Genome-wide characterization and expression analysis of GRAS gene family in pepper (Capsicum annuum L.)

Abstract

Plant-specific GRAS transcription factors regulate various biological processes in plant growth, development and stress responses. However, this important gene family was not fully characterized in pepper (Capsicum annuum L.), an economically important vegetable crop. Here, a total of 50 CaGRAS members were identified in pepper genome and renamed by their respective chromosomal distribution. Genomic organization revealed that most CaGRAS genes (84%) have no intron. Phylogenetic analysis divided pepper CaGRAS members into 10 subfamilies, with each having distinct conserved domains and functions. For the expansion of the GRAS genes in pepper, segmental duplication contributed more than tandem duplication did. Gene expression analysis in various tissues demonstrated that most of CaGRAS genes exhibited a tissue- and development stage-specific expression pattern, uncovering their potential functions in pepper growth and development. Moreover, 21 CaGRAS genes were differentially expressed under cold, drought, salt and gibberellin acid (GA) treatments, indicating that they may implicated in plant response to abiotic stress. Notably, GA responsive cis-elements were detected in the promoter regions of the majority of CaGRAS genes, suggesting that CaGRAS may involve in signal cross-talking. The first comprehensive analysis of GRAS gene family in pepper genome by this study provide insights into understanding the GRAS-mediated regulation network, benefiting the genetic improvements in pepper and some other relative plants.

Keywords: Abiotic stress; Duplication; GRAS genes; Gene expression; Pepper.

Figure 1. Exon-intron structure of CaGRAS genes.

Blue box indicates exon, and black line indicates intron. Y-axis represents the subfamily name of each CaGRAS genes. The lengths of the exons and introns were drawn to scale.

Figure 2. Positions of CaGRAS genes on pepper chromosomes.

Gray shading indicates tandem duplicated region.

Figure 3. Microsynteny analyses of GRAS genes among pepper (Ca), tomato (Sl), and Arabidopsis (At).

Red, yellow and blue lines connecting two chromosomal regions indicate syntenic regions between pepper and tomato, pepper and Arabidopsis, tomato and Arabidopsis chromosomes, respectively. Black lines denote segmental duplicated GRAS genes on the pepper chromosome.

Figure 4. Phylogenetic analyses of GRAS proteins from pepper, tomato, rice and Arabidopsis.

The phylogenetic tree was constructed using neighbor-joining (NJ) method by MEGA6.0. Subfamilies were indicated by different colors.

Figure 5. Distribution of conserved motifs in CaGRAS proteins.

(A) The phylogenetic tree and their classification were depicted using the neighbor-joining method in MEGA 6.0. (B) Motif distribution in each GRAS sequence. Motif 10 and 4 is in LHRI domain at N-terminus, followed by Motif 7 and 1 in VHIID domain, Motif 6 and 8 in LHRII domain, Motif 9, 3 and 11 in PFYRE domain, and Motif 2 and 5 in SAW domain at C-terminus.

Figure 6. The interaction network of CaGRAS proteins in pepper according to interologs from Arabidopsis.

Figure 7. Heatmap and hierarchical clustering of CaGRAS genes in leaf, stem, root and mature green (MG), breaker (B), five and 10 days post-breaker (B5, B10), six, 16, 25 days post-anthesis (6DPA, 16DPA, 25DPA) of pericarp (PC) and placenta (PL).

The expression values were calculated by RPKM measure and then were log2 transformed before generating heat maps.

Figure 8. Differential expression analyses of 21 GRAS genes under GA, drought, salt and cold treatment in pepper seedlings.

The color scale represents log2 expression values.