Evolution analysis of GH3 gene family in five Rosaceae species and FaGH3.17, FaGH3.18 improve drought tolerance in transgenic Arabidopsis

Abstract

Background: Gretchen Hagen 3 (GH3), one of the important auxin-responsive gene families, plays essential roles in plant growth, development process, and stress response by regulating hormone homeostasis. However, the evolutionary analysis of the GH3 gene family in Rosaceae species has not been well-studied and the specifc functions of Fragaria ananassa are not well-documented.

Results: In the current study, 64 members of the GH3 family genes were identifed from five Rosaceae species and divided into 8 groups. According to the comprehensive analysis of evolutionary relationship, collinearity, selection pressure and codon bias, the GH3 gene family was found to be highly conserved across these Rosaceae species, suggesting that purifying selection was a significant force in the evolution of GH3 genes, and the expansion of the GH3 gene family in Rosaceae species might be attributed to fragment duplication. Meanwhile, the codon bias of GH3s in subfamily G and K showed a relatively strong codon bias. Significantly, both FaGH3.17 and FaGH3.18 were localized in the cytoplasm and nucleus. Additionally, compared with the wild type (WT), the malondialdehyde (MDA) content and relative conductivity of FaGH3.17 and FaGH3.18 transgenic Arabidopsis were decreased, while the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities were increased under drought stress. The above results indicate that overexpression of FaGH3.17 and FaGH3.18 signifcantly enhanced the tolerance to drought in transgenic Arabidopsis.

Conclusions: The study provides crucial insights into the evolution of the GH3 gene family in Rosaceae species and provides a theoretical basis for further investigation on the function of FaGH3s.

Keywords: FaGH3.17; FaGH3.18; Drought stress; Evolution; Rosaceae species.

Fig. 1

Phylogenetic tree of the GH3 gene family of 5 Rosaceae species. Phylogenetic trees were constructed using GH3 protein sequences. Neighbor-joining (NJ) method was adopted, and the bootstrap value was set to be equal to 1 000

Fig. 2

Gene structure analysis and conserved motif prediction of FaGH3s, MdGH3s, PavGH3s, PpGH3s and PbrGH3s. A The results of gene structure analysis of polypeptide sequences cluster analysis tree using NJ method. CDS (coding sequence) regions were marked by red boxes, upstream and downstream regions with blue boxes, and introns with black lines. B The motifs represented by the boxes with different colors refer to the legend on the right. The scales at the bottom are used to measure the length of the sequence. C The conserved motif logos of GH3s

Fig. 3

Synteny analysis of the GH3 gene family of 5 species. A-E Synteny analysis of GH3 gene family in Fragaria ananassa, Malus domestica, Prunus avium, Prunus persica, and Pyrus x bretschneideri, respectively. F Collinearity analysis of GH3 gene family among 5 species

Fig. 4

Evolutionary selection pressure analysis of GH3s. Numbers (A-E) stand for species Fragaria ananassa, Malus domestica, Prunus avium, Prunus persica, and Pyrus x bretschneideri, respectively. The median line of the box represents the average level of the selection pressure, and the width indicates the degree of fluctuation

Fig. 5

Codon usage bias analysis of GH3 genes in 5 Rosaceae species. A Codon parameter analysis of GH3s.“A3s, G3s, C3s, and T3s” refer to synonymous codon corresponding base frequency on the third; “CAI” refers to codon adaptation index; “CBI” refers to codon bias index; “FOP” refers to frequency of optimal codons; “ENc” refers to the effective number of codon; “GC3s” refers to the amount of the third codon (G + C); “GC” refers to the count of genes (G + C). B The relative synonymous codon usage (RSCU) analysis of GH3 gene family in 5 Rosaceae species. A color gradient mapped from low (blue) to high (red) indicates an increase in RSCU. C Codon usage parameters correlation analysis

Fig. 6

Subcellular localization of FaGH3.17 and FaGH3.18. A Diagram of the FaGH3.17-pCAMBIA1300-GFP and FaGH3.18-pCAMBIA1300-GFP fusion construct. B Subcellular localization of FaGH3.17 and FaGH3.18 in tobacco

Fig. 7

Phenotypic investigation and expression analysis of corresponding genes in WT, FaGH3.17 and FaGH3.18 transgenic Arabidopsis. A-B Phenotype investigation of FaGH3.17 and FaGH3.18 transgenic Arabidopsis treated with natural drought for 10 days, respectively. Control represents seedlings before stress treatment. C Expression of the FaGH3.17 in FaGH3.17 transgenic Arabidopsis under drought stress. D Expression of the FaGH3.18 in FaGH3.18 transgenic Arabidopsis under drought stress

Fig. 8

Analysis of relative conductivity, MDA content and proline content after drought stress. Fig. A, C and E show the relative conductivity, MDA content and proline content of FaGH3.17 transgenic Arabidopsis, respectively. Fig. B, D and F show the relative conductivity, MDA content and proline content of FaGH3.18 transgenic Arabidopsis, respectively. Diferent small letters mean signifcant diference at p < 0.05

Fig. 9

Analysis of H₂O₂ content and POD, SOD and CAT activity after drought stress. A-B The H₂O₂ content of FaGH3.17 and FaGH3.18 transgenic Arabidopsis, respectively. C-D The POD activity of FaGH3.17 and FaGH3.18 transgenic Arabidopsis, respectively. E-F The SOD activity of FaGH3.17 and FaGH3.18 transgenic Arabidopsis, respectively. G-H The CAT activity of FaGH3.17 and FaGH3.18 transgenic Arabidopsis, respectively. Diferent small letters mean signifcant diference at p < 0.05

Fig. 10

A systematic analysis of GH3 gene family in five Rosaceae species and a working model of FaGH3.17 and FaGH3.18 response to drought stress. Red fonts represent up-regulated expression levels in WT vs. OE, respectively