Genome-wide identification of glutathione S-transferase gene family in pepper, its classification, and expression profiling under different anatomical and environmental conditions

Abstract

Glutathione S-transferases (GSTs) compose a family of multifunctional enzymes involved in the numerous aspects of regulating plant growth, development, and stress response. An in silico genome-wide analysis of pepper (Capsicum annuum L.) was performed to identify eighty-five GST genes that were annotated according to their chromosomal location. Segmental duplication contributed more than tandem duplication for the expansion of GST gene family in pepper. All the identified members belong to ten different classes which are highly conserved among Arabidopsis, rice, tomato and potato counterparts indicating the pre-dicot-monocot split diversification of GST classes. Gene structure, protein domain, and motif organization were found to be notably conserved over the distinct phylogenetic groups, which demonstrated the evolutionary significant role of each class. Expression of most of the CaGST transcripts as well as the total pepper GST activity was found to be significantly up-regulated in response to cold, heat, drought, salinity and osmotic stress conditions. Presence of various hormone and stress-responsive cis-elements on most of the putative CaGST promoter regions could be directly correlated with the alteration of their transcripts. All these findings might provide opportunities for future functional validation of this important gene family in pepper.

Figure 1

Phylogenetic and Structural analysis of CaGST genes. (A) Number at each node in the phylogenetic tree represents the bootstrap value higher than 50. The different class of GST genes forms separate clades. (B) The schematic diagram represents the gene structure of all 85 CaGST genes identified in this study. Exons are shown as red boxes; introns are shown as black lines, and the upstream/downstream regions are shown as blue boxes. The relative size of the full transcript, intron, exon, and upstream region could be inferred from the supplied scale in kilobase pair (kb).

Figure 2

Phylogenetic analysis of GST proteins. GST proteins from five species- pepper, tomato, potato, Arabidopsis, and rice were used to construct an unrooted phylogenetic tree using MEGA Maximum-likelihood method with 1000 bootstraps. GST members from tau, lambda, zeta, DHAR, theta, GHR, TCHQD, phi, MGST, EF1Bγ classes were marked with red, green, cyan, blue, violet, gray, coral, yellow, pink, royal blue; respectively. Members of each class formed different clades with few exceptions.

Figure 3

Copy number variation between Capsicum and tomato GST genes. The numbers presented inside the circles and rectangles represent the numbers of GST genes as common ancestral and species-specific, respectively. Numbers on the branches with plus and minus symbols represent the numbers of gene gains and losses, respectively throughout the evolution between these two species. The light gray boxes represent Capsicum, while dark gray boxes symbolize tomato.

Figure 4

Expression profiling of CaGST genes at different anatomical tissues. Expression of all the identified CaGST transcripts was analyzed in 57 developmentally diverse tissues using the RNA-seq data. All these tissues could be categorized into nine major stages, such as seedlings, flower, petal, ovary, anther, fruit, pericarp, seed, and placenta. Heatmap with hierarchical clustering was performed using the expression values in MeV software package with Manhattan correlation. The highest level of expression is represented by dark blue (100%), while the low level is presented as white (0%). Thus, the intensity of the color in the heatmap is directly proportioned to the transcript abundance of each member.

Figure 5

Alteration of CaGST transcripts in various abiotic stresses. Expression of all 85 CaGST transcripts was analyzed from the leaf (A) and root (B) samples treated with five major abiotic stresses, such as cold, heat, drought, salinity and oxidative. Relative fold change of transcript abundance in seven different time points of each stress (1 h, 1.5 h, 3 h, 6 h, 12 h, 24 h, and 48 h) was plotted with MeV software package. The color scale, depicted at the down of each heatmap, represents the intensity of alterations where green color indicates downregulation and red indicates upregulation.

Figure 6

Measurement of total GST activity in response to various abiotic stresses. Total GST enzyme activity was measured in response to various abiotic stresses such as cold, heat, drought, salinity, and oxidative at four different time points of stress exposure. The activity was represented as nmoles/min/mg protein. All the experiments were repeated thrice and represent as the average ± standard deviation (n = 3). The significance level of the paired student’s two-tailed t-test is represented as * and ** with a p-value less than 0.05 and 0.01; respectively.

Figure 7

Analysis of the putative promoter of CaGST genes. One kb 5′ upstream sequences of all the identified CaGST genes were retrieved from the genome database and analyzed through PlantCARE to identify the presence and number of various cis-acting regulatory elements. Different hormone-responsive and stress-related elements were identified and plotted against a bar diagram. The abundance of different regulatory elements on each of the promoter was represented with different colors.