In silico identification and expression analysis of superoxide dismutase (SOD) gene family in Medicago truncatula

Abstract

Superoxide dismutase (SOD) proteins are crucial antioxidant enzymes that play critical roles in plant growth, development, and response to various abiotic stresses. The SOD gene family has been characterized in various plant species, but not in Medicago truncatula yet. Here, a total of 7 MtSOD genes were first identified from the whole genome of M. truncatula, including 1 MnSOD, 2 FeSODs, and 4 Cu/ZnSODs, which are unevenly distributed in five out of the eight chromosomes. Phylogenetic analysis showed that SOD proteins from M. truncatula and other plant species could be classified into two main categories (Cu/ZnSODs and Fe-MnSODs), which could be further divided into eight subgroups, and members within the same subgroup tended to share the same subcellular localization. In addition, MtSOD genes together with AtSODs and OsSODs within the same subgroup also displayed similar motif compositions and exon-intron structures. Most MtSOD genes were ubiquitously expressed in various tissues, particularly in leaves, seeds and root nodules at different developmental stages. Moreover, microarray analysis and high-throughput sequencing showed that most MtSOD genes were differentially expressed under salt, drought, and cold treatments, indicating their pivotal roles in stress response of M. truncatula. These findings provide useful information for the functional characterization of SOD family genes for growth, development, and stress response of M. truncatula.

Keywords: Abiotic stress; Evolution; Expression patterns; Medicago truncatula; SOD gene family.

Fig. 1

Phylogenetic analysis of SOD proteins from M. truncatula and other plant species. Multiple sequence alignments of the full-length SOD protein sequences from M. truncatula, C. sativus, S. lycopersicum, O. sativa, S. bicolor, A. thaliana, B. distachyon, G. raimondii, and S. italica were performed with Clustal Omega, and the phylogenetic tree was constructed with MEGA 5.0 software by the neighbor-joining method using 1000 bootstrap replicates. The numbers indicated for each clade represent the bootstrap support values given as percentages

Fig. 2

Comparison of the amino acid sequences of MtSOD proteins. a Multiple sequence alignment of the deduced amino acid sequences of Cu/ZnSOD proteins. Two conserved Cu/ZnSOD signatures (GFH[VLI]H[EA][YL]GDTT and GNAG[GA]R[VL]ACG) are underlined. The metal-binding sites for Cu²⁺ and Zn²⁺ are marked with regular and inverted triangles, respectively. b Multiple sequence alignment of the deduced amino acid sequences of CCS proteins. The conserved metal-binding motifs (MXCXXC and CXC) are underlined. c Multiple sequence alignment of the deduced amino acid sequences of FeSOD proteins. The FeSOD signature ([AE][QEL][VA]WNH[DEH]FFWES) and conserved metal-binding domain (D[VL]WEHAYY) are underlined. Two conserved metal-binding His residues are marked with regular triangles. d Multiple sequence alignment of the deduced amino acid sequences of MnSOD proteins. The conserved metal-binding domain (DVWEHAYY) is underlined. Six conserved residues (His, Gln, and Asp) are marked with regular triangles

Fig. 3

Phylogenetic relationship and conserved motif analysis of SOD proteins from M. truncatula, Arabidopsis, and rice. Left: phylogenetic analysis for the SOD proteins from Arabidopsis, rice, and M. truncatula through neighbor-joining method with 1000 bootstrap replicates. The proteins can be further divided into eight subgroups as marked by different colors. Right: conserved motif analysis of the SOD proteins from Arabidopsis, rice, and M. truncatula using the online tool MEME. The ten motifs are assigned to differently colored boxes

Fig. 4

Gene structure analysis of SOD genes from M. truncatula, Arabidopsis, and rice based on phylogenetic relationship. Left: phylogenetic analysis for the SOD proteins from Arabidopsis, rice, and M. truncatula. Right: structural features of the SOD genes from Arabidopsis, rice, and M. truncatula were examined using the online tool GSDS. Lengths of introns, exons, and upstream/downstream of each SOD gene are proportionally presented

Fig. 5

Chromosomal distribution of MtSOD genes from M. truncatula. The chromosome number is indicated at the top of each chromosome

Fig. 6

Expression profiles of MtSOD genes in different developmental stages including panicle development (a) and root development (b). The average log signal values of MtSOD genes were downloaded from the Medicago truncatula gene expression Atlas (MtGEA) Project database (https://mtgea.noble.org/v3/). The color scale (representing log signal values) is shown at the bottom. dap, days after pollination

Fig. 7

Expression patterns of MtSOD genes in response to various abiotic stresses based on data of microarray (a) and transcriptome (b–d). a Two-week-old M. truncatula seedlings were grown in hydroponics media with 180 mM NaCl for 0, 6, 24, and 48 h (Li et al. 2009). The microarray data were retrieved from the Medicago truncatula gene expression Atlas (MtGEA) Project database (https://mtgea.noble.org/v3/), and represented as the relative signal intensity values. Transcriptome sequencing (RNA-seq) was performed to investigate expression profiles of MtSOD genes in response to salt (b), drought (c), and cold (d) as described in our previous study (Song et al. 2017). The FPKM-normalized values were represented by a color gradient from low (blue) to high expression (red)