Genome-Wide Characterization and Expression Profiling of the AP2/ERF Gene Family in Fragaria vesca L

Abstract

The wild strawberry (Fragaria vesca L.; F. vesca) represents a resilient and extensively studied model organism. While the AP2/ERF gene family plays a pivotal role in plant development, its exploration within F. vesca remains limited. In this study, we characterized the AP2/ERF gene family in wild strawberries using the recently released genomic data (F. vesca V6.0). We conducted an analysis of the gene family expansion pattern, we examined gene expression in stem segments and leaves under cold conditions, and we explored its functional attributes. Our investigation revealed that the FvAP2/ERF family comprises 86 genes distributed among four subfamilies: AP2 (17), RAV (6), ERF (62), and Soloist (1). Tandem and segmental duplications significantly contributed to the growth of this gene family. Furthermore, predictive analysis identified several cis-acting elements in the promoter region associated with meristematic tissue expression, hormone regulation, and resistance modulation. Transcriptomic analysis under cold stress unveiled diverse responses among multiple FvAP2/ERFs in stem segments and leaves. Real-time fluorescence quantitative reverse transcription PCR (RT-qPCR) results confirmed elevated expression levels of select genes following the cold treatment. Additionally, overexpression of FvERF23 in Arabidopsis enhanced cold tolerance, resulting in significantly increased fresh weight and root length compared to the wild-type control. These findings lay the foundation for further exploration into the functional roles of FvAP2/ERF genes.

Keywords: AP2/ERF; Fragaria vesca L.; abiotic stresses; cold; transcription factor.

Figure 1

AP2/ERF superfamily phylogenetic evolutionary tree of Arabidopsis thaliana and Fragaria vesca. The color blocks representing the AP2, ERF (I-XII), RAV, and Soloist subfamilies are depicted uniquely. Names are assigned based on their chromosomal positions. Fv, Fragaria vesca L.; At, Arabidopsis thaliana.

Figure 2

Distribution of AP2/ERF gene locations on chromosomes in Fragaria vesca.

Figure 3

Species collinearity analysis and repeat gene pairs in Fragaria vesca. The red line in the center of the figure represents the repeat gene pair, and the gray part represents the collinearity block.

Figure 4

Arabidopsis thaliana, F. × ananassa, and F. vesca interspecies collinearity analysis. The bright blue lines represent the collinearity gene pair (see Supplementary File S1), and the gray part represents the collinearity region blocks of the genome of different species.

Figure 5

Expression profile of FvERF genes in strawberry stem segments and leaves after room temperature (22 °C), freezing (−4 °C), and chilling (4 °C) treatments. Heat map of (A) ERF subfamily genes, (B) AP2 subfamily genes, and (C) RAV subfamily genes. Row data were clustered. The RNA-seq expression data of the corresponding genes, FPKM + 1, were converted with log2 as the base, and the heat map was plotted. Blue to red indicates low to high expression levels.

Figure 6

Expression levels of 10 FvERF genes (A–J) from strawberry stem and leaf tissues after different treatments were verified by RT-qPCR. CK: untreated plants; cold: 6 h at 4°C. Every data point was computed utilizing the 2^−ΔΔCt technique. The mean of three replicates ± SE is included in the experimental data. A student’s t-test was used. p ** < 0.01 indicates a highly significant difference.

Figure 7

Expression of four genes ((A) FvCBF1, (B) FvCBF3, (C) FvCOR15a, (D) FvRD29A) in F. vesca. was analyzed with the extension of the cold treatment duration. The expression levels of the genes were detected by RT-qPCR and the experiment consisted of three replicates. One-way ANOVA was used. p ** < 0.01 and p *** < 0.001 indicate a highly significant difference, p * < 0.05 indicates a significant difference.

Figure 8

Phenotypes of Arabidopsis after 7 days and 28 days of growth under cold stress. (A) Arabidopsis overexpressing the FvERF23 gene showed phenotypic differences from WT after cold stress. A student’s t-test was used to analyze root length (B) and fresh weight (C), proline content (D), and MDA content (E) after 28 days of growth compared with control WT. A student’s t-test was used. p ** < 0.01 indicates a highly significant difference, p * < 0.05 indicates a significant difference.

Figure 9

Phenotypic and antioxidant enzyme activity analysis of wild-type and transgenic Arabidopsis thaliana under different treatments. (A) −4 °C for 6 h to simulate low-temperature stress during its growth. Plants were irrigated with 300 mM NaCl, 10% PEG-6000, and 250 µM ABA solutions for 5 days to simulate salt stress, drought stress, and ABA stress, respectively. (B) POD activity and (C) and SOD activity were determined for the strains in all treatments. Significance markers were placed by using a one-way ANOVA. p ** < 0.01 indicates a highly significant difference, p * < 0.05 indicates a significant difference.