Introduction

Nitrogen, as one of the essential elements in plant growth and development, is a constituent of many biomolecules in plants, such as proteins, nucleic acids, and chlorophyll1. It plays an important role in biochemical reactions and metabolic processes in plants and is an important nutrient that affects the physiological performance and yield of plants2. Low nitrogen environment significantly affects plant growth and development, such as plant dwarfing, lateral branch reduction, root-shoot ratio increase, early flowering and senescence under low nitrogen stress3,4. In order to increase plant productivity, a large amount of nitrogen fertilizer are applied to the soil every year, while more than 75% of the nitrogen fertilizer are not absorbed by plants and run off the environment, resulting in the eutrophication of water bodies and the increase of nitrogen oxide gases in the air, which triggers a series of environmental and economic problems5,6,7. Therefore, it is of great theoretical and practical application value to study the mechanism of nitrogen uptake and utilization by plants to improve nitrogen utilization efficiency and reduce nitrogen fertilizer application.

For most dry-land plants, the main form of nitrogen absorbed and utilized is nitrate nitrogen. Nitrate is the most abundant inorganic nitrogen in soil and its uptake and translocation within plants have a significant influence on their nitrogen use efficiency8,9. Nitrate is not only the most important source of nitrogen for most plants, but also an important signaling molecule during plant growth and development10. Nitrate is actively taken up through the roots and leaves, and then transported within the plant by various nitrate transporters, each of which has different features11,12. The Nitrate Transporter 1 (NRT1)/Peptide Transporter (PTR) family (NPF), NRT2 family, Chloride Channel (CLC) family and Slow Anion Channel (SLAC) protein family are the four protein families that fulfill key functions about nitrate transport13,14. These interactions form an intricate regulatory network. Two types of nitrate transport systems have been found in higher plants: high-affinity nitrate transport system (HATS) and low-affinity nitrate transport system (LATS)15. When the concentration of nitrate in the environment was lower than 1 mM, plants absorb nitrate from the external environment mainly through HAST. On the other hand, the LAST system was activated to take up nitrate from the external environment when the nitrate concentration was higher than 1 mM in the environment16. Both systems have inducible and constitutive components. Among them, NPF mediates LATS and NRT2 mediates HATS17.

NRT2 homologous proteins are typical high-affinity nitrate transporters in the plant kingdom and are responsible for the nitrate uptake process. In general, NRT2 proteins (NRT2s) have a typical membrane topology connected by a cytosolic loop, including 1 major facilitator superfamily (MFS) domain that exhibits dual affinities for nitrate18 and 12 transmembrane domains19, which are usually located on the cell plasma membrane20. Lots of NRT2 members have been identified in Arabidopsis thaliana (7)15, Brassica napus (17)21, Oryza sativa (4)22, Zea mays (4)23, Solanum lycopersicum (4)24, Manihot esculenta (6)25, and so on25,26,27. Molecular biology studies have shown that different members of NRT2 have abundant biological functions. Studies of the NRT2 gene family have mostly focused on A. thaliana. 7 NRT2 genes were identified in A. thaliana. The most representative members were AtNRT2.1 and AtNRT2.2 genes, which were extremely important for nitrate transport and compensate each other28. AtNRT2.3 gene was mainly expressed periodically in young leaves. AtNRT2.4 is responsible for translocating nitrate from roots to shoot29. AtNRT2.5 plays a major role in the secondary veins of mature leaves. The growth analysis of the mutant showed that AtNRT2.5 was a necessary condition to support the growth of N-deficient adult plants, and it cooperated with AtNRT2.1, AtNRT2.2 and AtNRT2.4 to ensure the effective absorption of nitrate by loading nitrate into phloem during nitrate remobilization30,31. AtNRT2.6 gene was mainly expressed in roots and young leaves. AtNRT2.7 gene was mainly expressed in seeds. In addition, AtNRT2.7 is involved in the regulation of nitrate content and dormancy in seeds. It was shown that AtNRT2.7 is specifically expressed in seeds and is uniquely involved in nitrogen oxide (NO3−) storage in vesicular membranes32. Notably, except for AtNRT2.7, all of the other AtNRT2 transporters could interact with AtNAR2.1, enhancing the nitrate uptake capacity of AtNRT2s33. Similarly, the homologs of AtNRT2s in other plants were widely demonstrated to perform numerous roles in nitrate uptake, transport, and utilization processes across developmental stages. Together, NRT2 homologous genes play key roles in nitrate uptake and even utilization in plants. Thus, systematically identifying the NRT2 gene families in plant genomes and exploring their roles involved in nitrate utilization processes may contribute to promoting nitrogen utilization efficiency and crop yields without resorting to excessive nitrogen fertilizer.

Alfalfa (Medicago sativa) is the legume forage grass with the longest cultivation history and the widest area in the world. Compared with gramineous crops, its absorption and utilization of nitrogen is more complex due to its high protein content. It has become an indispensable protein feed in the development of animal husbandry.

Nitrogen application is one of the most common methods used in crop production to increase crop yields. However, nitrogen fertilizer application prolongs the flowering period and delays the maturity of alfalfa, thereby increasing the risk of yield loss. Therefore, it is important to study the NRT gene to improve nitrogen use efficiency in alfalfa34. Given the important roles in nitrogen utilization-related processes, we identified NRT2 members at the genome-wide level in alfalfa. Herein, a total of 3 NRT2 genes were characterized from alfalfa. Additionally, we performed systematic analyses of phylogenetic relationships, conserved motifs, gene structures and chromosome locations. Therefore, the present experimental data will be a reference and provide a basis for the functional analysis of the NRT2s in alfalfa.

Results

Identification of MsNRT2 genes in alfalfa

Using HMM of SHMT domain, we identified 3 NRT2 genes in alfalfa. The NRT2 genes were named from MsNRT2.1 to MsNRT2.3 according to their chromosomal locations (Table 1). All NTR2 genes contain PF07690 domain structure and their protein lengths ranged from 449 to 526 amino acid residues. The molecular weights (MW) of different MsNRT2 genes range from 48.52 kDa to 57.07 kDa. The isoelectric point (pI) of all MsNRT2s were alkaline, indicating that the NRT2s of alfalfa were rich in alkaline amino acids. Among them, the PI of MsNRT2.1 and MsNRT2.3 were equal at 9.21. The predicted subcellular localization result showed that all the 3 MsNRT2s were located in the cell membrane. The instability index of MsNRT2.1, MsNRT2.2 and MsNRT2.3 were 38.2, 36.59 and 39.29 respectively, which suggested that their properties were relatively stable. The grand average of hydropathicity (GRAVY) index ranges from 0.353 (MsNRT2.1) to 0.558 (MsNRT2.3), indicating that they were all hydrophilic proteins (Table 1).

Table 1 The characteristics of the MsNRT2 genes in alfalfa.
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Phylogeny analysis of MsNRT2 genes

In order to decipher the evolutionary history and functional associations of alfalfa NRT2 proteins, a Neighbor-Joining phylogenetic tree was constructed using the full-length amino acid sequences of NRT2s from M. sativa (3), A. thaliana (7) and O. sativa (4). As can be seen in Fig. 1, MsNRT2.2 and MsNRT2.3 belong to the same subfamily, while MsNRT2.1 belongs to another subfamily. They have high homology with the NRT2 genes of A. thaliana. This may be due to that the fact that both are dicotyledons, whereas O. sativa is a monocotyledon.

Fig. 1
figure 1

Phylogenetic relationship analysis of M. sativa, O. sativa and A. thaliana. Different colors represent different clades, green squares mark NRT2 genes in A. thaliana, red stars mark NRT2 genes members in M. sativa, and blue circles mark NRT2 genes members in O. sativa.

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Gene structure and chromosomal location analysis of MsNRT2 genes

For study the conservation and diversity of alfalfa MsNRT2 proteins during long-term evolution, we analyzed their chromosome location, gene structure, and conservative motifs. There were 3 extrons in MsNRT2.1, 2 in MsNRT2.2, and 4 in MsNRT2.3 (Fig. 2A). We analyzed 10 conserved motifs of 3 MsNRT2 proteins using an online MEME program. In this study, MsNRT2.2 and MsNRT2.3 proteins contain all the motifs except MsNRT2.1 protein which lacks motif 10 (Fig. 2B). It was obvious that motif 1 ~ motif 10 had the same arrangement in the MsNRT2 protein family. Therefore, members of the MsNRT2 protein family are relatively conserved, and it is speculated that motif1-motif10 are likely to be closely related to the execution of nitrate uptake by MsNRT2.

In order to clarify the distribution of MsNRT2 on the chromosomes, we used the alfalfa genome annotation information and TBtools software to visualize the distribution of chromosomes (Fig. 2C). The results showed that the 3 MsNRT2s map to alfalfa chromosomes 4, and 8.

Fig. 2
figure 2

Gene structure of the NRT2 gene in alfalfa. (A) Exon–intron structures of the NRT2 family genes. (B) Conserved motifs of alfalfa NRT2. The motifs were identified using the MEME program. Boxes of different colors represent motif 1 to 10, respectively. The length of amino acid sequences can be estimated by the scale at the bottom. (C) Chromosomal location of NRT2 genes. The length of chromosomes can be estimated using the scale on the left.

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Cis-acting regulatory elements analysis in the promoter region of MsNRT2 genes

The study subsequently predicted cis-acting elements on the 2000 bp upstream promoter regions of the 3 MsNRT2s by PlantCARE online software. The study found a total of 89 main cis-acting elements which were divided into 3 major types, including hormone response elements (28), light-responsive elements (31) and stress-related elements (30) (Fig. 3A). The cis-elements were associated with responses to various plant hormones, such as auxin (IAA), abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA) and ethylene (ETH). Among these, the most prevalent cis-acting elements were TCA-element and ERE, accounting for 21.43%, followed by ABRE at 17.86% (Fig. 3B). In the light signal responses group (Fig. 3C), 6 main types of cis-acting elements: Box 4 (32.26%), GATA-motif (19.35%), GT1-motif (16.13%), G-box (16.13%), TCT-motif (12.90%) and MRE (3.23%). Among the cis-acting elements involved in stress responses, some functioned in response to specific stress conditions. For instance, drought and salt response elements (MYB and MYC) were essential for abiotic stress, accounting for 30.00% and 26.67%. Additionally, anaerobic induced response element (ARE), wound responsiveness element (WRE3), low-temperature responsiveness (LTR), and defense and stress response element (TC-rich repeats) were also identified as stress responses (Fig. 3D). In summary, our findings suggest that the three NRT2 genes play important roles in various biological processes, especially light signal response, hormone response, and stress response.

Fig. 3
figure 3

Promoter cis-acting elements analysis of MsNRT2 genes. (A) The identified cis-acting elements were divided into three functional groups and represented by red, green and blue shades of colors. The numbers indicate the quantity of corresponding cis-acting elements in each NRT2 genes promoter region. Distribution of cis-acting elements with different categories within each group of cis-acting elements with different specific functions, including hormone response (B), light signal (C), and stress response (D).

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Expression patterns of MsNRT2 genes in different tissues

To further determine the expression specificity of alfalfa NRT2 genes in different tissues, we analyzed the expression levels of alfalfa in roots, nodules, leaves, elongating stem internodes (ES), post-elongation stem internodes (PES) and flowers based on RNA sequencing data (Fig. 4). MsNRT2.1 and MsNRT2.2 were not expressed in the other four tissues except in root and nodule, and the expression of MsNRT2.1 was higher in nodule and MsNRT2.2 in root. Unlike MsNRT2.1 and MsNRT2.2, MsNRT2.3 was expressed in every tissue and showed specificity. The expression level of MsNRT2.3 was much higher in root and leave than in other tissues. These results suggest that MsNRT2 genes displayed diverse expression patterns and might play special roles in different plant organs.

Fig. 4
figure 4

Expression patterns of 3 MsNRT2 genes in 6 tissues of alfalfa. The expression level was based on the RNA sequencing data from SRA database of NCBI (Accession number SRP055547). Color bars represent expression values, and different colors indicate different expression levels. The abbreviations in the figure represent ES, elongating stem internodes; PES, post-elongation stem internodes.

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Expression profile analysis of MsNRT2 genes in leaves under hormonal and abiotic stress

qRT-PCR was used to detect the relative expression of the MsNRT2 genes in alfalfa seedling leaves and to understand the potential function of MsSNRT2 in responding to exogenous factors during the alfalfa seedling stage (Figs. 5 and 6). The MsNRT2.1 gene was not expressed in leaves. MsNRT2.2 and MsNRT2.3 genes could be induced by various treatments. Under ABA treatment (Fig. 5A), the expression of MsNRT2.2 gene increased first and then decreased with the increase of treatment time, reaching the maximum value at 12 h. The transcript level of MsNRT2.3 was significantly inhibited and the expression level was lowest at 48 h. Under SA treatment (Fig. 5B), the expression of the MsNRT2.2 gene was significantly higher at 12 h and 48 h than at 0 h, indicating that MsNRT2.2 gene responded to SA at 12 h and 48 h. The expression level of the MsNRT2.3 gene was significantly suppressed before 24 h, but was significantly higher at 48 h than at 0 h, suggesting that MsNRT2.2 and MsNRT2.3 coordinated with each other at 48 h to promote the SA response.

As shown in Fig. 6A, under PEG stress, the expression patterns of MsNRT2.2 and MsNRT2.3 genes were different in various treatment periods. The expression of the MsNRT2.2 gene was significantly increased, reaching the maximum at 24 h, which increased 9.58-fold compared to 0 h. However, the expression of MsNRT2.3 was significantly suppressed and showed the lowest value at 12 h. Under NaCl stress, the expression level of MsNRT2.3 gradually decreased with the extension of NaCl treatment time (Fig. 6B). Taken together, the abiotic stress treatments induced changes in the abundance of MsNRT2.2 and MsNRT2.3 genes transcripts, and the expression patterns of MsNRT2 genes suggested that MsNRT2 genes might play a role in abiotic stress responses in alfalfa leaves.

Fig. 5
figure 5

Expression profiles of the MsNRT2 genes in leaves treated with (A) abscisic acid (ABA), and (B) salicylic acid (SA). Error bars represent the standard deviation (SD) of three replicates. The relative expression of each gene in different treatments is expressed as mean ± SD (n = 3). Bars with different lowercase letters were significantly different by Duncan’s multiple range tests (P<0.05), the same as below.

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Fig. 6
figure 6

Expression profiles of the MsNRT2 genes in leaves treated with (A) PEG, and (B) NaCl.

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Expression profile analysis of MsNRT2 genes in roots under hormonal and abiotic stress

To clarify the role of MsNRT2s in response to environmental stimuli, the expression patterns of 3 MsSNRT2 genes in the roots of alfalfa seedlings under ABA, SA, PEG, and NaCl treatments were examined using qRT-PCR assays (Figs. 7 and 8). Under ABA and SA treatment (Fig. 7A and B), the expression of MsNRT2.1 gene increased first and then decreased with the increase of treatment time, reaching the maximum value at 12 h. The transcript levels of MsNRT2.2 showed different expression patterns under ABA and SA treatments. The expression of MsNRT2.2 reached its maximum at 12 h under ABA treatment and at 6 h under SA treatment. Compared with 0 h, they increased by 5.94 and 1.97-fold respectively. The expression level of the MsNRT2.3 gene was significantly suppressed after treatment.

Fig. 7
figure 7

Expression profiles of the MsNRT2 genes in roots treated with (A) abscisic acid (ABA), and (B) salicylic acid (SA).

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As shown in Fig. 8A, under PEG stress, the expression patterns of 3 MsNRT2 genes were different in various treatment periods. The expression level of MsNRT2.1 was significantly higher than at 0 h except at 12 h and 24 h. Except for 6 h, all treatments induced MsNRT2.2 up-regulated and reached the highest value after 48 h of treatment. However, the expression of MsNRT2.3 was significantly suppressed at 12 h. Under NaCl stress, the expression level of MsNRT2.1 gradually increased with the extension of NaCl treatment time (Fig. 8B). All treatments induced MsNRT2.2 up-regulation except for 6 h. The transcription level of MsNRT2.3 was down-regulated after NaCl treatment.

Fig. 8
figure 8

Expression profiles of the MsNRT2 genes in roots treated with (A) PEG, and (B) NaCl.

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Fig. 9
figure 9

Effect of hormones and abiotic stress on nitrate uptake and assimilation processes in the root system of alfalfa. AA, amino acids; GS2, plastidial glutamine synthetase; NiR, nitrite reductase; NR, nitrate reductase; GOGAT, glutamate synthase; Gln, glutamine; Glu, glutamate; ABA, abscisic acid; SA, salicylic acid.

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Discussion

NRT2, as a high-affinity nitrate transporter protein, plays an important role in nitrate uptake and transport in plants, and it is expressed in roots, phloem, epidermis, leaves, seeds, and other organs of plants35. The NRT2 genes has been studied in many species, however, little is known about the NRT2 gene family in alfalfa. In this study, we identified three NRT2 genes using bioinformatics methods. It has been found that seven, four, five, and four NRT2 genes were identified from A. thaliana36, O. sativa37, G. max38, and Populus tomentosa39, respectively. The reason for the difference in the number of genes among species may be due to the difference in the degree of gene amplification among species40. Three genes were distributed on chromosomes 4 and 8 (Fig. 2C). Despite alfalfa is better adapted to infertile soils, the number of NRT2 was not significantly expanded.

To explore the evolutionary relationships of MsNRT2 members with other species, this study analyzed the phylogenetic trees of NRT2 members of M. sativa, A. thaliana and O. sativa. Phylogenetic analysis of these genes revealed that these genes can be classified into three categories, while MsNRT2.2 and MsNRT2.3 in alfalfa were classified into the same category, and MsNRT2.1 was classified into another branch closer to A. thaliana NRT2s (Fig. 1). Based on a phylogenetic tree analysis of the MsNRT2 genes revealed that different evolutionary branches of the MsNRT2 genes contain common or specific motifs. Although the number of motif members varies across branches, the group motif patterns are strongly conserved. Comparison of novel functional structural domains or motif sequences across multiple homologous proteins has become a widely used method for predicting protein function based on evolutionary conservation41. Protein motif analysis showed that all MsNRT2 genes contain nine conserved elements that constitute the MFS_1 structural domain. The existence of these conserved motifs provides a guarantee for MsNRT2 protein family to function and play a role in nitrate absorption and transport, which is of great significance. For example, when following Atnrt2.1/ Atnrt2.2 double mutants, the function of the protein can be significantly affected by disruption of the conserved motifs, resulting in reduced nitrate uptake42. However, MsNRT2.1 motifs differ mainly in motif 10, and it is hypothesized that the difference in protein motifs may be the main reason for the functional differences between MsNRT2.2/2.3 and MsNRT2.1 (Fig. 2B). These results suggest that during the evolution of the MsNRT2 family, internal structural differentiation may have led to functional differentiation.

When plants are stimulated by external factors, some transcription factors are activated, and the activated factors combine with the cis-acting elements of the downstream target gene promoters to change the expression pattern of genes. The study of gene cis-acting elements is particularly important for mining the potential function of genes. To date, many cis-acting elements have been well characterized and divided into distinct groups43. In Malus domestica, all NRT2s were found to contain jasmonic (JA) and ABA motifs except MdNRT2.4 and MdNRT 2.544. The cis-acting regulatory element of Brassica napus NRT2 genes can be categorized into four groups: abiotic stress response elements, developmental regulatory elements, MYB binding sites, and oilseed rape-associated elements21. By analyzing the action elements in the promoter region of alfalfa NRT2 family members, a large number of light-responsive and stress elements were found (Fig. 3A, C and D), which suggests that the expression of MsNRT2 genes may be regulated by light and stress. In addition, many hormone-responsive elements were found, such as ABRE, AuxRR-core, TATC-box and TCA-element. These results indicate that the MsNRT2 genes might respond to a variety of hormone signals and involved in alfalfa growth and developmental activities (Fig. 3B). Reported studies have found that phytohormones play a role in nitrogen regulation and signaling, including auxin (IAA), cytokinin (CTK), as well as abscisic acid (ABA) and epibrassinolide (BR)45. Broad spectrum cis-acting regulatory elements possessed by the NRT2 genes may support an active role for the gene family in cellular metabolic pathways, particularly hormone signaling pathways.

To explore the role of MsNRT2 genes in the growth and development of alfalfa, the expression levels of MsNRT2 genes in different tissues of alfalfa were analyzed, and it was found that the spatiotemporal and temporal expression patterns of MsNRT2 genes differed in various tissues of alfalfa. For example, MsNRT2.1 was expressed only in roots and rhizomes. MsNRT2.3 was expressed in all tissues with differences (Fig. 4). Although the present study did not knockout experiments to demonstrate that the MsNRT2 genes could enhance the stress tolerance of alfalfa, the changes in the expression of these genes under stress suggested that they might be involved in the stress response. Zhao et al.46 found that the Cl/NO3 co-transport system is critical for salt tolerance in plants, and that this system may be controlled through NRT2s, especially in Cl-sensitive plants. The SlNRT2 gene also influences Solanum lycopersicum response to drought and salinity. The SlNRT2 gene responds to drought and salt stress in a variety of ways, with neither stress specificity nor tissue specificity24. In rice, the expression of OsNRT2.3 gene were increased under high temperature stress, thereby improving high temperature tolerance and nitrogen absorption levels47. Overall, it can be concluded that NRT2 genes play active roles in different tissues, organs and developmental stages.

Based on tissue-specific and cis-acting element analysis studies, we analyzed the expression of the alfalfa MsNRT2 genes in roots and leaves using ABA, SA, PEG, and NaCl treatment (Figs. 5, 6, 7 and 8). The results showed that the MsNRT2 genes were mainly expressed in roots (Figs. 7 and 8). ABA and SA treatment increased the expression of MsNRT2.1 and MsNRT2.2 and decreased the expression level of MsNRT2.3, suggesting that ABA and SA stimulated the up-regulation of MsNRT2.1 and MsNRT2.2 activities and increased nitrogen assimilation during nitrate uptake in alfalfa. Several studies indicate nitrate can regulate biosynthesis, de-conjugation, degradation, transport, and signaling of different phytohormones, adjusting nitrogen availability and plant growth and development48,49,50. In contrast, hormonal signaling feedback controls nitrate regulatory networks and metabolism51,52,53. Apparently, nitrate and hormone signaling are closely related in coordinating plant morphology and function. Hormone signaling exerts feedback control on nitrate regulatory networks and metabolism to align plant nutrient-adapted growth and development with existing environmental conditions (Fig. 9). Tong et al. found that the expression of BnNRT2.7 and BnNRT2.1a in the NRT2 family of oilseed rape was up-regulated under drought stress21. In this study, the expression of MsNRT2.1 and MsNRT2.2 was also up-regulated under drought and salt stress, suggesting that the expression of MsNRT2 genes was associated with abiotic stress response and affected nitrogen uptake and translocation. Therefore, the specific functions of MsNRT2 genes require further in-depth research. Figure 9 shows the potential regulatory model of MsNRT2s. Here, we highlight the link between different hormones and abiotic stress and nitrate signaling pathways. External conditions affect the expression of key genes involved in nitrogen uptake, transport, assimilation and signal transduction, thereby affecting nitrogen uptake efficiency in alfalfa. NRT2.1, NRT2.2, and NRT2.3 may also regulate the expression of hormone- and abiotic stress-related genes that they affect nitrogen uptake efficiency. In summary, the study of MsNRT2 gene will provide excellent candidate genes for further genetic improvement of nitrogen utilization efficiency in alfalfa.

Conclusions

In this study, the identified 3 alfalfa NRT2 genes were comprehensively studied. The evolutionary relationship of MsNRT2 genes in alfalfa and other species were established, showing that these subfamilies were evolutionarily conserved in both structure and expression. Moreover, the cis-acting elements, tissue-specific analysis of MsNRT2 family were also determined. Last but not least, we provided evidence that alfalfa NRT2s were involved in alleviating abiotic stress and hormonal responses. The comprehensive bioinformatics analysis of the MsNRT2 genes lays a solid foundation for further studies on the structure and function of the MsNRT2 genes. In addition, these results will also help to expand our knowledge to identify candidate genes that improve plant architecture under stressful conditions, and open up possibilities for breeding and genetic improvement of other forage grasses.

Materials and methods

Identification and phylogenetic analysis of the MsNRT2 genes in alfalfa

The genome data, CDS sequences and protein sequences information file of A. thaliana and M. sativa were obtained from TAIR database (http://www.arabidopsis.org) and figshare projects (https://figshare.com/projects/whole_genome_sequencing_and_assembly_of_Medicago_sativa/66380)54, respectively. Protein sequences of the A. thaliana NRT2 family were used as probes to analyze the genome sequence of alfalfa. Local BLASTP (E-value-20) search was performed based on the Hidden Markov Model (HMM) mapping of the structural domains of the NRT2 genes in the Pfam database (http://pfam.janelia.org/)55. We removed redundant and incomplete sequences, and the remaining sequences containing the protein kinase domain (PF07690) were considered candidate NRT2 members. To further ensure the existence of NRT2 conserved structures, we reconfirmed the structures of the candidate members using the online software SMART (http://smart.embl-heidelberg.de/)56, HMMER (https://www.ebi.ac.uk/Tools/hmmer/)57 and Pfam (http://pfam.xfam.org/). Finally, the study obtained the members of the M. sativa NRT2 gene family, and analyzed the physicochemical properties of these genes using ExPASy (https://web.expasy.org/protparam/)58. The subcellular localization of MsNRT2S protein was predicted using the online software PlantmPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/).

The ClustalX software was used for multiple sequence alignment of MsNRT2 proteins. Based on the alignments, the phylogenetic tree of MsNRT2 between alfalfa, Arabidopsis, and rice was constructed using MEGA 7 software with the neighbor-joining method with 1,000 replicates of bootstrap values59. The EvolView tool (http://www.evolgenius.info) was used to draw the phylogenetic tree.

Gene structure and protein motif analysis

The distribution of introns and exons phase patterns were evaluated by the online tool Gene Structure Display Server (GSDS; http://gsds.cbi.pku.edu.cn)60. The online software Multiple Expectation Maximization for Motif Elicitation (MEME; http://meme-suite.org/tools/meme)61 was used to analyze the conserved motifs, and the number of conserved motifs was defined as 10. TBtools software62 was used to visualize the gene structure and protein motif.

Chromosomal location and cis-acting elements analysis of MsNRT2 genes

This research used BLAST (Basic Local Alignment Search Tool; https://blast.ncbi.nlm.nih.gov/Blast.cgi) to extract the chromosomal location of the MsNRT2 genes information, and the distribution of the chromosomes was drawn using the MG2C v.2 program (http://mg2c.iask.in/mg2cv2.0/).

The up-stream 2000 bp DNA sequences of MsNRT2 genes was extracted from the alfalfa database using TBtools software. Then PlantCARE Database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)63 was used to identify the cis-acting regulatory elements in the promoter sequences. The number of the cis-acting elements were counted in excel.

Expression analysis of MsNRT2 genes in different plant tissues

We downloaded the expression of NRT2 genes data from NCBI’s Short Read Archive (SRA) database to study expression profiles in different tissues of alfalfa. The obtained RNA-Seq data were analyzed according to the method of Wei et al.64. Gene expression levels were calculated with Log (RPKM + 1). Then we used TBtools software to construct the heat map of the expression profile of MsNRT2s.

Plant materials and treatments

Alfalfa seeds were provided by Pratacultural College, Gansu Agricultural University, Lanzhou, China. The experiment was conducted using nutrient solution sand culture method. Full and consistent seeds were sterilized with 5% NaClO for 10 min, rinsed them with deionized water, and 20 seeds were planted evenly in 9 cm diameter and 12 cm high culture cups filled with sterilized sand. The environment of the growth room was controlled to have a photoperiod of 14/10 h (light/dark), an air temperature of 28/20℃ (day/night), a light intensity of 260–350 µmol·m− 2·s− 1. Seedlings were watered with 1×Hoagland nutrient solution 7 d after germination, and the solution was changed every 7 days. At the age of 4 weeks, the seedlings were treated with 10% (w/v) PEG-6000, 100 mM NaCl, 100 µM ABA, 1.5 mM SA, the samples were collected at 0, 3, 6, 12, 24, and 48 h after treatment, with three replicates of each treatment and 10 healthy and uniformly growing seedlings in each replicate, and then placed in − 80 °C cryopreservation for subsequent experiments.

RNA extraction and quantitative RT-PCR

According to the method provided by the manufacturer, total RNA was extracted using an RNA extraction kit (Tiangen, Beijing, China). The total RNA was then reverse-transcribed into cDNA using a cDNA synthesis kit (Tiangen, Beijing, China). We used the LightCycler 480 Real-Time PCR System (Roche Applied Science) and SYBR® Green Premix Pro Taq HS qPCR Kit for qRT-PCR experiment65. There were three biological replicates per gene. The primers were designed with Primer 5 and their specificities were confirmed by a BLAST search. As shown in Table 2, the alfalfa MsActin gene was used to normalize relative expression levels. The relative expression of each MsNRT2 gene was analyzed by 2−∆∆CT method66.

Table 2 Primer sequence of MsNRT2 for quantitative RT-PCR.
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Statistical analysis

SPSS 22.0 software was used to perform one-way analysis of variance (ANOVA) on the experimental data. Excel 2010 was used to analyze and calculate the data. Duncan method was used to analyze the difference significance (P < 0.05).