Comparative analysis of POD gene family and expression differentiation under NaCl, H2O2 and PEG stresses in sorghum

Abstract

Background: Sorghum has a strong tolerance to multi-abiotic stresses such as drought, heat, poor soil fertility, and salinity, yet its growth and development are significantly affected by extreme abiotic stresses, ultimately reducing grain yield. Peroxidase (POD) proteins, which are members of the oxidoreductase enzyme family, play an important role in protecting plants against multi-abiotic stress. However, this gene family has rarely been studied in sorghum.

Results: In this study, 114 POD gene members (SbPODs) were identified in sorghum and distributed unevenly throughout 10 chromosomes. Phylogenetic analysis clustered these genes into 12 subgroups. Gene structure and motif analyses showed the structural and functional diversity among the subgroups. These genes have undergone 16 segmental duplication and 32 tandem duplication events, indicating that both segmental and tandem duplications are major contributors to the expansion of the SbPOD gene family. Most duplicated SbPODs have undergone purifying selection with limited functional divergence during duplication events. Compared with maize, rice, and Arabidopsis, SbPOD gene family not only occurred the duplication on the whole-genome level, but also existed independent duplication events. The qRT-PCR results showed twenty SbPOD genes exhibited differential expression patterns in leaves under NaCl, H₂O₂ and PEG stress treatments. Of twenty SbPOD genes, most SbPOD genes showed consistently significant downregulation under NaCl, H₂O₂, and 30% PEG6000 stress treatments, except for SbPOD81 and SbPOD26. Under NaCl stress, the expression level of SbPOD81 showed a down-up regulation trend. Under H₂O₂ stress, the expression level of SbPOD81 showed a down regulation trend. Under 30% PEG6000 stress, SbPOD81 maintained down-up regulation trend but quite different under NaCl stress. In contrast, the expression level of SbPOD26 showed consistent down-up regulation trend under both NaCl and H₂O₂ stress, while after 3 h of adaptation under 30% PEG6000 stress, the expression level of SbPOD26 continued to increase. The analysis of cis-elements and conserved domain showed that SbPOD81 contains one W-box, four MYB, and three MYC cis-elements, while SbPOD26 has two W-boxes, four MBS, four MYB, and eight MYC cis-elements. Additionally, SbPOD26 possesses one PAT1 domain, which is absent in SbPOD81 and other SbPOD genes. This structural distinction not only would explain the observed expression divergence but suggests SbPOD26 may perform specialized biological functions distinct from other SbPOD genes.

Conclusions: Totally, 114 POD gene members were identified in sorghum by a series of bioinformatics analysis. The expression patterns of some members of SbPOD gene family exhibited significantly difference under various stress conditions. These findings not only provide valuable insights into the evolutionary dynamics of the SbPOD gene family, but also lay a foundation for further studies on the functions of POD genes.

Keywords: SbPOD candidate genes; Abiotic stress; Expression pattern; Peroxidases; Sorghum.

Fig. 1

Distribution and phylogenetic analyses of SbPOD genes. A Distribution of the 114 SbPOD genes on 10 chromosomes. B Phylogenetic analyses of 114 SbPOD genes using the neighbor-joining (NJ) method with 1000 bootstraps. The color indicates the different group

Fig. 2

The prediction of subcellular localization for SbPOD proteins in sorghum. The color indicate the values of reliable index of the prediction results

Fig. 3

Conserved motif and gene structure of SbPOD genes. A Conserved motifs in amino acid sequence of 12 sub-groups. The colored boxes on the right represent diverse conserved motifs. B Exon-intron analysis of 12 subgroups of SbPOD genes. Yellow boxes represent exons and green boxes represent 5’-UTR and 3’-UTR

Fig. 4

Analysis of cis-acting elements in 2.0 kb promoter regions of SbPOD genes. Different colored short lines represent various cis-acting elements. MBS, DRE, and DRE core are associated with drought stress. MYB and MYC are related to responses to abiotic stresses. ABRE is involved in abscisic acid responses. CGTCA-motif and TGACG-motif are linked to MeJA responsiveness. STRE is associated with heat shock activation. as-1 and TCA-element are related to salicylic acid responses. WUN-motif and WRE3 are involved in wound responses. TC-rich repeats are associated with defense and stress responses. P-box is related to gibberellin signaling. TGA-box and TGA-element are auxin-responsive elements. W-box is associated with salt stress, and LTR is involved in low-temperature stress responsiveness

Fig. 5

Distribution of segmental duplicated POD genes in the sorghum genome generated by Circos

Fig. 6

Syntenic analysis of POD genes between sorghum and three representative plant species. A Syntenic analysis of POD genes between sorghum and Arabidopsis. B Syntenic analysis of POD genes between sorghum and rice. C Syntenic analysis of POD genes between sorghum and maize. Gray lines in the background indicate the collinear blocks within sorghum and other plant genomes, whereas red lines highlight syntenic POD gene pairs

Fig. 7

The alignment of 12 SbPOD protein sequences shows conserved secondary structure domains indicated by red lines

Fig. 8

Prediction of three-dimensional structure of POD proteins in sorghum

Fig. 9

The expression profiles of SbPOD genes in different tissues and under various stress treatments

Fig. 10

qRT-PCR analysis of 8 SbPODs under NaCl, H₂O₂, and 30% PEG6000 stresses. *, **, and ***, represent significant differences at P < 0.05, P < 0.01, and P < 0.001, respectively. One-way ANOVA was used to analyze the significance of relative expression difference of 8 SbPODs at 0, 1, 3, 6, 9, and 12 h under NaCl, H₂O₂, and PEG stresses