Genome-wide identification of high-affinity nitrate transporter 2 (NRT2) gene family under phytohormones and abiotic stresses in alfalfa (Medicago sativa)

Abstract

The high-affinity nitrate transporter 2 (NRT2) protein plays an important role in nitrate uptake and transport in plants. In this study, the NRT2s gene family were systematically analyzed in alfalfa. We identified three MsNRT2 genes from the genomic database. They were named MsNRT2.1-2.3 based on their chromosomal location. The phylogenetic tree revealed that NRT2 proteins were categorized into two main subgroups, which were further confirmed by their gene structure and conserved motifs. Three MsNRT2 genes distributed on 2 chromosomes. Furthermore, we studied the expression patterns of MsNRT2 genes in six tissues based on RNA-sequencing data from the Short Read Archive (SRA) database of NCBI, and the results showed that MsNRT2 genes were widely expressed in six tissues. After leaves and roots were treated with drought, salt, abscisic acid (ABA) and salicylic acid (SA) for 0-48 h, and we used quantitative RT-PCR to analyze the expression levels of MsNRT2 genes and the results showed that most of the MsNRT2 genes responded to these stresses. However, there are specific genes that play a role under specific treatment conditions. This result provides a basis for further research on the target genes. In summary, MsNRT2s play an irreplaceable role in the growth, development and stress response of alfalfa, and this study provides valuable information and theoretical basis for future research on MsNRT2 function.

Keywords: Medicago sativa; NRT2; Gene family; Hormones; Nitrogen.

Fig. 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.

Fig. 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.

Fig. 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).

Fig. 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.

Fig. 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.

Fig. 6

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

Fig. 7

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

Fig. 8

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

Fig. 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.