Genome-wide analysis of the CaHsp20 gene family in pepper: comprehensive sequence and expression profile analysis under heat stress

Abstract

The Hsp20 genes are present in all plant species and play important roles in alleviating heat stress and enhancing plant thermotolerance by preventing the irreversible aggregation of denaturing proteins. However, very little is known about the CaHsp20 gene family in pepper (Capsicum annuum L.), an important vegetable crop with character of temperate but thermosensitive. In this study, a total of 35 putative pepper Hsp20 genes (CaHsp20s) were identified and renamed on the basis of their molecular weight, and then their gene structure, genome location, gene duplication, phylogenetic relationship, and interaction network were also analyzed. The expression patterns of CaHsp20 genes in four different tissues (root, stem, leaf, and flower) from the thermotolerant line R9 under heat stress condition were measured using semi-quantitative RT-PCR. The transcripts of most CaHsp20 genes maintained a low level in all of the four tissues under normal temperature condition, but were highly induced by heat stress, while the expression of CaHsp16.6b, 16.7, and 23.8 were only detected in specific tissues and were not so sensitive to heat stress like other CaHsp20 genes. In addition, compared to those in thermotolerant line R9, the expression peak of most CaHsp20 genes in thermosensitive line B6 under heat stress was hysteretic, and several CaHsp20 genes (CaHsp16.4, 18.2a, 18.7, 21.2, 22.0, 25.8, and 25.9) showed higher expression levels in both line B6 and R9. These data suggest that the CaHsp20 genes may be involved in heat stress and defense responses in pepper, which provides the basis for further functional analyses of CaHsp20s in the formation of pepper acquired thermotoleance.

Keywords: CaHsp20 genes; Capsicum annuum L.; expression profile; gene family; heat stress.

Figure 1

Distribution of conserved motifs in the CaHsp20 family members. Ten putative motifs are represented by a number in colored box. Names of all members and combined p-values are shown on the left side of the figure and motif sizes are indicated at the bottom of the figure. For details of motifs refer to Table 2.

Figure 2

Alignment the ACDs of CaHsp20s. (A) Alignment of the ACD from the MEME results for the CaHsp20 with Hsp20 from Pfam. The Motif 2, 4, 3, and 1 obtained via MEME analysis formed the putative CaHsp20 ACD, and the HMM logo from Pfam representing the Hsp20 domain (PF00011). The amino acids with red color indicate the matches between the MEME motifs and HMM sequences. (B) Alignment of the ACDs of CaHsp20s from pepper. Names of all members are shown on the left side of the figure. The alignment is performed by the Promals3D structural alignment program. Each predicted β-plated sheets are in blue. The primary structure of the ACD, including the conserved regions I (CRI), II (CRII), and β6-loop, is shown at the bottom of the figure.

Figure 3

Phylogenetic tree of pepper, tomato, and Arabidopsis Hsp20 proteins. The Hsp20 proteins are used for construction of the phylogenetic tree using the MEGA 5.10 program. Unrooted Neighbor-Joining analysis is performed with pairwise deletion and Poisson correction. For the tomato (prefixed by Sl), Arabidopsis (prefixed by At), rice (prefixed by Os), and maize (prefixed by Zm) Hsp20 proteins, both gene names and subfamily names are used, and marked with green, black, blue, and cyan triangles, respectively. CaHsp20 proteins are marked with red triangles. The 12 subfamilies are marked by different colors, and the Hsp20 proteins unclassified have been colored in gray.

Figure 4

Location and duplications of CaHsp20 paralogs on pepper chromosomes. The scale represents megabases (Mb). The chromosome numbers are indicated at the top of each bar. The CaHsp20 genes present on duplicated chromosomal segments are connected by gray dotted lines and tandem duplicated genes are marked by gray rectangles.

Figure 5

The interaction network of CaHsp20 genes in pepper according to the orthologs in Arabidopsis. The green circles represent the CaHsp20 genes, and pink circles represent the pepper genes interaction with CaHsp20 genes. The red lines represent the PCC > 0 (Pearson Correlation Coefficient), the blue lines mean the PCC < 0, and the green lines represent the PCC is not calculated.

Figure 6

RT-PCR analysis of expression pattern of 35 CaHsp20 genes in four pepper tissues under normal and HS conditions. R, root; S, stem; L, leaf; F, flower. Normal condition: 26°C for 2 h; HS condition: 40°C for 2 h. The pepper ubiquitin-conjugating protein gene (CaUBI-3) is used as an internal control.

Figure 7

Heat map showing CaHsp20 genes expression pattern in pepper thermosensitive line B6 and thermotolerant line R9 under HS condition. Leaves of B6 and R9 seedlings (6–8 true leaves) are used to test the changes of CaHsp20 genes expression level at different time points (0, 0.5, 1, 2, and 4 h) with HS treatment (40°C). CaUBI-3 is used as an internal control. qRT-PCR data are shown relative to 0 h. The relative expression levels were calculated using the 2^−ΔΔCT method, and normalized using log10. The heat map was created using MeV.

Figure 8

Predicted cis-elements in the promoter regions of CaHsp20 genes. Promoter sequences (–500 bp) for 30 CaHsp20 genes (promoter regions of CaHsp18.4a, 21.1, 21.2, 22.1, and 28.4 were absent) are analyzed. The names of the promoters of CaHsp20 genes are shown on the left side of the figure. The number at the bottom indicates the number of nucleotides upstream to the translation initiation codon, ATG. The blue triangles stand for the heat shock elements (HSEs), the pink cylinders for the TATA-box elements, the sky blue arrows for the TC-rich repeats, the black rectangles for the CCAAT elements, and the red pentagons for the W-box elements.