Genome-Wide Identification and Functional Characterization of the Cation Proton Antiporter (CPA) Family Related to Salt Stress Response in Radish (Raphanus sativus L.)

Abstract

The CPA (cation proton antiporter) family plays an essential role during plant stress tolerance by regulating ionic and pH homeostasis of the cell. Radish fleshy roots are susceptible to abiotic stress during growth and development, especially salt stress. To date, CPA family genes have not yet been identified in radish and the biological functions remain unclear. In this study, 60 CPA candidate genes in radish were identified on the whole genome level, which were divided into three subfamilies including the Na⁺/H⁺ exchanger (NHX), K⁺ efflux antiporter (KEA), and cation/H⁺ exchanger (CHX) families. In total, 58 of the 60 RsCPA genes were localized to the nine chromosomes. RNA-seq. data showed that 60 RsCPA genes had various expression levels in the leaves, roots, cortex, cambium, and xylem at different development stages, as well as under different abiotic stresses. RT-qPCR analysis indicated that all nine RsNHXs genes showed up regulated trends after 250 mM NaCl exposure at 3, 6, 12, and 24h. The RsCPA31 (RsNHX1) gene, which might be the most important members of the RsNHX subfamily, exhibited obvious increased expression levels during 24h salt stress treatment. Heterologous over-and inhibited-expression of RsNHX1 in Arabidopsis showed that RsNHX1 had a positive function in salt tolerance. Furthermore, a turnip yellow mosaic virus (TYMV)-induced gene silence (VIGS) system was firstly used to functionally characterize the candidate gene in radish, which showed that plant with the silence of endogenous RsNHX1 was more susceptible to the salt stress. According to our results we provide insights into the complexity of the RsCPA gene family and a valuable resource to explore the potential functions of RsCPA genes in radish.

Keywords: CPA gene family; RsNHX1; over-expression; radish; salt resistance; virus-induced gene silence.

Figure 1

Phylogenetic relationship of RsCPA, BraCPA, and AtCPA members.

Figure 2

Conserved motif and gene structure distribution of RsCPA proteins. (a) Phylogenetic tree of RsCPA proteins. The scale bar indicates 200 aa; (b) Conserved motif distribution of RsCPA proteins; (c) Exon-intron structure of CPA genes in radish. The scale bar indicates 1 kb.

Figure 3

Cis-acting elements on promoters of RsCPA genes. Distribution of cis-acting elements on promoters of RsCPA genes.

Figure 4

Chromosomal distribution and chromosomal relationships of RsCPA genes. (a) Chromosomal distribution of RsCPA genes; (b) Genome distribution and collinearity of the RsCPA family. Red lines indicate the collinear relationship among genes.

Figure 5

Evolution analysis of the RsCPA gene family. Synteny blocks of CPA genes between radish and Arabidopsis. Colored lines connecting two chromosomal regions indicate syntenic regions between Arabidopsis (Chr1–5) and radish (R1–9) chromosomes.

Figure 6

Expression profile of RsCPA genes in different stages and tissues. (a) RsCPA genes expression heatmap in six stages (7, 14, 20, 40, 60, and 90 days) and five tissues (cortical, cambium, xylem, root tip, and leaf). Expression values were calculated by (reads per kilobase per million) RPKM. The scale represents relative expression values; (b) Number of RsCPAs with a high transcriptional abundance level (RPKM > 10) in each tissue; (c) Venn diagram of overlapping RsCPAs that are abundantly expressed (RPKM > 10) in different tissues.

Figure 7

Phenotypic identification of over-expression and inhibit-expression RsNHX1 transgenic lines responding to salt stress in Arabidopsis. (a) Morphological comparison between wild-type (WT), OE–RsNHX1 and amiR–RsNHX1 transgenic lines; (b) Morphological comparison of germination between WT, OE–RsNHX1 and amiR–RsNHX1 transgenic seedlings with different concentrations of NaCl; (c) Statistical analysis of germination ratio after salt stress. Each bar shows the mean ± SD of the double assay. Values with different letters indicate significant differences at p < 0.05 according to Duncan’s multiple range tests; (d) Morphological comparison of root length between WT, OE–RsNHX1, and amiR–RsNHX1 transgenic seedlings with different concentrations of NaCl; (e) Statistical analysis of root length after salt stress. Each bar shows the mean ± SD of the triplicate assay. Values with different letters indicate a significant difference at p < 0.05 according to Duncan’s multiple range tests.

Figure 8

Functional analysis of RsNHX1 gene in radish during the salt stress. (a) Phenotype of virus symptoms on leaves of the entire plant; (b) Electrophoresis identification of pTY–S. M–marker, WT (line 1–3), pTY–S (line 4 and 5), pTY–RsPDS (line 6–9), pTY–RsNHX1 (line 10–14); (c) Relative expression levels of the RsNHX1 gene. The data represented are means of the triplicate assay and error bars represent the standard deviation of means. Different letters above bars indicate significant differences (p < 0.05) between plants; (d) Phenotype of plants after salt stress for seven days.