was involved in the absorption of nitrate by the roots and the transport of nitrate from roots to stems in Arabidopsis [12]. Recent studies have shown that AtNRT1.1 can activate a variety of nitrate sensing and signaling mechanisms that regulate nitrate response in Arabidopsis roots [2,13], but it also affects a variety of biological processes, including root development and configuration, auxin transport, seed dormancy, flowering time, and stomata movement [14,15,16]. AtNRT2.1 is mainly located on the plasma membrane of epidermal cells and cortical cells in Arabidopsis root, and plays an important role in regulating root development under low concentrations of NO3− [17,18,19]. In most plants, the regulation of NRT expression levels (mRNA and protein) by external conditions determines its nitrate absorption capacity [20]. Many molecular mechanisms regulating the absorption of NO3− have been confirmed. The expression of NRT is regulated by many factors, such as the absorption of substrates (NO3−, NO2−, NH4+), as well as other environmental conditions (e.g., pH and light) [21]. The response of transcription level is very rapid after NO3− treatment; the change can be detected soon in plants [22,23]. The analysis results of the transcriptional response of nitrate-treated roots were summarized and found that more than half of the genes responded to nitrate, this highlighted the environmental specificity and complexity of the nitrate response [24].
NO3− absorption is a highly integrated process, depending on the availability of nitrate and the demand for nitrogen. In addition, ammonium and nitrate have a synergistic effect on promoting plant growth and can stimulate plant growth by affecting N uptake, distribution, assimilation, and signal transduction [25]. For example, nutrient solutions with different NH4+/NO3− ratios significantly increased the accumulation of soluble protein in cotton leaves and roots under controlled hydroponic conditions, especially 75/25 [26]. The nutrient solution with the NH4+/NO3− ratio of 50:50 can improve the absorption of nutrients and maintain proper N assimilation and storage ratio in cabbage [27]. In Chinese kale, a hydroponic environment with an NH4+/NO3− ratio of 50/50 increased the glucosinolates content of the bolting stems [28]. Many previous studies have shown that a proper amount of NH4+ can increase crop yields. For example, the grain yield of two spring wheat varieties Len and Inbar increased by 28% and 78% under the condition of increased NH4+, respectively [29]. It is also reported that the total dry weight and the number of tubers per plant grown of potato under increased NH4+ conditions are higher than any single nitrogen form nutrition [30]. Other plants, such as lettuce significantly improved their root vitality in the solution with 25/75 of NH4+/NO3−. Therefore, it can be seen that the presence of nitrate reduces the inhibitory effect of NH4+ on root growth, while a certain concentration of NH4+ increases the thickness of secondary roots and the number of lateral roots [31]. However, excessive addition of NH4+ may cause plant rhizosphere acidification and ammonia poisoning, thereby inhibiting plant growth [32]. The expression of the NRT gene also changed under the condition of increased NH4+ in the root. Compared with a single NO3− nutrition, the expression of LeNRT1.2 is not significantly affected when the increase in NH4+ is 25%, but the expression of LeNRT1.2 was significantly inhibited when the NH4+ increase intensity reaches 50% [33]. Therefore, applying an appropriate NH4+/NO3− ratio is an important way to improve the NUE of plants [34].
Flowering Chinese cabbage (Brassica campestris), a characteristic vegetable that originates from southern China, is a variant of a subspecies of pakchoi. It can be sown in all seasons of the year and is now introduced and cultivated all over the world. The flowering Chinese cabbage prefers to absorb nitrate nitrogen and tends to accumulate excessive nitrate in the product organs [35]. Our understanding of the nitrate-nitrogen absorption law and regulation mechanism of Chinese cabbage is of great value for improving the nitrogen utilization efficiency (NUE) and the supply of healthy products. Although there are some studies on the effects of different NH4+/NO3− ratios on the growth of various plants, little information is available on the combined analysis of nitrogen uptake and N-related genes in flowering Chinese cabbage. To do this, we cloned three important nitrate transporter-encoding genes from the Brassica campestris genome and analyzed protein physical and chemical properties of them using bioinformatics methods. Then, the N absorption capacity and NRT genes expression characteristics of Chinese cabbage were explored under different treatments. The potential role and molecular mechanism of BcNRTs in root development and N absorption need to be revealed, and more available genetic resources are provided for improving the NUE of flowering Chinese cabbage.

2. Methods

2.1. Plant Material and Treatments

The experiment was carried out in the greenhouse of the College of Horticulture, South China Agricultural University. Flowering Chinese cabbage seeds (cultivar ‘Youlv 501’) were provided by Guangzhou Academy of Agriculture Science, Guangdong Province, China. Seedlings grow on sponge blocks, and three seedlings with developed third true leaves were selected and transferred to 1/2 improved Hoagland nutrient solution for 10 d. The nutrient solution formula was as follows: 4.0 mmol/L NaNO3, 2.0 mmol/L KH2PO4, 2.0 mmol/L KCl, 2.0 mmol/L MgSO4, 0.5 mmol/L CaCl2, 0.1 mmol/L Fe-EDTA, 50 μM H3BO3, 12 μM MnSO4, 1 μM ZnC12, 1 μM CuSO4, 0.2 μM Na2MoO4, 30 mg/L ampicillin. Ampicillin is an antibiotic, which can be used in nutrient solutions to prevent microorganisms from oxidizing ammonium into nitrate. The nutrient solution was changed every 3 days, and the pH value of the nutrient solution was adjusted to about 6.2 every day. The ventilation was performed every hour for 15 min during the trial period. Roots and leaves were collected separately in the 2-leaf stage, 6-leaf stage, bolting stage, late bolting stage, and flowering stage. We performed nitrogen starvation treatment for 48 h on 10-day-old seedlings (i.e., removed NaNO3 from the nutrient solution), and then treated the growing flowering Chinese cabbage with different concentrations of NaNO3 (2, 3, 4, and 8 mM) and NH4Cl (1, 3, 4, and 8 mM). The content of other mineral nutrients remained unchanged in each experimental treatment, except for the change in N source supply. The roots and leaves were collected after treatment 0, 0.5, 2, 8, and 24 h, respectively. All samples were stored at −80 °C for later use.

2.2. Molecular Cloning of NRT Genes

We isolated total RNA using TRIzol. Reverse transcriptase reaction was performed with a reverse transcription kit (TaKaRa Bio, Tokyo, Japan) to obtain cDNA. The specific primers of three BcNRT genes (BcNRT1.1, BcNRT1.2, and BcNRT2.1) are designed based on the sequence of Arabidopsis (AtNRT1.1 Gene ID: 837763, AtNRT1.2 Gene ID: 843321, AtNRT2.1 Gene ID: 837327) (Table S1). The CDS sequence of BcNRT genes was amplified using PCR with specific primers. The NRT cloned products were recovered with a DNA gel recovery kit (Shanghai Shenggong) and NRT gene fragments were connected to the T-vector using the pMD19-T Vector Kit (TaKaRa Bio, Tokyo, Japan). We obtained BcNRT1.1, BcNRT1.2, and BcNRT2.1 gene sequences for subsequent analysis after they were sent to Invitrogen (Shanghai, China) Corporation for sequencing. A phylogenetic tree was constructed using Mega7.0 software, and DNAMAN software was used for homology comparison [36].

2.3. Effects of Different N Treatments on N Absorption and NRT Genes Expression in Flowering Chinese Cabbage

To explore the effect of different NH4+/NO3− ratios on the expression of BcNRTs, the content of other mineral nutrients remained unchanged except for the nitrogen. The formulas of the trace elements in the nutrient solution treated with different NH4+/NO3− ratios are shown in Table 1. The flowering Chinese cabbage was grown in a nutrient solution with different NH4+/NO3− ratios (0/100, 25/75, 50/50) after the nitrogen deficiency treatment. We call the NH4+/NO3− ratio of 0:100 as the control treatment (CK), because the nitrogen source is all nitrate nitrogen in most nutrient solution formulations, that is, 100% NO3−. Then, we carried out different ratios of NH4+/NO3− to increase the NH4+ and reduce the NO3−, which was called T1 and T2 treatments. There were 5 replicates for each treatment and each replicate had one plant. The roots and leaves were taken at 0.5, 2, 8, 24, and 48 h after treatment. All the roots were washed with deionized water, and the leaves were wiped dry with tissues and stored in the refrigerator at −80 °C for later use.

2.4. Parameter Measurements

The NO3− absorption of flowering Chinese cabbage during different growth periods was determined using the conventional depletion method. The NO3− contents of the nutrient solution were determined by the hydrazine sulfate method [37]. The NH4+ content was determined by the indophenol blue method [38]. The total N content was determined by the H2SO4-H2O2 Kjeldahl method [39]. The ion concentration in the nutrient solution was measured at each time point after plant samples were taken. Then, the amount of specific ions in the nutrient solution was calculated at that time according to the ion concentration. We analyzed the N uptake of the whole plant by subtracting the nitrogen content in the nutrient solution from the total N content of the whole plant.

2.5. Quantitative Real Time PCR

The total RNA of flowering Chinese cabbage was extracted, and its concentration and quality were detected by a nucleic acid protein analyzer and agarose gel electrophoresis. Reverse transcription was performed with a reverse transcription kit (TaKaRa Bio, Tokyo, Japan) to obtain the first strand of cDNA. According to the nucleotide sequences of BcNRT1.1, BcNRT1.2, and BcNRT2.1, three pairs of expression primers were designed (Table S1). The qPCR was performed with SYBR Green (TaKaRa Bio, Tokyo, Japan). Actin2 and GAPDH were used as internal controls. Three biological replicates were used to calculate relative gene expression levels by the 2-∆∆Ct method [40].

2.6. Data Analysis

The data were analyzed by one-way analysis of variance (ANOVA) using SPSS 19.0. Correlation analysis was performed by Pearson’s correlation coefficient analysis, then plot the heatmap with TBtools [41]. The differences between treatments were compared using the least significant difference (LSD) with a significance level of p < 0.05. The tables and figures were created using Excel 2010 and Adobe Illustrator 2020.

3. Results

3.1. Cloning and Molecular Characterization of NRT Genes

We identified three important BcNRT genes whose expression changes were evident under ammonium and nitrate nutrition from our transcriptomic data [35]. Three NRT genes were amplified using cDNA as a template. The CDS of BcNRT genes were sequenced by Shanghai Invitrogen Corporation, and the sequencing results showed that the full-length open reading frames (ORF) of BcNRT1.1, BcNRT1.2, and BcNRT2.1 were 1770 bp, 1638 bp, and 1533 bp, respectively (Figure S1). Protein sequence analysis showed that the amino acid lengths of BcNRT1.1, BcNRT1.2, and BcNRT2.1 were 589, 545, and 510 aa, respectively (Figure S2).
To understand the evolutionary relationship of these NRT genes, we constructed a phylogenetic tree by aligning the full-length protein sequences of 46 NRTS from various species. Phylogenetic analysis suggested that these 46 NRTs were clustered on their big branches (Figure 1), which were named NRT1, NRT2, and NRT3, respectively. BcNRT1.1 and BcNRT1.2 belong to the NRT1 subfamily while BcNRT2.1 belongs to the NRT2 subfamily. The NRTs shared highly homologous between Chinese cabbage and flowering Chinese cabbage but had many amino acid mutations (Figure S2). The results above indicate that the flowering Chinese cabbage as a subspecies of Chinese cabbage has undergone slight variations during the domestication process. Compared with other species, flowering Chinese cabbage has a closer evolutionary relationship with Arabidopsis and Camelina sativa, both from the cruciferous family.

3.2. N Absorption Characteristics and NRT Genes Expression Pattern of Flowering Chinese Cabbage during Different Growth Periods

To understand the N uptake characteristics, we measured the NO3− absorption rate in flowering Chinese cabbage roots at five stages. The result showed that the root dry weight gradually increased with the growth of flowering Chinese cabbage and reached a maximum of 0.47 ± 0.03 g/plant at the flowering stage (Figure 2a). The NO3− absorption rate is a trend that increased first and then decreased and reached a maximum of 0.4 mmolg−1 h−1 at S3, which is the critical period for the rapid elongation of stalks of flowering Chinese cabbage. This suggested that the demand for NO3− in the bolting stage may be higher than that in other periods (Figure 2b). The expression pattern of BcNRT1.1, BcNRT1.2, and BcNRT2.1 was examined in roots and leaves of flowering Chinese cabbage. The results showed that the BcNRT1.1 expression first increased and then decreased in the root and leaf, reaching the maximum at S3. In addition, the BcNRT1.1 expression trend is consistent with the NO3− absorption, both of which are highest at S3 (Figure 2b,c). The BcNRT1.2 expression level was relatively stable in the roots, while the transcripts abundance of BcNRT2.1 rises and then falls. From the two-leaf stage, the BcNRT1.2 expression continued to increase in the leaves and reached the maximum at the flowering period. However, the expression level of BcNRT1.2 in roots and leaves was the same at the beginning of bolting, and subsequently, BcNRT1.2 was mainly expressed in leaves (Figure 2c). The expression of BcNRT2.1 showed an overall downward trend in leaves from the two-leaf stage, and the expression level of BcNRT2.1 in roots is much higher than that in leaves (Figure 2c).

3.3. Effects of Different N Sources on the N Absorption Characteristics and NRT Genes Expression of Flowering Chinese Cabbage

The absorption of NO3− by the roots per unit time increases with the elevation of the concentration of nitrate nitrogen in the nutrient solution (Figure 3a). Within 0.5–24 h, the absorption of the nutrient solution with NO3− concentration of 8 mM by flowering Chinese cabbage was significantly higher than that of other NO3− treatments (2, 3, and 4 mM). Within 2 h after treatment, the NO3− absorption was no significant difference in 2, 3, and 4 mM nitrate nitrogen by flowering Chinese cabbage.
We tested the gene expression of three BcNRTs in flowering Chinese cabbage at different NO3− concentrations and observed that their expression showed an upward trend over time. For BcNRT1.1, a nitrate-inducible gene, its expression first increased then decreased due to the induction of nitrate in the root of flowering Chinese cabbage after nitrogen deficiency treatment. The BcNRT1.1 expression increased the fastest, reaching the maximum at 2 h under 8 mM NO3−. The expression level of BcNRT1.2 was increased under the conditions of 4 mM and 8 mM NO3−, but there was no increase under the conditions of 2 mM and 3 mM. Under the 4 mM and 8 mM NO3−, the gene expression of BcNRT1.2 reached the maximum at 8 h after treatment. Strangely, BcNRT2.1 was significantly induced by low concentration NO3− (2 mM), which showed the opposite result to BcNRT1.1 and BcNRT1.2 (Figure 3c). In addition, lower concentrations of NH4+ increased the expression of the three BcNRTs. (Figure 3d).
In order to observe the effect of single N sources with different concentrations on the N absorption rate of flowering Chinese cabbage, we calculated the absorption rate per hour in different time periods after treatment (Table 2). The rate of NH4+ uptake was higher in the 8 mM NH4+ treatment than in the other three NH4+ treatments at 0–0.5 h after treatment. However, the rate of NH4+ uptake at 4 mM treatment was highest after 0.5 h of treatment. During 24 h of NH4+ treatment, the rate of NH4+ uptake reached the maximum at 4 mM, followed by 8 mM, but there was no significant difference between the two treatments. The flowering Chinese cabbage had the highest rate of NO3− uptake at 8 mM, except for 2–8 h. Overall, the average absorption rate to NO3− with various concentrations had no significant difference between 0–8 h, but there was a significant difference between 0–24. We concluded that the optimal concentration of flowering Chinese cabbage was higher for absorbing NO3− than that for absorbing NH4+, indicating that flowering Chinese cabbage had greater absorption saturation for NO3−.

3.4. Effect of Different NH4+/NO3− Ratios on the N Absorption and BcNRTs Expression

To elucidate the effect of different NH4+/NO3− ratios on N absorption, we measured the total N content and total N absorption of flowering Chinese cabbage. From Figure 4a, it can be seen that the total N content was T1 > T2 > CK at 24 and 48 h after treatment. The total N content in T1 was significantly higher than T2 and CK by 4.56% and 7.62%, but there was no significant difference in total N content between T2 and CK throughout the trial period. Nitrogen absorption was calculated using the subtraction method. The results showed that the total N uptake in T1 was always higher than that in T2 and CK throughout the trial period. However, there was no significant difference between T2 and CK (Figure 4b).
To clarify the effect of different NH4+/NO3− ratios on BcNRT genes in flowering Chinese cabbage, we dynamically monitored the expression levels of three key BcNRT genes (BcNRT1.1, BcNRT1.2, and BcNRT2.1) during the experimental period. The three BcNRTs transcripts differed as the concentration of NH4+ in the nutrient solution increased among the three treatments (Figure 4c). Compared with the CK treatment, the increase in the NH4+ concentration reduced the expression of BcNRT1.2, and BcNRT2.1, which was the lowest in the T2 treatment (CK > T1 > T2). On the contrary, the expression level of BcNRT1.1 increased with increased NH4+ concentration in the T1 treatment, which was 19.38% and 86.67% higher than that of T2 and CK at 8 h after treatment, respectively. As time went on, the gap in BcNRT1.2 expression gradually widened among the three treatments. Regarding BcNRT2.1, the expression level in the control treatment was 1.75 and 2.33 times that in T1 and T2 at 2 h, respectively. After that, the gap between the CK and T1 treatment gradually narrowed, and the expression level turned.

3.5. Correlation Analysis between N Absorption and BcNRT Genes Expression in Flowering Chinese Cabbage

To verify the relationship between N absorption and NRT genes, we conducted a correlation analysis on the NO3− absorption rate and the roots BcNRT genes expression throughout the growth period. The result showed that BcNRT1.1 and BcNRT1.2 expression were significantly correlated with the NO3− absorption capacity in the roots of flowering Chinese cabbage (Pearson’s correlation coefficients are 0.84 and 0.95, respectively), especially BcNRT1.2. (Figure 5a). The NH4+ uptake rate was positively correlated with BcNRT2.1 gene expression at different time intervals under NH4+ with different concentrations. On the contrary, the absorption rate of NO3− was generally negatively correlated with BcNRT genes in flowering Chinese cabbage under NO3− with different concentrations (Figure 5b). Then, we measured the correlation between the total N uptake and the expression of BcNRT of flowering Chinese cabbage under the mixture of NH4+ and NO3−. We found that the total N uptake of flowering Chinese cabbage had a significant positive correlation with BcNRT1.2 under three treatments of different NH NO3− ratios (Figure 5c).

4. Discussion

In this study, we obtained three nitrate transporter genes (BcNRT1.1, BcNRT1.2, and BcNRT2.1) from flowering Chinese cabbage. They encode 589, 546, and 510 amino acids, respectively, which is genetically similar to Arabidopsis and Chinese cabbage (Figures S1 and S2). The NRT family is divided into subfamilies in these plants. The phylogenetic tree of three BcNRTs and the NRT genes from other plants showed that BcNRT1.1 and BcNRT1.2 belong to high-affinity transporter family NRT1, and BcNRT2.1 belongs to the low-affinity transporter family NRT2 (Figure 1). The NO3− absorption capacity of the roots for plant growth greatly varied during different growth stages, which is the result of coordination between the internal factors for plant growth and external N supply [42]. The N uptake has a regular trend that first rises to the maximum and then declines throughout the growth period in some horticultural crops, such as cowpea, mung bean, soybean, and corn [42,43]. We found that the NO3− absorption capacity first increased and then decreased throughout the growth cycle and reached a maximum value at S3 in flowering Chinese cabbage (Figure 2a). This result is consistent with the results of previous studies, indicating that the N absorption capacity declines in the late growth stage are compatible with the decline in external nitrogen concentration.
The NRT genes are an indispensable part of nitrate nitrogen absorption in plants, and their expression is usually closely related to the growth period [44]. Roots and leaves are crucial organs in the system of NO3− uptake and transport, and the difference of the NRT genes expression between roots and leaves also indicates the different roles that the genes assume [45]. In this study, the three BcNRTs expressed all showed a regular trend of increasing first and then decreasing in the root. Compared with BcNRT1.1, the changing trend of BcNRT1.2 expression is relatively flat as the BcNRT1.2 is a constitutive gene (Figure 2c). In addition, the expression level of BcNRT2.1 was the same as that of AtNRT2.1 in Arabidopsis roots (Figure 2c) [46]. We also found that the expression level of BcNRT1.1 in leaves was always lower than that in roots except for the two-leaf stage. This showed that BcNRT1.1, as the main member of the NPF gene families, plays an important role in the NO3− transport in roots. Many studies have shown that BcNRT1.2 was mainly expressed in the root cortex and root hairs, which was similar to the expression during the vegetative period of flowering Chinese cabbage. However, the expression of BcNRT1.2 was mainly concentrated in the leaves from the beginning of bolting. The higher expression of BcNRT1.2 may be closely related to the NO3− transport in the leaves of flowering Chinese cabbage during the reproductive period. The expression level of NRT2.1 in roots was much higher than that in leaves in non-heading Chinese cabbage [47], and was also mainly concentrated in Arabidopsis roots [45]. We found that the expression of BcNRT2.1 in the roots was much higher than that in the leaves at each stage (Figure 2c). This showed that among the three BcNRTs, the expression difference in the BcNRT2.1 was the largest between roots and leaves. It is suggested that BcNRT2.1 may mainly function in the roots of flowering Chinese cabbage.
There are many factors that cause great differences in nitrogen absorption capacity, such as root vitality and morphology, and the NRT genes expression. In this study, the change in the expression of BcNRT1.1 and BcNRT1.2 had the greatest correlation with NO3− uptake rate during the entire growth period (Figure 5a). Other studies have shown that the expression of NRT1 genes also changes with changes in the N uptake rate in Arabidopsis roots [48]. In view of the fact that the BcNRT1.1 expression had a similar trend to the absorption rate of NO3−, we think that the expression changes of NRT1 genes, especially BcNRT1.1, are compatible with the NO3− absorption capacity of flowering Chinese cabbage. Moreover, there is a growing body of evidence confirming that NRT1.1 is essential for both high- and low-affinity nitrate uptake in many species [49]. These findings can be used to study the effects of different ammonium and nitrate ratios on N absorption in roots. The absorption system of NO3− by roots is composed of HATS and LATS, in which HATS is saturated by substrate concentrations but LATS is not. This combination makes the absorption of NO3− both increased with the continuous increase in the concentration and the increase rate tends to decrease [46]. Many studies have shown that although plants have a stronger affinity for NH4+ under mixed conditions of ammonium and nitrate, this does not mean that their absorption will increase [50,51]. In this study, the absorption of NO3− was greater than that of NH4+ in flowering Chinese cabbage (Figure 3b,c). Moreover, the NO3− absorption capacity and total N content increased with the increase in NO3− concentration. The NH4+ absorption capacity no longer increases after the NH4+ reaches a certain concentration. However, the total N content does not change significantly, which shows that the NH4+ absorption has greater saturation.
The expression of NRT1.1 and NRT2;1 genes was induced by nitrate nitrogen in many plants roots, such as AtNRT2;1 (Arabidopsis) [48] and NpNRT1.2 (tobacco) [52]. The NRT2;1 gene expression increased rapidly after treatment with a low concentration of NO3− as NRT2 may be an NO3− sensor in Lotus japonicus [53] and Brassica napus [54]. In Chinese cabbage, the BrNRT2.1 expression level was higher and the duration was longer after treatment with higher concentrations of NO3−, which may be regulated by the low-affinity nitrate transporter gene NRT1.1 [55]. Some studies have found that the response of NRT to NO3− depends on the concentration of NO3− and the induction time [56]. In this study, the expression of BcNRT1.1 and BcNRT1.2 increased as the concentration of NO3− increased in the roots of flowering Chinese cabbage, and the induction effect gradually diminished. However, BcNRT2.1 was significantly up-regulated mainly at lower concentrations of NO3− (Figure 3c). Similar results were found in tobacco; the NpNRT1.2 gene was often inhibited by reduced forms of N (such as NH4+ and glutamate) [57,58]. In summary, this study shows that NH4+ and NO3− have different effects on the expression of NRT genes. The three BcNRTs in the roots of flowering Chinese cabbage were all induced by higher concentrations of NO3−, which upregulated BcNRT1.1 and BcNRT1.2 while inhibiting BcNRT2.1. In addition, the three BcNRTs expressions were upregulated at lower concentrations of NH4+ but suppressed at higher concentrations of NH4+ (Figure 3d). These results suggested that BcNRT1.1 and BcNRT1.2 play dominant roles at high concentrations of NO3−. We also found that the optimal concentration of flowering Chinese cabbage to absorb nitrate nitrogen is higher than that of NH4+, indicating that flowering Chinese cabbage prefers to absorb NO3− and thus easily accumulate excess nitrate during growth, which is what needs to be solved in production (Table 2). Therefore, we can reduce the ratio of NO3− or rationally match NH4+/NO3− nutrition during production to create the best nitrogen fertilizer application formula for the production of Chinese cabbage.
Under the mixed condition of NH4+ and NO3−, the N absorption process is more complicated than that under a single N source in plants. NO3− and NH4+ are not only used as substrates to be absorbed by plants but also as signal substances that affect the nitrogen absorption of plants [59]. Three treatments were set up in this experiment, the NH4+/NO3− ratios were 0/100 (CK), 25/75 (T1), and 50/50 (T2). We found that the total N uptake in T1 and T2 by flowering Chinese cabbage was significantly higher than that of the control treatment (Figure 4b). Correspondingly, the total N content in the T1 treatment was significantly higher than that in the CK and T2 treatments (Figure 4a). However, the NO3− absorption in the control treatment was significantly higher than that in the T1 and T2 treatments. The decrease in NO3− absorption may be caused by the coexistence of two N sources and the decrease in NO3− content under the mixed condition of NH4+ and NO3−. It showed that the coexistence of NH4+ and NO3− was beneficial to the absorption of NO3− in the flowering Chinese cabbage within a short period of time, which may cause more NO3− accumulation in the body, after all the N sources were used to replace the NO3− source. However, the concentration of NH4+ increased relatively with the extension of the cultivation time, and the rate of NO3− entering the plant body decreased, while the reduction rate of original accumulation and newly absorbed nitrate nitrogen continued to increase, resulting in a decrease in the NO3− content of flowering Chinese cabbage. Therefore, we believe that the increase in total N uptake by roots under T1 conditions is due to the fact that the plant’s absorption of NH4+ effectively compensates for the decrease in nitrate absorption. The addition of excessive NH4+ will not increase the absorption of NH4+, but will further reduce the absorption of nitrate. This is also the reason why the total N absorption with an NH4+/NO3− ratio of 50:50 is lower than that in NH4+/NO3− ratio of 25:75 (Figure 4b).
Many studies have shown that the NRT1.2 gene has an important relationship with the absorption of NH4+ and NO3− [60]. In this study, the expression of BcNRT1.2 was induced by NH4+ and NO3− and was positively correlated with the total N absorption (Figure 5c). In addition, the roots BcNRT1.2 expression was CK > T1 > T2 under the three treatments of different NH4+/NO3− ratios after treatment 8 h, which was always positively correlated with the NO3− content in the nutrient solution (Figure 4c). In rape, BnNRT1.1 expression was higher under NO3− conditions than that in NH4+, and NRT2.1 was more inhibited by NH4+ [61,62]. In Arabidopsis, a strong correlation was also found between the transcription level of AtNRT2.1 and NO3− uptake. However, our results showed that the expression of BcNRT2.1 has a greater positive correlation with the NH4+ uptake rate of the flowering Chinese cabbage under different concentrations of NH4+. Furthermore, BcNRT2.1 has a negative correlation with the NO3− uptake rate under different concentration of NO3−. There was a significant correlation between the NO3− absorption rate of flowering Chinese cabbage and the BcNRT1.1 and BcNRT1.2 expression (Figure 5a). In addition, the total N absorption was also significantly correlated with BcNRT1.2 under the mixed treatment of NH4+ and NO3−, and the correlation coefficients were all above 0.9 (Figure 5c). These results suggest that the expression of BcNRT1.1 and BcNRT1.2 is crucial for the absorption and efflux of NO3− in flowering Chinese cabbage and is beneficial to the absorption of total nitrogen. In addition, the transcriptional regulation of NRT2.1 expression may play a major role in controlling high-affinity NO3− uptake in roots.
In this study, we cloned three important NRT genes from flowering Chinese cabbage, and clarified their response patterns under different N forms and different ratios of NH4+/NO3−. However, there are still many doubts about their role in the nitrogen utilization mechanism in plants. For example: (1) How do NRT genes modify their expression level to regulate plant biomass accumulation? (2) Which transcription factors regulate the differential expression level of NRT related to nitrogen absorption? More questions need to be answered in future studies. Ultimately, these studies will potentially lead to the development of new technologies to increase crop yields and reduce nitrogen pollution in modern agriculture.

5. Conclusions

Three key BcNRT genes (BcNRT1.1, BcNRT1.2, and BcNRT2.1) were isolated from the flowering Chinese cabbage genome. BcNRT1.1 and BcNRT2.1 were mainly expressed in roots, while BcNRT1.2 was more expressed in roots than in leaves during vegetative growth and was mainly expressed in leaves during reproductive growth. The NO3− absorption capacity had a significant correlation with the BcNRT1.1 and BcNRT1.2 expression in roots in different growth stages. The total N content also increased with the increase in the NO3− concentration in flowering Chinese cabbage. The NH4+ uptake was slightly induced by NH4+, and the total N content had no significant difference under the four treatments with different concentrations of NH4+. The lower concentrations of NH4+ promoted the expression of BcNRT1.1 and BcNRT1.2, while inhibiting the expression of BcNRT2.1 in the roots of flowering Chinese cabbage. The amount of total N uptake in the treatment with 25/75 of NH4+/NO3− was significantly higher than that of the other two treatments (0/100 and 50/50). In addition, the total N uptake was significantly correlated with BcNRT1.2 expression under a mixture of NH4+ and NO3−. These results lay a theoretical foundation for our more in-depth research on the influence of these genes on N absorption and utilization in the future.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae8030188/s1, Table S1: Sequences of primers used for this study; Table S2: Sequence information of NRT proteins from different species; Figure S1: PCR amplification of NRT genes from Brassica campestris; Figure S2: Multiple sequence alignment of NRT protein sequences from Brassica Campestris (Bc), Arabidopsis thaliana (At), and Brassica rapa (Br).

Author Contributions

Conceptualization, Y.Z. and S.Z.; methodology, W.S.; software, Y.W.; validation, S.Z., Y.Z. and G.S.; formal analysis, G.S. and W.S.; investigation, Y.H.; resources, R.C., H.L. and S.S.; data curation, S.Z. and Y.Z.; writing—original draft preparation, S.Z.; writing—review and editing, S.S., Y.H. and Y.W.; visualization, S.Z.; supervision, S.S.; project administration, R.C.; funding acquisition, S.S. and R.C. 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; 32072656), the Key-Area Research and Development Program of Guangdong Province (2020B0202010006), the Guangdong Provincial Special Fund for Modern Agriculture Industry Technology Innovation Teams (2021KJ131), and the China Agriculture Research System of MOF and MARA.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogeny tree of NRTs from different species. At: Arabidopsis thaliana, Bc: Brassica campestris, Sl: Solanum lycopersicum, Ta: Triticum aestivum, Os: Oryza sativa, Nt: Nicotiana tabacum, Gm: Glycine max, Cs: Camelina sativa, Br: Brassica rapa, Cm: Chrysanthemum × morifolium. The numbers on the branches represent evolutionary distances. The sequence information for each protein is provided in Table S2.
Figure 1. Phylogeny tree of NRTs from different species. At: Arabidopsis thaliana, Bc: Brassica campestris, Sl: Solanum lycopersicum, Ta: Triticum aestivum, Os: Oryza sativa, Nt: Nicotiana tabacum, Gm: Glycine max, Cs: Camelina sativa, Br: Brassica rapa, Cm: Chrysanthemum × morifolium. The numbers on the branches represent evolutionary distances. The sequence information for each protein is provided in Table S2.
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Figure 2. Roots dry weight (a), N absorption characteristics (b) and NRT genes expression pattern (c) of flowering Chinese cabbage during different growth periods. S1, two-leaf; S2, six-leaf; S3, bolting; S4, late bolting; S5, flowering. The housekeeping gene GAPDH was used as an internal reference gene. The data represent the mean ± SE (n = 3). Different letters in (a,b) indicate significant differences at p < 0.05.
Figure 2. Roots dry weight (a), N absorption characteristics (b) and NRT genes expression pattern (c) of flowering Chinese cabbage during different growth periods. S1, two-leaf; S2, six-leaf; S3, bolting; S4, late bolting; S5, flowering. The housekeeping gene GAPDH was used as an internal reference gene. The data represent the mean ± SE (n = 3). Different letters in (a,b) indicate significant differences at p < 0.05.
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Figure 3. Effects of different N sources on the N absorption characteristics and NRT genes expression of flowering Chinese cabbage. (a,b) Effects of different N sources on the N absorption characteristics. (c,d) The roots BcNRT genes expression, as affected by different NO3− (c) and NH4+ (d) concentrations. The housekeeping gene GAPDH was used as an internal reference gene. The data represent the mean ± SE (n = 3). Different letters in (a,b) indicate significant differences at p < 0.05.
Figure 3. Effects of different N sources on the N absorption characteristics and NRT genes expression of flowering Chinese cabbage. (a,b) Effects of different N sources on the N absorption characteristics. (c,d) The roots BcNRT genes expression, as affected by different NO3− (c) and NH4+ (d) concentrations. The housekeeping gene GAPDH was used as an internal reference gene. The data represent the mean ± SE (n = 3). Different letters in (a,b) indicate significant differences at p < 0.05.
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Figure 4. Influence of different NH4+/NO3− ratios in response to N absorption (a,b) and roots NRT genes expression (c) of flowering Chinese cabbage, Control= 0/100, T1 = 25/75, T2 = 50/50. The β-actin gene was used as an internal reference gene. The data represent the mean ± SE (n = 3). Different letters in (a,b) indicate significant differences at p < 0.05.
Figure 4. Influence of different NH4+/NO3− ratios in response to N absorption (a,b) and roots NRT genes expression (c) of flowering Chinese cabbage, Control= 0/100, T1 = 25/75, T2 = 50/50. The β-actin gene was used as an internal reference gene. The data represent the mean ± SE (n = 3). Different letters in (a,b) indicate significant differences at p < 0.05.
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Figure 5. Correlation analysis between N absorption and BcNRT genes expression in flowering Chinese cabbage. (a) Correlation analysis between NO3− absorption rate and BcNRT genes. (b) Correlation between phasic N uptake rate and BcNRT genes expression under different concentrations of N sources. (c) Correlation between total N uptake and BcNRT genes expression under different NH4+/NO3− ratios. Red: Positive correlation; blue: negative correlation; white: no correlation. White asterisks indicate significance (*: p < 0.05, **: p < 0.01).
Figure 5. Correlation analysis between N absorption and BcNRT genes expression in flowering Chinese cabbage. (a) Correlation analysis between NO3− absorption rate and BcNRT genes. (b) Correlation between phasic N uptake rate and BcNRT genes expression under different concentrations of N sources. (c) Correlation between total N uptake and BcNRT genes expression under different NH4+/NO3− ratios. Red: Positive correlation; blue: negative correlation; white: no correlation. White asterisks indicate significance (*: p < 0.05, **: p < 0.01).
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Table
Table 1. Nutrient solution with different NH4+/NO3− ratios (unit: mM).
Table 1. Nutrient solution with different NH4+/NO3− ratios (unit: mM).
TreatmentsNH4+/NO3−NaNO3KH2PO4MgSO4·7H2O(NH4)2SO4KClCaCl2
CK0/100422-20.5
T125/753220.520.5
T250/50222120.5
Table
Table 2. Effects of different N sources on the N uptake rate of flowering Chinese cabbage (unit: mmol L−1 h−1).
Table 2. Effects of different N sources on the N uptake rate of flowering Chinese cabbage (unit: mmol L−1 h−1).
TreatmentsConcentration0–0.5 h0.5–2 h2–8 h8–24 h0–8 h0–24 h
NH4+1 mM0.696 ± 0.217c0.911 ± 0.067b0.323 ± 0.012a0.041 ± 0.002c0.456 ± 0.008b0.179 ± 0.001b
2 mM2.035 ± 0.134b0.840 ± 0.038b0.260 ± 0.025a0.045 ± 0.001c0.479 ± 0.022b0.251 ± 0.004b
4 mM2.379 ± 0.308b1.622 ± 0.165a0.352 ± 0.020a0.137 ± 0.004a0.717 ± 0.005a0.269 ± 0.002a
8 mM4.886 ± 0.131a0.365 ± 0.131c0.350 ± 0.080a0.086 ± 0.011b0.659 ± 0.030a0.277 ± 0.004a
NO3−2 mM1.698 ± 0.001c0.110 ± 0.014b0.196 ± 0.023a0.109 ± 0.007b0.233 ± 0.016a0.150 ± 0.002d
3 mM1.718 ± 0.030b0.135 ± 0.128ab0.168 ± 0.059a0.138 ± 0.033b0.259 ± 0.043a0.178 ± 0.010c
4 mM1.498 ± 0.004d0.318 ± 0.316ab0.092 ± 0.017a0.239 ± 0.029a0.222 ± 0.050a0.233 ± 0.005b
8 mM1.758 ± 0.312a0.695 ± 0.238a0.104 ± 0.133a0.298 ± 0.031a0.318 ± 0.055a0.305 ± 0.004a
Data are presented as the mean ± SE (n = 3). Different letters indicate significant differences at p < 0.05.
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Zhang, S.; Zhang, Y.; Wang, Y.; Hao, Y.; Su, W.; Sun, G.; Liu, H.; Chen, R.; Song, S. Nitrogen Absorption Pattern Detection and Expression Analysis of Nitrate Transporters in Flowering Chinese Cabbage. Horticulturae 2022, 8, 188. https://doi.org/10.3390/horticulturae8030188

AMA Style

Zhang S, Zhang Y, Wang Y, Hao Y, Su W, Sun G, Liu H, Chen R, Song S. Nitrogen Absorption Pattern Detection and Expression Analysis of Nitrate Transporters in Flowering Chinese Cabbage. Horticulturae. 2022; 8(3):188. https://doi.org/10.3390/horticulturae8030188

Chicago/Turabian Style

Zhang, Shuaiwei, Yuepeng Zhang, Yudan Wang, Yanwei Hao, Wei Su, Guangwen Sun, Houcheng Liu, Riyuan Chen, and Shiwei Song. 2022. "Nitrogen Absorption Pattern Detection and Expression Analysis of Nitrate Transporters in Flowering Chinese Cabbage" Horticulturae 8, no. 3: 188. https://doi.org/10.3390/horticulturae8030188

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