Genome-wide identification and analysis of ascorbate peroxidase (APX) gene family in hemp (Cannabis sativa L.) under various abiotic stresses

Abstract

Ascorbate peroxidase (APX) plays a critical role in molecular mechanisms such as plant development and defense against abiotic stresses. As an important economic crop, hemp (Cannabis sativa L.) is vulnerable to adverse environmental conditions, such as drought, cold, salt, and oxidative stress, which lead to a decline in yield and quality. Although APX genes have been characterized in a variety of plants, members of the APX gene family in hemp have not been completely identified. In this study, we (1) identified eight members of the CsAPX gene family in hemp and mapped their locations on the chromosomes using bioinformatics analysis; (2) examined the physicochemical characteristics of the proteins encoded by these CsAPX gene family members; (3) investigated their intraspecific collinearity, gene structure, conserved domains, conserved motifs, and cis-acting elements; (4) constructed a phylogenetic tree and analyzed interspecific collinearity; and (5) ascertained expression differences in leaf tissue subjected to cold, drought, salt, and oxidative stresses using quantitative real-time-PCR (qRT-PCR). Under all four stresses, CsAPX6, CsAPX7, and CsAPX8 consistently exhibited significant upregulation, whereas CsAPX2 displayed notably higher expression levels under drought stress than under the other stresses. Taken together, the results of this study provide basic genomic information on the expression of the APX gene family and pave the way for studying the role of APX genes in abiotic stress.

Keywords: Abiotic stresses; Bioinformatics analysis; CsAPX gene family; Expression patterns; Hemp.

Figure 1. Phylogenetic tree analysis of the APX in Arabidopsis thaliana, hemp (Cannabis sativa L.), rice (Oryza sativa L.) and corn (Zea mays L.).

The phylogenetic relationships between APX genes and their homologous counterparts. There are six AtAPXs, seven CsAPXs, eight OsAPXs, and seven ZmAPXs. The colors indicate three clusters that represent Organeller APXs (purple), Peroxisomal APXs (blue), and Cytoplasmatic APXs (green).

Figure 2. Phylogenetic relationships, gene structure, conserved domains, and conserved motifs analysis of CsAPXs.

(A) Phylogenetic tree of all CsAPX proteins constructed using maximum-likelihood method; (B) Motif distribution of CsAPXs; Motifs 1–9 are shown as rectangular boxes of different colors; (C) Conserved domain distribution of CsAPX proteins; the brown box indicates the Plant-peroxidase domain in the corresponding amino acid sequence, the yellow box indicates the Ascorbate-peroxidase domain in the corresponding amino acid sequence, the purple box indicates the PLN02608 domain in the corresponding amino acid sequence; (D) Gene structures of CsAPXs arranged according to phylogenetic relationship; green boxes represent 5′ UTR and 3′ UTR, yellow boxes represent exons, and gray lines represent introns.

Figure 3. Distribution of CsAPXs on the Cannabis sativa chromosomes.

The scale on the left side estimates chromosome length. Members of the CsAPX gene family are sequentially numbered from 1 to 8, indicated in black. The black values on the chromosome’s left side represent the chromosome numbers. A color gradient from blue to red visually depicts gene density on the chromosomes.

Figure 4. Collinearity analysis of CsAPX gene family members.

From the outside in, different colors represent chromosomes 1-10, the red line indicates the genome’s GC ratio, and the innermost circle’s heatmap shows gene density. In the center, gray lines represent synteny blocks in the Cannabis sativa genome, and pink lines between chromosomes mark segmental duplication gene pairs.

Figure 5. Interspecific collinearity relationships of the CsAPX genes among Arabidopsis, hemp (Cannabis sativa L.), and corn (Zea mays L.).

Homologous APX gene pairs exist between Arabidopsis and hemp (AtAPX1-CsAPX7, AtsAPX-CsAPX4, AtAPX3-CsAPX3, and AtAPX5-CsAPX3), and between hemp and corn (CsAPX7-ZmAPX2). Chromosomes from Arabidopsis (blue), hemp (yellow), and corn (green) are color-coded for distinction, with chromosome numbers indicated either above, below, or inside each chromosome. Gray lines represent hemp gene blocks orthologous to other genomes, while blue and green lines delineate syntenic APX gene pairs. The location of CsAPX genes is marked by a yellow triangle.

Figure 6. Analysis of cis-acting elements in the promoter region of CsAPX genes.

(A) The distribution of cis-acting elements in the promoter region of CsAPX genes (−2000 bp) is represented by different colors. The ruler at the bottom indicates the sequence’s direction and length; (B) The classification and statistical analysis of cis-acting elements are presented. Twenty-nine types of cis-acting elements, excluding light-responsive elements, are categorized into three groups: abiotic and biotic stress elements (blue), phytohormone responsive elements (brown), and plant growth and development elements (green). In the left grid, each element’s count is represented numerically, with a color gradient from white to green indicating an increase in element count. The bar chart on the right counts the total number of different categories of cis-acting elements in each CsAPX gene.

Figure 7. The relative expression levels of CsAPX genes were evaluated under various stress conditions: cold stress (A), drought stress (B), salt stress (C), and oxidative stress (D), at time points of 0, 3, 6, 12, 24, and 48 hours.

Values are presented as the mean ± standard deviation (SD) of three independent biological replicates, relative to the 0-hour control. Asterisks indicate statistical significance compared to the control (0 h), as determined by a two-tailed Student’s t-test. Asterisks indication of statistical significance: ** p < 0.01, and * p < 0.05.