Genomic Analysis of the Glutathione S-Transferase Family in Pear (Pyrus communis) and Functional Identification of PcGST57 in Anthocyanin Accumulation

Abstract

Anthocyanin accumulation in vacuoles results in red coloration in pear peels. Glutathione S-transferase (GST) proteins have emerged as important regulators of anthocyanin accumulation. Here, a total of 57 PcGST genes were identified in the European pear 'Bartlett' (Pyrus communis) through comprehensive genomic analysis. Phylogenetic analysis showed that PcGST genes were divided into 10 subfamilies. The gene structure, chromosomal localization, collinearity relationship, cis-elements in the promoter region, and conserved motifs of PcGST genes were analyzed. Further research indicated that glutamic acid (Glu) can significantly improve anthocyanin accumulation in pear peels. RNA sequencing (RNA-seq) analysis showed that Glu induced the expression of most PcGST genes, among which PcGST57 was most significantly induced. Further phylogenetic analysis indicated that PcGST57 was closely related to GST genes identified in other species, which were involved in anthocyanin accumulation. Transcript analysis indicated that PcGST57 was expressed in various tissues, other than flesh, and associated with peel coloration at different developmental stages. Silencing of PcGST57 by virus-induced gene silencing (VIGS) inhibited the expression of PcGST57 and reduced the anthocyanin content in pear fruit. In contrast, overexpression of PcGST57 improved anthocyanin accumulation. Collectively, our results demonstrated that PcGST57 was involved in anthocyanin accumulation in pear and provided candidate genes for red pear breeding.

Keywords: PcGST57; anthocyanins; glutathione S-transferase; pear (Pyrus communis).

Figure 1

Phylogenetic analysis of GST genes from European pear and Arabidopsis. An unrooted phylogenetic tree was constructed using the full-length protein sequences of GST by MEGAX using neighbor-joining method, with 1000 bootstrap replicates. The branches with different colors indicates 10 subfamilies of GST proteins.

Figure 2

The chromosomal locations of PcGST genes in European pear. The position of PcGST genes was mapped to pear chromosomes based on the location information obtained from the genome of ‘Bartlett’ DH.

Figure 3

Collinearity analysis of the pear PcGST gene family. (A) Collinearity relationships of PcGST genes in pear genome. The panel exhibited 17 pear chromosomes in a circle with red lines connecting PcGST genes with WGD/segmental duplication events. Chromosome numbers were indicated on the circle with blue color. The PcGST genes were mapped to the chromosomes outside the circle. (B) Collinearity relationships of PcGST genes in pear, Arabidopsis, and apple. The blue lines indicated the PcGST genes with homologous relationship with Arabidopsis or apple.

Figure 4

Structure and conserved domain analysis of PcGST genes. (A) Phylogenetic relationship and conserved motif analysis of PcGST genes. The boxes with different colors indicated different motifs. (B) Exon–intron structure and conserved domain analysis of PcGST genes. The conserved GST domains were exhibited with indicated colors, and the introns were shown as black lines.

Figure 5

Cis-elements analysis in the promoter region of PcGST genes. The 2-kb of 5′ flanking sequence upstream from the start codon was obtained for analysis, and the boxes with different colors indicated different cis-elements.

Figure 6

Heatmap of transcript profiles of PcGST genes in response to Glu treatment at indicated time points. The transcript abundance of PcGST genes was represented by different colors. The red and blue color on the panel indicate high and low expression, respectively. The data was obtained from three biological replicates.

Figure 7

Phylogenetic analysis and sequence alignment of PcGST57 and its paralogs. (A) Phylogenetic relationships of PcGST57 paralogs was shown by phylogenetic tree. GST proteins from Pyrus communis, Malus domestica, Arabidopsis, Prunus persica, Fragaria ananassa, and several other species were used for the construction of a neighbor-joining tree. (B) Sequence alignment of PcGST57 and its paralogs. The GST domain with accession number of cl31543 was indicated by blue box, the red box indicated as GST-N domain with accession number of cd03058, and green box indicated as GST-C domain with accession number of cd03185. The accession number of GST members used in the analysis were as follows, MdGSTF6 (MD17G1272100), PpGST1 (Prupe.3G013600.1), FvRAP (gene31672), VviGST4 (AAX81329), LcGST4 (ALY05893), PfGST1 (BAG14300), CkmGST3 (BAM14584), PhAN9 (CAA68993), IbGSTF4 (MG873448), AtGSTF12 (AED92398), ZmBZ2 (AAA50245), and GmGST26A (NP_001238439).

Figure 8

The transcript patterns of PcGST57. (A) Transcript analysis of PcGST57 in different pears. Pears, including ‘Zaosu’, ‘Mansoo’, ‘Danxiahong’, ‘Red Clapp’s Favorite’, and ‘Red Zaosu’, with different colors were selected for analysis. (B) Transcript analysis of PcGST57 in various pear tissues of ‘Danxiahong’. Various pear tissues of ‘Danxiahong’, including floral shoot, floral receptacle, leaf, petal, and anther at 0 DAF, and peel, flesh of 100 DAF were sampled for transcript analysis. (C) Transcript analysis of PcGST57 in pear peels of ‘Danxiahong’ at different developmental periods. (D) Transcript analysis of PcGST57 in pear flesh of ‘Danxiahong’ at different developmental periods. PcTubulin was used as the internal control for gene expression analysis. All the data indicate means ± SD of three biological replicates.

Figure 9

Functional identification of PcGST57 by transient expression in ‘Danxiahong’ pear peels. Transient overexpression (A) and TRV-mediated silencing (B) assays of PcGST57 in ‘Danxiahong’ pear peels. Transcript analysis of PcGST57 in overexpressing peels (C) and silencing peels (F). Measurement of anthocyanin content in PcGST57 overexpressing peels (D) and silencing peels (G). Measurement of total phenol content in PcGST57 overexpressing peels (E) and silencing peels (H). The photograph were captured 15-day post injection, and the peels around the injection sites were sampled for expression analysis and measurement of anthocyanin content and total phenol content. Pctubulin was used as the internal control for gene expression analysis. All the data indicate means ± SD of three biological replicates. Asterisks indicate statistical significance (*, p < 0.05; and **, p < 0.01) determined by student’s t-test compared with corresponding controls.