Genome-Wide Identification of Glutathione S-Transferase Genes in Eggplant (Solanum melongena L.) Reveals Their Potential Role in Anthocyanin Accumulation on the Fruit Peel

Abstract

Anthocyanins are ubiquitous pigments derived from the phenylpropanoid compound conferring red, purple and blue pigmentations to various organs of horticultural crops. The metabolism of flavonoids in the cytoplasm leads to the biosynthesis of anthocyanin, which is then conveyed to the vacuoles for storage by plant glutathione S-transferases (GST). Although GST is important for transporting anthocyanin in plants, its identification and characterization in eggplant (Solanum melongena L.) remains obscure. In this study, a total of 40 GST genes were obtained in the eggplant genome and classified into seven distinct chief groups based on the evolutionary relationship with Arabidopsis thaliana GST genes. The seven subgroups of eggplant GST genes (SmGST) comprise: dehydroascorbate reductase (DHAR), elongation factor 1Bγ (EF1Bγ), Zeta (Z), Theta(T), Phi(F), Tau(U) and tetra-chlorohydroquinone dehalogenase TCHQD. The 40 GST genes were unevenly distributed throughout the 10 eggplant chromosomes and were predominantly located in the cytoplasm. Structural gene analysis showed similarity in exons and introns within a GST subgroup. Six pairs of both tandem and segmental duplications have been identified, making them the primary factors contributing to the evolution of the SmGST. Light-related cis-regulatory elements were dominant, followed by stress-related and hormone-responsive elements. The syntenic analysis of orthologous genes indicated that eggplant, Arabidopsis and tomato (Solanum lycopersicum L.) counterpart genes seemed to be derived from a common ancestry. RNA-seq data analyses showed high expression of 13 SmGST genes with SmGSTF1 being glaringly upregulated on the peel of purple eggplant but showed no or low expression on eggplant varieties with green or white peel. Subsequently, SmGSTF1 had a strong positive correlation with anthocyanin content and with anthocyanin structural genes like SmUFGT (r = 0.9), SmANS (r = 0.85), SmF3H (r = 0.82) and SmCHI2 (r = 0.7). The suppression of SmGSTF1 through virus-induced gene silencing (VIGs) resulted in a decrease in anthocyanin on the infiltrated fruit surface. In a nutshell, results from this study established that SmGSTF1 has the potential of anthocyanin accumulation in eggplant peel and offers viable candidate genes for the improvement of purple eggplant. The comprehensive studies of the SmGST family genes provide the foundation for deciphering molecular investigations into the functional analysis of SmGST genes in eggplant.

Keywords: SmGSTF1; anthocyanin; eggplant; glutathione S-transferase.

Figure 1

Phylogenetic tree of the 185 GST protein sequences from Arabidopsis, eggplant and tomato. Different subgroups—Tau, Phi, Lambda, TCHQD, Theta, Maxin, Zeta, DHAR, EF1Bγ, GHR and MGST—are designated with different colors.

Figure 2

Phylogenetic relationships, gene structures of SmGST and conserved motifs of SmGST genes. (A) The phylogenetic tree of 40 SmGST family genes. SmGST subfamilies were assigned unique colors for identification. (B) Exon and intron structure of SmGST genes. Yellow rectangles are exons, while black lines are introns and green rectangles represent untranslated regions. (C) Different rectangular-shaped colors show the conserved motif patterns.

Figure 3

Chromosomal distribution and duplication of SmGST. (A) Representation of SmGST distributions on the chromosomes. Blue vertical bars are the chromosomes and their number is indicated to the left (black) while the gene number (red) is indicated to the right of each chromosome. (B) Illustration of the inter-chromosomal connections of SmGST. The blue lines denote segmental duplications while the red lines show tandem duplications of SmGST gene pairs.

Figure 4

The collinearity analysis of GST genes among eggplant, Arabidopsis and tomato. The gray lines in the background are collinearity blocks, whereas the blue lines show the collinearity of GST gene pairs. The red triangles represent corresponding collinear SmGST genes.

Figure 5

Promoter analysis of SmGST. (A) The main cis-acting elements in the promoter of SmGST genes. The cis-elements are represented by different colors. (B) The relative proportion of each group of promoter elements of light, plant growth, stress and phytohormones.

Figure 6

Expression profile of SmGST and anthocyanin content. (A) Expression profiles for SmGST genes in the peel of different eggplant varieties. FPKM values obtained from RNA-seq data and the levels of expression are plotted on Log 2-transformed (FPKM+1). Dark-purple cultivar; A1, green; A2, black-purple with green calyx cultivar; A3, white cultivar; A4 and reddish-purple cultivar; A5. (B) qRT-PCR expression patterns of 9 SmGST in the peel, anther, petal, fruit calyx and calyx, of EP26 and EP28 eggplant cultivars examined by qRT-PCR. Error bars denote standard error. (C) Correlation analysis of the expression profiles of SmGST genes, anthocyanin content and anthocyanin structural genes (ASGs) and relation with total anthocyanin content at p < 0.05. Expression profiles for SmGST genes obtained from RNA-seq data of the peel of different eggplant varieties. PAL (Smechr0500713), phenylalanine ammonia-lyase; C4H (Smechr0603018), cinnamate 4-hydroxylase; 4CL (Smechr0302347), 4-coumarate-CoA ligase; CHS (Smechr0500409), chalcone synthase; CHI (Smechr0500261; Smechr1001863), chalcone isomerase; F3H (Smechr0202240), flavanone 3-hydroxyl enzyme; FLS (Smechr1001394), flavonol synthase; DFR (Smechr0202337), dihydroflavonol reductase; ANS (Smechr1002343), anthocyanin synthase; UFGT (Smechr0502047), flavonoid 3-O-glucosyl transferase.

Figure 7

The effect of SmGSTF1 silencing in VIGS eggplant fruits. Peel color change of eggplant fruit peels infused with Agrobacterium cells (TRV2; SmGSTF1-TRV2). A devoid TRV vector was the control. The injection was done underneath the calyx and the calyx was removed afterwards.