Genome-Wide Identification of APX Gene Family in Citrus maxima and Expression Analysis at Different Postharvest Preservation Times

Abstract

Ascorbate peroxidase (APX) is a crucial enzyme involved in cellular antioxidant defense and plays a pivotal role in modulating reactive oxygen species (ROS) levels under various environmental stresses in plants. This study utilized bioinformatics methods to identify and analyze the APX gene family of pomelo, while quantitative real-time PCR (qRT-PCR) was employed to validate and analyze the expression of CmAPXs at different stages of fruit postharvest. This study identified 96 members of the CmAPX family in the entire pomelo genome, with uneven distribution across nine chromosomes and occurrences of gene fragment replication. The subcellular localization includes peroxisome, cytoplasm, chloroplasts, and mitochondria. The CmAPX family exhibits a similar gene structure, predominantly consisting of two exons. An analysis of the upstream promoter regions revealed a significant presence of cis-acting elements associated with light (Box 4, G-Box), hormones (ABRE, TCA-element), and stress-related (MBS, LTR, ARE) responses. Phylogenetic and collinearity analyses revealed that the CmAPX gene family can be classified into three subclasses, with seven collinear gene pairs. Furthermore, CmAPXs are closely related to citrus, pomelo, and lemon, followed by Arabidopsis, and exhibit low homology with rice. Additionally, the transcriptomic heat map and qPCR results revealed that the expression levels of CmAPX57, CmAPX34, CmAPX50, CmAPX4, CmAPX5, and CmAPX81 were positively correlated with granulation degree, indicating the activation of the endogenous stress resistance system in pomelo cells by these genes, thereby conferring resistance to ROS. This finding is consistent with the results of GO enrichment analysis. Furthermore, 38 miRNAs were identified as potential regulators targeting the CmAPX family for post-transcriptional regulation. Thus, this study has preliminarily characterized members of the APX gene family in pomelo and provided valuable insights for further research on their antioxidant function and molecular mechanism.

Keywords: APX gene family; Citrus maxima; ascorbic acid; postharvest preservation; reactive oxygen species.

Figure 1

Chromosome location of the APX gene in pomelo; the bar scale on the left indicates chromosome length.

Figure 2

Phylogenetic analysis of the amino acid sequence of APX from C. maxima (five-pointed star), C. sinensis (triangle), A. thaliana (circle), and O. sativa (square).

Figure 3

Gene structures and conservative motif analysis of the APX gene family in pomelo. (A) phylogenetic tree of CmAPX proteins; (B) conserved motifs of CmAPX proteins; (C) gene structure of CmAPX genes.

Figure 4

Analysis of the cis-acting elements of the APX gene promoter in pomelo. The depth of color in the figure represents the amount, with light green colors indicating a smaller or zero quantity, and dark green colors indicating a larger quantity.

Figure 5

Collinearity analysis of APX genes in C. maxima (A) and between C. maxima and A. thaliana (B), O. sativa (C), C. sinensis (D), C. maxima cv Cuipi Majiayou (E), and C. limon (F). Genes of APX subfamilies with gene duplication relationships are connected by lines in red.

Figure 6

CmAPX GO function classification diagram.

Figure 7

Network of miRNA–gene interaction of APX genes. Blue color represents the APX gene and yellow color represents the csi-miRNA associated with APX.

Figure 8

Expression profiling of CmAPX genes at different stages of development of pomelo fruit sacs (A) and under different storage times after harvest (B); one-way ANOVA was used to compare the expression level. Data represent mean values ± SD, n = 3. Different letters mean p < 0.05.