Genome-Wide Identification of Na+/H+ Antiporter (NHX) Genes in Sugar Beet (Beta vulgaris L.) and Their Regulated Expression under Salt Stress

Abstract

Salinity is one of the major environment factors that limits the growth of plants and the productivity of crops worldwide. It has been shown that Na⁺ transporters play a central role in salt tolerance and development of plants. The objective of this study was to identify Na⁺/H⁺ antiporter (NHX) genes and investigate their expression patterns in sugar beet (Beta vulgaris L.) subjected to various concentrations of NaCl. A total of five putative NHX genes were identified and distributed on four chromosomes in sugar beet. Phylogenetic analysis revealed that these BvNHX genes are grouped into three major classes, viz Vac- (BvNHX1, -2 and -3), Endo- (BvNHX4), and PM-class NHX (BvNHX5/BvSOS1), and within each class the exon/intron structures are conserved. The amiloride-binding site is found in TM3 at N-terminus of Vac-class NHX proteins. Protein-protein interaction (PPI) prediction suggested that only BvNHX5 putatively interacts with calcineurin B-like proteins (CBL) and CBL-interacting protein kinases (CIPK), implying it might be the primary NHX involved in CBL-CIPK pathway under saline condition. It was also found that BvNHX5 contains one abscisic acid (ABA)-responsive element (ABRE), suggesting that BvNHX5 might be involved in ABA signal responsiveness. Additionally, the qRT-PCR analysis showed that all the BvNHX genes in both roots and leaves are significantly up-regulated by salt, and the transcription levels under high salinity are significantly higher than those under either low or moderate salinity. Taken together, this work gives a detailed overview of the BvNHX genes and their expression patterns under salt stress. Our findings also provide useful information for elucidating the molecular mechanisms of Na⁺ homeostasis and further functional identification of the BvNHX genes in sugar beet.

Keywords: Na+ compartmentalization; Na+ exclusion; Na+/H+ antiporter; amiloride-binding site; salt tolerance; sugar beet.

Figure 1

Phylogenetic tree of the NHX genes from Beta vulgaris (Bv), Arabidopsis thaliana (At), Cucurbita maxima (Cm), Eutrema halophilum (Eh), Hordeum vulgare (Hv), Gossypium hirsutum (Gh), O. sativa (Os), Solanum lycopersicum (Sl), Sorghum bicolor (Sb), Spinacia oleracea (So), Solanum tuberosum (St), Triticum aestivum (Ta) and Vitis vinifera (Vv). The tree was determined by the Neighbor-Joining method (NJ) with 1000 bootstrap replicates using MEGA7.0. According to the clustering of the NHX proteins, we divided proteins into three different classes, viz Vac-, Endo-, and PM-class. Proteins from sugar beet and Arabidopsis are denoted by blue and red diamonds, respectively. Details of NHXs from 13 species are listed in Supplementary Table S1.

Figure 2

Physical mapping and structure analysis of the BvNHX gene. (A) The distribution of the BvNHX genes of sugar beet on 9 chromosomes. The number of chromosomes is shown at the top of each chromosome. The numbers of BvNHX genes are indicated on the left of each chromosome. The scale of the genome size is given on the left. (B) Gene structures of the BvNHX genes. Exons are indicated by yellow boxes. Introns are indicated by black lines. Upstream and downstream are indicated by blue boxes. The scale of genes length is given at the bottom. CDS: Coding sequence; Mb: million bases; kb: kilo bases.

Figure 3

Motif analysis of the BvNHX proteins. Distribution of conserved motifs in BvNHXs was analyzed by the online tool MEME. Details of different motifs indicated by different colors are shown in Supplementary Figure S2. The scale of proteins length was given at the bottom. aa: amino acid.

Figure 4

Structural analysis of five BvNHX modeled proteins. The best PDB structural analog for each transporter is shown in Table 6. Details of secondary structure of BvNHX proteins are shown in Supplementary Figure S3.

Figure 5

Protein-protein interaction (PPI) network of BvNHXs. Line thickness indicates the strength of data support. Network is clustered to 3 clusters, which are represented with red, green, and blue nodes, respectively. Details of string analysis for individual BvNHX protein are shown in Supplementary Figure S4.

Figure 6

Relative expression levels of BvNHX genes in leaf and root of sugar beet seedlings subjected to 50, 100, 200, and 300 mM NaCl for 0, 3, 6, 12, 24, and 48 h. Expression of the BvNHX genes normalized to those of BvACTIN and shown relative to the expression at 0 h. The 2^−ΔΔCt method was used to calculate the expression levels of BvNHX genes at different time. Experiments were repeated at least 3 times. Values are means ± SE and bars indicate SE (n = 3). Columns with different letters represent significant differences at p < 0.05 level (Duncan’s test).