Genome-Wide Identification, Evolutionary Analysis, and Functional Studies of APX Genes in Melon (Cucuis melo L.)

Abstract

The antioxidative enzyme ascorbate peroxidase (APX) exerts a critically important function through scavenging reactive oxygen species (ROS), alleviating oxidative damage in plants, and enhancing their tolerance to salinity. Here, we identified 28 CmAPX genes that display an uneven distribution pattern throughout the 12 chromosomes of the melon genome by carrying out a bioinformatics analysis. Phylogenetic analyses revealed that the CmAPX gene family comprised seven different clades, with each clade of genes exhibiting comparable motifs and structures. We cloned 28 CmAPX genes to infer their encoded protein sequences; we then compared these sequences with proteins encoded by rice APX proteins (OsAPX2), Puccinellia tenuiflora APX proteins (PutAPX) and with pea APX proteins. We found that the CmAPX17, CmAPX24, and CmAPX27 genes in Clade I were closely related, and their structures were highly conserved. CmAPX27 (MELO3C020719.2.1) was found to promote resistance to 150 mM NaCl salt stress, according to quantitative real-time fluorescence PCR. Transcriptome data revealed that CmAPX27 was differentially expressed among tissues, and the observed differences in expression were significant. Virus-induced gene silencing of CmAPX27 significantly decreased salinity tolerance, and CmAPX27 exhibited differential expression in the leaf, stem, and root tissues of melon plants. This finding demonstrates that CmAPX27 exerts a key function in melon's tolerance to salt stress. Generally, CmAPX27 could be a target in molecular breeding efforts aimed at improving the salt tolerance of melon; further studies of CmAPX27 could unveil novel physiological mechanisms through which antioxidant enzymes mitigate the deleterious effects of ROS stress.

Keywords: APX gene family; functional validation; melon; salt stress.

Figure 1

Conserved motif and structure analyses for the CmAPX genes. (A) Schematic diagram of the conserved motifs predicted in CmAPX proteins. Each conserved motif is shown in a specific color. The length of the amino acid sequence can be inferred using a ruler at the bottom. (B) Structure of CmAPX genes. (C) Length and amino acids of the 10 conserved motifs identified among the CmAPX proteins. Letter size corresponds to the frequency of the amino acid.

Figure 1

Conserved motif and structure analyses for the CmAPX genes. (A) Schematic diagram of the conserved motifs predicted in CmAPX proteins. Each conserved motif is shown in a specific color. The length of the amino acid sequence can be inferred using a ruler at the bottom. (B) Structure of CmAPX genes. (C) Length and amino acids of the 10 conserved motifs identified among the CmAPX proteins. Letter size corresponds to the frequency of the amino acid.

Figure 2

The distribution pattern of CmAPX genes on melon chromosomes.

Figure 3

Collinearity analysis of CmAPX genes. Orange, red, and yellow lines denote sequence homology between the melon and rice, melon and Arabidopsis, and Arabidopsis and rice genomes, respectively; blue lines indicate sequence homology among melon chromosomes. The segmental duplication pairs among chromosomes are linked by lines.

Figure 4

Tissue expression profiles of CmAPX genes.

Figure 5

Schematic diagram of CmAPX genes in the phylogenetic tree.

Figure 6

Amino acid sequence alignment diagram.

Figure 7

RT-qPCR analyses of the relative mRNA abundances of CmAPX17, CmAPX24, and CmAPX27 genes in melon leaves after salt stress treatment for different periods (as indicated by the x-axis). Error bars indicate the standard error derived from three independent experiments. The lowercase letters denote that the differences among groups reach a statistically significant level of p < 0.05 in Duncan’s multiple range test.

Figure 8

Phenotypes of melon plants after gene silencing and expression analysis. (A) The phenotypes of CmAPX27-silenced and non-silenced melon plants were determined following exposure to 150 mM NaCl stress in plants. NS, non-silenced control; pV190-EV, plants infected with the pV190 empty vector; pV190-PDS, plants infected with pV190-PDS; pV190-CmAPX27, plants infected with pV190-CmAPX27. (B) Analysis of pV190-PDS and pV190-CmAPX27 expression patterns after gene silencing. The lowercase letters denote that the differences among groups reach a statistically significant level of p < 0.05 in Duncan’s multiple range test. (C) Gene expression patterns of NS, pV190-EV, and pV190-CmAPX27 plants in rhizomes and leaves under 150 mM NaCl stress. The lowercase letters denote that the differences among groups reach a statistically significant level of p < 0.05 in Duncan’s multiple range test.

Figure 9

Variation in APX activity in melon plants. The lowercase letters denote significant differences in APX activity among groups (p < 0.05) according to Duncan’s multiple range test. (A) Determination of APX activity in melon tissues in the absence of salt stress. (B) APX activity of melon leaves at different times under 150 mM NaCl. (C) APX activity in leaves of NS plants and pV190-CmAPX27 plants in the absence of salt stress. (D) APX activity in various tissues of NS plants and pV190-CmAPX27 plants under 150 mM NaCl.

Figure 10

Schematic diagram of the ascorbate–glutathione pathway.