Genome-Wide Characterization and Expression Analysis of the HD-ZIP Gene Family in Response to Salt Stress in Pepper

Abstract

HD-ZIP is a unique type of transcription factor in plants, which are closely linked to the regulation of plant growth and development, the response to abiotic stress, and disease resistance. However, there is little known about the HD-ZIP gene family of pepper. In this study, 40 HD-ZIP family members were analyzed in the pepper genome. The analysis indicated that the introns number of Ca-HD-ZIP varied from 1 to 17; the number of amino acids was between 119 and 841; the theoretical isoelectric point was between 4.54 and 9.85; the molecular weight was between 14.04 and 92.56; most of them were unstable proteins. The phylogenetic tree divided CaHD-ZIP into 4 subfamilies; 40 CaHD-ZIP genes were located on different chromosomes, and all of them contained the motif 1; two pairs of CaHD-ZIP parallel genes of six paralogism genes were fragment duplications which occurred in 58.28~88.24 million years ago. There were multiple pressure-related action elements upstream of the start codon of the HD-Z-IP family. Protein interaction network proved to be coexpression phenomenon between ATML1 (CaH-DZ22, CaHDZ32) and At4g048909 (CaHDZ12, CaHDZ31), and three regions of them were highly homology. The expression level of CaHD-ZIP gene was different with tissues and developmental stages, which suggested that CaHD-ZIP may be involved in biological functions during pepper progress. In addition, Pepper HD-ZIP I and II genes played a major role in salt stress. CaHDZ03, CaHDZ 10, CaHDZ17, CaHDZ25, CaHDZ34, and CaHDZ35 were significantly induced in response to salt stress. Notably, the expression of CaHDZ07, CaHDZ17, CaHDZ26, and CaHDZ30, homologs of Arabidopsis AtHB12 and AtHB7 genes, was significantly upregulated by salt stresses. CaHDZ03 possesses two closely linked ABA action elements, and its expression level increased significantly at 4 h under salt stress. qRT-P-CR and transcription analysis showed that the expression of CaHDZ03 and CaHDZ10 was upregulated under short-term salt stress, but CaHDZ10 was downregulated with long-term salt stress, which provided a theoretical basis for research the function of Ca-HDZIP in response to abiotic stress.

Figure 1

Unrooted neighbor-joining phylogenetic tree and conserved motif analysis of CaHD-ZIP proteins. (a) The phylogenetic tree was generated based on the protein sequences of CaHD-ZIP proteins. (b) Conserved motif analysis of CaHD-ZIP proteins. Different color boxes represent different types of motifs.

Figure 2

Chromosomal locations and duplication events of CaHD-ZIP genes of pepper. Locations of CaHD-ZIP were based on physical locations. The numbers on the top represent the chromosome number. The chromosome 00 (Chr00) means two different scaffolds containing unassigned CaHD-ZIP to any of the 12 pepper chromosomes. Red lines indicate 2 pairs of paralogism genome duplication events.

Figure 3

Cis analysis of CaHD-ZIP promoter related to stress response. Different cis-elements with the same or similar functions are shown in the same shape and color.

Figure 4

Predicted protein-protein interaction network of CaHD-ZIP proteins. The colors of the line indicate different data sources.

Figure 5

(a) Pepper HD-ZIPgenes expression of different tissues and developmental stages. Raw data were taken from RNA-seq data of CM334. The tissues included root, stem, leaf, pericarp (PC), and placenta (PL) at 6, 16, and 25 days postanthesis (DPA), PC and PL at mature green (MG) and at breaker (B) stages, PC and PL at 5 (B5) and 10 (B10) days after postbreaker, respectively. (b) The heat map of CaHD-ZIP genes under NaCl treatment (VPC represents CK, VPY represents NaCl stress). Blue and red colors represent relatively low and high expression (log 2 RPKM value).

Figure 6

The heat map of the CaHD-ZIP genes under NaCl treatment (VPC represents CK, VPY represents NaCl stress). Expression patterns of 9 CaHDZ genes under salt stress. Salt stress (100 mM NaCl) was carried out on pepper seedlings at the six-leaf stage. The relative expression level of CaHDZ gene was analyzed by qRT-PCR, using sesame VPC4h gene as the internal control.

Figure 7

Phylogenetic trees of 132 HD-ZIP proteins in pepper (blue square), Arabidopsis (red square), and rice (purple square). The HD-ZIP gene is divided into four subfamilies (groups I, II, III, and IV, with yellow, purple, green, and blue branches, respectively).