Genome-Wide Identification and Expression Analysis of Auxin-Responsive GH3 Gene Family in Pepper (Capsicum annuum L.)

Abstract

As an auxin-responsive gene, Gretchen Hagen 3 (GH3) maintains hormonal homeostasis by conjugating excess auxin with amino acids in plant stress-related signaling pathways. GH3 genes have been characterized in many plant species, but the characteristics of pepper (Capsicum annuum L.) GH3 (CaGH3) gene family members in response to multiple stimulants are largely unknown. In this study, we systematically identified the CaGH3 gene family at the genome level and identified eight members on four chromosomes in pepper. CaGH3s were divided into two groups (I and III) and shared conserved motifs, domains, and gene structures. Moreover, CaGH3s had close evolutionary relationships with tomato (Solanum lycopersicum L.), and the promoters of most CaGH3 genes contained hormone and abiotic stress response elements. A protein interaction prediction analysis demonstrated that the CaGH3-3/3-6/3-7/3-8 proteins were possibly core members of the CaGH3 family interaction. In addition, qRT-PCR results showed that CaGH3 genes were differentially expressed in pepper tissues and could be induced by phytohormones (IAA, ABA, and MeJA) and abiotic stresses (salt, low temperature, and drought) with different patterns. In addition, CaGH3-5 and CaGH3-7 were cloned, and the sequences showed a high degree of conservation. Moreover, the results of subcellular localization indicated that they were located in the membrane and chloroplast. Notably, after overexpressing CaGH3-7 in tomato, RNA-seq was performed on wild-type and transgenic lines, and the differentially expressed genes were mainly enriched in response to external stimuli. This study not only lays the foundation for a comprehensive understanding of the function of the CaGH3 gene family during plant growth and stress responses but also provides potential genetic resources for pepper resistance breeding.

Keywords: Gretchen Hagen 3 gene family; auxin; expression pattern; pepper.

Figure 1

Structural analysis of GH3 family proteins of Capsicum annuum. (A) Secondary structure analysis of CaGH3 proteins. Blue line, α-helix; green line, β-turn; purple line, extended strand; orange line, random coil. (B) Tertiary structure analysis of CaGH3 proteins.

Figure 2

Collinear analysis of Capsicum annuum and five other plants (Arabidopsis thaliana, Oryza sativa, Solanum tuberosum, Solanum lycopersicum, and Brassica rapa). The gray line represents the collinear blocks of the pepper genome and other plant genomes, and the red curve represents the CaGH3 gene collinearity.

Figure 3

Phylogenetic analysis of GH3 homolog proteins from Capsicum annuum (Ca), Arabidopsis thaliana (At), and Solanum lycopersicum (Sl).

Figure 4

Phylogenetic relationships (A), conserved motifs (B), conserved domains (C), and gene structures (D) of CaGH3s.

Figure 5

Cis-regulatory element analysis of CaGH3 family genes. The number of each cis-acting element is shown in the heatmap box, ranging from blue to red, with white boxes indicating that there are no corresponding cis-acting elements.

Figure 6

Expression patterns of CaGH3 gene family in different tissues. All data points are means ± standard errors. Different lowercase superscripts indicate significant differences, as determined using Duncan’s new multiple range test (p-value < 0.05).

Figure 7

Expression patterns of CaGH3 gene family under different hormone treatments. All data points are means ± standard errors. Different lowercase superscripts indicate significant differences, as determined using Duncan’s new multiple range test (p-value < 0.05).

Figure 8

Expression patterns of CaGH3 gene family under different abiotic stresses. All data points are means ± standard errors. Different lowercase superscripts indicate significant differences, as determined using Duncan’s new multiple range test (p-value < 0.05).

Figure 9

The protein interaction network of pepper CaGH3 family members. The nodes in the protein interaction network indicate all proteins produced by a protein-coding locus, and different colors indicate different degrees of interaction. The deeper the color of the lines between the nodes, the higher the intensity of the interaction. The thinner lines indicate weaker interactions between the proteins.

Figure 10

Multiple alignments of GH3 proteins for C. annuum (Ca), S. lycopersicum (Sl), and A. thaliana (At). Conserved residues are highlighted with red boxes, with similar residues shown in a lighter color. The underlying amino acid sequence indicates the GH3 domain.

Figure 11

Subcellular localization of CaGH3-5 and CaGH3-7 in Nicotiana benthamiana. GFP fluorescence was observed with a fluorescence microscope. PIP2-mCherry was used for membrane localization. Images were taken in a dark field for green fluorescence, while the cell outlines were photographed in a bright field. Bars = 25 µm.

Figure 12

Transcriptome-based enrichment analysis of differentially expressed genes. (A) Gene Ontology (GO) classification. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) classification. The numbers after the bars indicate the number and percentage of genes annotated.