Genome-Wide Identification of Glutathione S-Transferase and Expression Analysis in Response to Anthocyanin Transport in the Flesh of the New Teinturier Grape Germplasm 'Zhongshan-HongYu'

Abstract

Anthocyanins are synthesized in the endoplasmic reticulum and then transported to the vacuole in plants. Glutathione S-transferases (GSTs) are thought to play a key role in anthocyanin transport. To clarify the mechanism of GST genes in the accumulation and transport of anthocyanin in the early fruit stage, we analyzed and characterized the GST family in the flesh of 'Zhongshan-HongYu' (ZS-HY) based on the transcriptome. In this study, the 92 GST genes identified through a comprehensive bioinformatics analysis were unevenly present in all chromosomes of grapes, except chromosomes 3, 9 and 10. Through the analysis of the chromosomal location, gene structure, conserved domains, phylogenetic relationships and cis-acting elements of GST family genes, the phylogenetic tree divided the GST genes into 9 subfamilies. Eighteen GST genes were screened and identified from grape berries via a transcriptome sequencing analysis, of which 4 belonged to the phi subfamily and 14 to the tau subfamily, and the expression levels of these GST genes were not tissue-specific. The phylogenetic analysis indicated that VvGST4 was closely related to PhAN9 and AtTT19. This study provides a foundation for the analysis of the GST gene family and insight into the roles of GSTs in grape anthocyanin transport.

Keywords: GST; anthocyanin; gene family; genome-wide; teinturier grape.

Figure 1

Phylogenetic tree constructed with 92 GST protein sequences with the GST genes from Arabidopsis thaliana and Malus domestica. Different colors represent different subfamilies of GST genes, different shapes indicate different species. The MEGA X software was used to construct the tree using the neighbor-joining method.

Figure 2

Chromosome localization of grape GST family members.

Figure 3

The collinearity analysis of the GST gene family in grape. The GST genes were mapped onto different chromosomes. The red lines indicate segmental duplicated gene pairs of grape GST genes, while the gray lines delineate synteny blocks in the grape genome.

Figure 4

The synteny analysis of the GST genes between grape, Arabidopsis thaliana, Malus domestica, Fragaria vesca and Actindia chienesis. The blue lines indicate segmental duplicated gene pairs of grape GST genes with other species, while the gray lines delineate synteny blocks in the grape genome.

Figure 5

Protein motifs and DNA structures of GST genes family in grapes. (a) The protein motifs in the GST members. Different colored boxes indicate different motifs. (b) The DNA structures of the GST gene family in grapes. Different colored boxes delineate different structures.

Figure 6

Prediction of cis-acting elements in the GST promoters. The 2000 bp sequence upstream of the promoters was analyzed and different regulatory elements are shown in different colors.

Figure 7

Change in appearance of the flesh color in grapes for 3 consecutive days.

Figure 8

Heat map of the expression patterns of GST genes and relative expression of GST genes in six tissues. (a) Heat map of the expression patterns of the GST genes. The expression levels of genes are shown using different colors from white (−1.5) to red (2), with the values representing the log2 FPKM values. A redder color indicates a higher gene expression level. (b) Relative expression of GST genes in six tissues. The left y-axis represents the relative gene expression levels analyzed by qRT-PCR.

Figure 9

Phylogenetic relationship between 18 GST genes and the PhAN9, AtTT19, ZmBZ2 and VviGST1 proteins. The tree was constructed according to the neighbor-joining method in MEGA X, and the Poisson model, partial deletion and 1000 bootstrap replicates were used.

Figure 10

Verification of relative expression levels of genes in RNA-seq by qRT-PCR. The left y-axis represents the relative gene expression levels analyzed by qRT-PCR.