Genome-wide identification and characterization of the soybean SOD family during alkaline stress

Abstract

Background: Superoxide dismutase (SOD) proteins, as one kind of the antioxidant enzymes, play critical roles in plant response to various environment stresses. Even though its functions in the oxidative stress were very well characterized, the roles of SOD family genes in regulating alkaline stress response are not fully reported.

Methods: We identified the potential family members by using Hidden Markov model and soybean genome database. The neighbor-joining phylogenetic tree and exon-intron structures were generated by using software MEGA 5.0 and GSDS online server, respectively. Furthermore, the conserved motifs were analyzed by MEME online server. The syntenic analysis was conducted using Circos-0.69. Additionally, the expression levels of soybean SOD genes under alkaline stress were identified by qRT-PCR.

Results: In this study, we identified 13 potential SOD genes in soybean genome. Phylogenetic analysis suggested that SOD genes could be classified into three subfamilies, including MnSODs (GmMSD1-2), FeSODs (GmFSD1-5) and Cu/ZnSODs (GmCSD1-6). We further investigated the gene structure, chromosomal locations and gene-duplication, conserved domains and promoter cis-elements of the soybean SOD genes. We also explored the expression profiles of soybean SOD genes in different tissues and alkaline, salt and cold stresses, based on the transcriptome data. In addition, we detected their expression patterns in roots and leaves by qRT-PCR under alkaline stress, and found that different SOD subfamily genes may play different roles in response to alkaline stress. These results also confirmed the hypothesis that the great evolutionary divergence may contribute to the potential functional diversity in soybean SOD genes. Taken together, we established a foundation for further functional characterization of soybean SOD genes in response to alkaline stress in the future.

Keywords: Alkaline; Evolution; Expression patterns; Phylogenetic analysis; Soybean.

Figure 1. Phylogenetic analysis of SOD genes in soybean, Arabidopsis, Medicago truncatula and Solanum lycopersicum.

The phylogenetic tree was constructed by the neighbor-joining method using MEGA 5.0. The bootstrap values were 1,000 replications for major branches. SOD family genes have been divided into three subfamilies.

Figure 2. Phylogenetic and exon-intron structure analyses of SOD genes.

(A) The phylogenetic tree was produced by the neighbor-joining method using MEGA 5.0. The bootstrap values were 1,000 replications for major branches. (B) Exon-intron structure analysis of soybean SOD genes by using GSDS online tools. The CDSs, untranslated regions and introns are described by yellow boxes, light blue boxes and black lines, respectively.

Figure 3. Conserved domain analysis of SOD family proteins.

The conserved motifs were predicted using the MEME online server. The different conserved motifs were marked by different colors. The protein sequences of eight different motifs commonly observed by MEME online server.

Figure 4. Syntenic analysis of SOD family genes in soybean.

The chromosomes are indicated as a circle. The duplication pairs are connected by lines.

Figure 5. Analysis of cis-acting elements of putative SOD promoters related to stress responses.

The different cis-elements are present with different colors.

Figure 6. Expression patterns of SOD family genes in different soybean tissues.

The RNA-seq data (GSE29163) were downloaded from the NCBI. All expression data were analyzed by Log₂ scaled. The heat map was generated using TM4: MeV4.9 software. The color scale represents the expression values: blue indicates low levels and yellow represents high levels.

Figure 7. Expression analysis of soybean SOD genes in response to various abiotic stresses.

The wild soybean transcriptome sequencing data were used to investigate the expression pattern of soybean SOD genes under alkaline stress. Expression of soybean SOD genes were downloaded from the soybean transcriptome sequencing data under salt (GSE57252) and cold (GSE57252) stresses. The heat map was generated using TM4: MeV4.9 software. The color scale represents the expression values: blue indicates low levels and yellow represents high levels (|Log₂ fold change| > 1, P < 0.05).

Figure 8. Expression analysis of soybean SOD genes in response to alkaline stress in leaves (A) and roots (B) using qRT-PCR assays.

Twelve days soybean seedlings were treated with 50 mm NaHCO₃ for 0, 6 and 12 h. The mean values were from three independent biological replicates. Statistical analyses were performed by Student’s t-test (*P < 0.05 and **P < 0.01).