Bioinformatics Analysis Reveals the Evolutionary Characteristics of the Phoebe bournei ARF Gene Family and Its Expression Patterns in Stress Adaptation

Abstract

Auxin response factors (ARFs) are pivotal transcription factors that regulate plant growth, development, and stress responses. Yet, the genomic characteristics and functions of ARFs in Phoebe bournei remain undefined. In this study, 25 PbARF genes were identified for the first time across the entire genome of P. bournei. Phylogenetic analysis categorized these genes into five subfamilies, with members of each subfamily displaying similar conserved motifs and gene structures. Notably, Classes III and V contained the largest number of members. Collinearity analysis suggested that segmental duplication events were the primary drivers of PbARF gene family expansion. Structural analysis revealed that all PbARF genes possess a conserved B3 binding domain and an auxin response element, while additional motifs varied among different classes. Promoter cis-acting element analysis revealed that PbARF genes are extensively involved in hormonal responses-particularly to abscisic acid and jasmonic acid and abiotic stresses-as well as abiotic stresses, including heat, drought, light, and dark. Tissue-specific expression analysis showed that PbARF25, PbARF23, PbARF19, PbARF22, and PbARF20 genes (class III), and PbARF18 and PbARF11 genes (class V) consistently exhibited high expression levels in the five tissues. In addition, five representative PbARF genes were analyzed using qRT-PCR. The results demonstrated significant differences in the expression of PbARF genes under various abiotic stress conditions (drought, salt stress, light, and dark), indicating their important roles in stress response. This study laid a foundation for elucidating the molecular evolution mechanism of ARF genes in P. bournei and for determining the candidate genes for stress-resistance breeding.

Keywords: ARF; Phoebe bournei; expansion; gene family; stress response.

Figure 1

Chromosomal distribution map of ARF genes in Phoebe bournei. The left side of each bar shows the chromosome number, and the left scale of the bars indicates the relative length of a chromosome. The left scale unit is Mb, and the short line shows the approximate location of the PbARF gene on the corresponding chromosome.

Figure 2

Phylogenetic tree of 86 ARF genes from Arabidopsis thaliana (At) and Oryza sativa (Os) and Phoebe bournei (Pb). The phylogenetic tree was constructed using the neighbor-joining method with MEGA11.0. All ARFs were categorized into five classes, each class represented by a different color: CLASS I is shown in blue, CLASS II in yellow, CLASS III in red, CLASS IV in dark green, and CLASS V in light green.

Figure 3

Schematic diagram of PbARF conserved motifs and gene structure. (A) Motifs in PbARF proteins, from motif 1 to motif 10, are marked by different colors. Black lines indicate non-conserved protein sequences. (B) Conserved domains of PbARF genes are depicted in different colors. (C) Gene structure of PbARFs. The yellow boxes represent coding sequences (CDS), and the green boxes indicate untranslated regions (UTRs) of PbARFs. The relative positions are consistently represented according to the kilobase scale at the bottom of the figures.

Figure 4

Schematic representation of important cis-acting elements in the promoter regions of PbARF genes, correlated with the phylogenetic classification of their encoded PbARF proteins. Different types of promoters are shown in different colors.

Figure 5

Intraspecific synteny of the ARF gene family in Phoebe bournei. The two outer rings represent the gene density per chromosome, and the grey line represents the synteny block in the genome. Lines of the same color represent duplicated PbARF gene pairs, and the chromosome number is shown in a rectangular box for each chromosome.

Figure 6

Interspecific synteny analysis of ARF genes in Arabidopsis, rice, and Phoebe bournei. Gray lines indicate covariance between the genomes of Phoebe bournei and other plants, and red lines indicate covariance of the PbARF genes with ARF genes in other plants.

Figure 7

Tissue-specific gene expression patterns of 25 PbARF genes were analyzed across root bark, root xylem, stem bark, stem xylem, and leaf tissues. Red indicates high abundance (higher FPKM value), blue indicates low abundance (lower FPKM value), and the size of each point increases with the value of high abundance.

Figure 8

The expression profiles of the five selected PbARF genes in Phoebe bournei were detected by qRT-PCR under heat, PEG, light, and darkness conditions. (A) Relative gene expression levels under heat treatment over periods of 4, 8, 12, and 24 h. (B) Relative gene expression levels under drought stress over the same periods (4, 8, 12, and 24 h). (C) Relative gene expression levels under treatment of light conditions over the same periods (4, 8, 12, and 24 h). (D) Relative gene expression levels under conditions without light (* p < 0.05, ** p < 0.01, and **** p < 0.0001).