Identification and Expression of the CorA/MRS2/ALR Type Magnesium Transporters in Tomato

Abstract

Magnesium (Mg²⁺) is the most abundant divalent ion in plants, participating in numerous metabolic processes in growth and development. CorA/MRS2/ALR type Mg²⁺ transporters are essential for maintaining Mg²⁺ homeostasis in plants. However, the candidate protein and its potential functions in the tomato plant have not been fully understood. In this study, we identified seven MGT genes (SlMRS2) in tomato based on sequence similarity, domain analysis, conserved motif identification, and structure prediction. Two SlMRS2 genes were analyzed in the bacterial strain MM281, and a functional complementary assay demonstrated their high-affinity transport of Mg²⁺. Quantitative real-time PCR analysis revealed that the expressions of these Mg²⁺ transporters were down-regulated in leaves under Mg²⁺ limitation, with a greater impact on lower and middle leaves compared to young leaves. Conversely, under Mg²⁺ toxicity, several genes were up-regulated in leaves with a circadian rhythm. Our findings indicate that members of the SlMRS2 family function as Mg²⁺ transporters and lay the groundwork for further analysis of their distinct functions in tomato.

Keywords: RT-PCR; genomic analysis; magnesium transporter; qRT-PCR; tomato (Solanum lycopersicum L.).

Figure 1

Multiple sequence alignment analysis of Mg²⁺ transporters in tomato. The alignment map was prepared using DNAMAN, wherein the same sequence was marked black, sequence similarity of more than 75% was marked as rose, sequences less than 50% were colorless, and the remaining sequence was marked cyan. Two conserved transmembrane domains were marked with black lines, and a red box was used to indicate the conserved tripeptide GMN.

Figure 2

The phylogenetic tree of Mg²⁺ transporters of tomato, maize, rice, and Arabidopsis. The neighbor-joining tree was contrasted using Mega X. These Mg²⁺ transporters were divided into 5 clusters (I–V), in different colors. The red branches represent Mg²⁺ transporters in tomato with the prefix Sl.

Figure 3

Gene structure analysis of Mg²⁺ transporters in tomato and Arabidopsis according to their phylogenetic relationship. All sequences were classified into 5 groups with different colors (left). The yellow boxes represent exons, the thin solid black lines represent introns, and the blue boxes indicate upstream or downstream sequences (right).

Figure 4

Conserved motifs in tomato Mg²⁺ transporters. The phylogenetic tree and the motif distribution are shown. A total of 10 motifs were detected. The detailed peptide sequences of each motif are also shown.

Figure 5

Cis-acting element prediction of tomato Mg²⁺ transporter genes. These cis-acting elements were analyzed using PLACE based on the sequence 2000 bp upstream of the initiation codon. A total of 11 cis-acting elements are shown here, and these elements were divided into 4 types. A: light-induced elements; B: plant hormone regulatory elements; C: pollen developmental elements; D: dehydration response elements.

Figure 6

Chromosomal location, gene distribution density, and interchromosomal relationships of SlMRS2 genes. The innermost showed the collinear analysis of the tomato genome. The middle circles showed the gene distribution density in the form of heat map, and the outermost circle showed the locations of Mg²⁺ transporters on different chromosomes of tomato.

Figure 7

Spatial structure prediction of SlMRS2-11. (a) Side view of protein complex; (b) vertical view of protein complex; (c) the structure of the monomer and location of conserved tripeptide GMN; (d) the condensed structural formulas of GMN.

Figure 8

The expression pattern of Mg²⁺ transporters in tomato. (a) Heatmap of Mg²⁺ transporter gene expression in different parts of tomato cultivars LA1589 and LA4345; (b) gel electrophoresis of semi-quantitative RT-PCR analysis of Mg²⁺ transporter genes in leaves, cotyledon, stem, and root of tomato. L1–L6 indicated leaves from full maturity to youngest.

Figure 9

The expression of SlMRS2 genes in the leaves, stems, and roots of tomato after 0, 6, 12, 24, 4, and 96 h of Mg²⁺ treatment. L: leaves; S: stem; R: root. CK: 1 mM Mg²⁺ as a control; MgD: 0 mM Mg²⁺ as Mg²⁺ deficiency; MgT: 5 mM Mg²⁺ as Mg²⁺ toxicity. The actin gene was used as an internal control to normalize the expression data, and the error bars represent ±SD (n = 3). Different letters on bars indicate significant differences using Tukey’s HSD test at p ≤ 0.05.

Figure 10

Effects of Mg²⁺ stress on the relative expression of SlMRS2 genes in leaves, cotyledon, stem, and root of tomato. L1–L6 indicated leaves from fully mature to youngest. CK: 1 mM Mg²⁺ as a control; MgD: 0 mM Mg²⁺ as Mg²⁺ deficiency; MgT: 5 mM Mg²⁺ as Mg²⁺ toxicity. The actin gene was used as an internal control to normalize the expression data, and the error bars represent ±SD (n = 3). Different letters on bars indicate significant differences using Tukey’s HSD test at p ≤ 0.05.

Figure 11

Complementation of Salmonella typhimurium mutant MM281 by SlMRS2-3 and SlMRS2-11. The mutant MM281 transformed with an empty vector was taken as negative control and wild strain MM1927 as a positive control. (a) Complementation assay on solid medium containing 0.01, 0.1, 0.5, 1, 2, 5, 10, and 20 mM MgSO₄. The horizontal lines show 10-fold dilutions of bacterial culture in a series. (b–e) Growth curve of MM281 strain expressing SlMRS2-3 grown on the liquid medium containing 0.1, 0.5, 1, and 10 mM MgSO₄. (f–i) Growth curve of MM281 strain expressing SlMRS2-11 grown on the liquid medium containing 0.1, 0.5, 1, and 10 mM MgSO₄. Growth was monitored every 5 h as OD₆₀₀. The error bars represent ±SD (n = 3).