Genome-wide identification, phylogeny, and expression analysis of PEBP gene family in Castanea mollissima

Abstract

The phosphatidylethanolamine binding protein (PEBP) family plays an important part in growth and development of plants. Castanea mollissima is an economic plant with significant financial value and has become an important food source in the Northern Hemisphere. However, the PEBP genes in C. mollissima have not been studied yet. In this study, six PEBP genes (CmPEBP1 ∼ CmPEBP6) were identified in C. mollissima and comprehensively analyzed in terms of physicochemical properties, phylogeny, gene structures, cis-regulatory elements (CREs), transcription factor interaction, and expression profiles. The six CmPEBP genes were categorized into three subfamilies according to the phylogeny analysis, and all of them share extremely similar gene and protein structures. A total of 136 CREs were identified in the promoter regions of the CmPEBP genes, mainly related to growth and development, environmental stress, hormone response, and light response. Comparative genomic analysis indicated that the expansion of the CmPEBP genes was mainly driven by dispersed duplication, and the CmPEBP3/CmPEBP5 derived from eudicot common hexaploidization (ECH) events retained orthologous genes in all species studied. A total of 259 transcription factors (TFs) belonging to 39 families were predicted to be regulators of CmPEBP genes, and CmPEBP4 was predicted to interact with the most TFs. The RNA-seq data analysis indicated the potential roles of CmPEBP genes in the ovule, bud, and flower development of C. mollissima, as well as in the response to temperature stress, drought stress, and the gall wasp Dryocosmus kuriphilus (GWDK) infestation. Additionally, the expression of CmPEBP genes in C. mollissima seed kernel development and their response to temperature stress were confirmed by RT-qPCR assays. This study gives references and directions for future in-depth studies of PEBP genes.

Keywords: Castanea mollissima; PEBP gene family; RT-qPCR; expression analysis; phylogeny.

FIGURE 1

The phylogenetic tree of 117 PEBP proteins of Arabidopsis thaliana (6), Malus domestica (8), Oryza sativa (19), Sorghum bicolor (19), Brachypodium distachyon (18), Solanum lycopersicum (13), Vitis vinifera (5), Zea mays (23) and C. mollissima (6). MEGA 7.0 was used to construct the phylogenetic tree based on the protein sequences with the maximum likelihood method. The proteins were clustered into three groups.

FIGURE 2

Chromosome distribution, gene structure, and conserved motifs of CmPEBP genes. (A) Chromosome distribution of CmPEBP genes. The color of segments in the chromosomes shows the gene density of the corresponding region. (B) Intron exon structure of CmPEBP genes. The phylogenetic tree containing only six CmPEBP genes is placed on the left side, constructed by MEGA 7.0 based on the maximum likelihood method. (C) Distribution of conserved motifs in CmPEBP proteins. (D) The sequence of eight conserved motifs in CmPEBP proteins.

FIGURE 3

Prediction of cis-regulatory elements in the promoters of PEBP genes in C. mollissima. (A) Cis-regulatory elements in the promoters of six CmPEBP genes. Various color symbols present different elements, and their position in the figure indicates their relative position on the promoter. (B) The relative proportions of different cis-regulatory elements in the promoters of six CmPEBP genes are indicated in the chart. The same color represents cis-regulatory elements sharing identical or similar functions. (C) The number of various cis-regulatory elements in the promoters of each CmPEBP genes.

FIGURE 4

Collinearity analyses of PEBP genes within C. mollissima genome, and between the PEBP genes of C. mollissima and seven representative plant species (Quercus, Pyrus, V. vinifera, A. thaliana, S. lycopersicum, O. sativa, and Z. mays).

FIGURE 5

Homologous collinear dot-plot within the C. mollissima genome. The boxes in the figure represent collinear regions within the C. mollissima genome, in which the dark or light highlighted boxes indicate regions formed by WGD event containing CmPEBP homologous gene pairs and complementary fragments forming more significant homologous regions, respectively.

FIGURE 6

Genes expression of CmPEBP genes in different tissues of C. mollissima. (A) Genes expression in fertile and abortive ovules on 15-July, 20-July and 25-July. (B) Genes expression in first and second female flowering, first and second male flowering. FFF: First flowering (female), SFF: Secondary flowering (female), FFM: First flowering (male), SFM: Secondary flowering (male). (C) Genes expression in nuts of the cultivar “Yanshanzaofeng” and “Yanlong” 60, 70, 80, 90, and 100 days after flowering. (D) Genes expression in buds 20, 25, and 30 days after flowering.

FIGURE 7

Genes expression of CmPEBP genes under different stresses in C. mollissima. (A) The expression profiles of CmPEBP genes under low (−15°C) and high (45°C) temperature stress at various periods. CK: control sample. G4h, G8h, G12h: high-temperature stress treatment for 4, 8, and 12 h, respectively. D5h, D10h, D15h: low-temperature stress treatment for 5, 10, and 15 h, respectively. (B) Genes expression in leaves of cultivar “Dabanhong” (DBH) and “Yanshanzaofeng” (YSZF) treated with drought for 0, 10, 20, 30, and 40 days. (C) Genes expression in leaves together with gall on 7-April, 10-April, 15-April, 26-April. (D) Genes expression in leaves with galls of cultivar “HongLi” (HL) (susceptible to GWDK infestation) and “Shuhe_Wuyingli” (SH) (partially resistant to GWDK infestation) infested with GWDK on 7-April, 15-April, 26-April. (E) Genes expression in leaves and insect galls induced by GWDK.

FIGURE 8

RT-qPCR of CmPEBP genes. (A) “Yanshanzaofeng” C. mollissima seed kernels, 1–5: seed kernels at 60, 70, 80, 90, and 100 days after flowering, respectively. (B) C. mollissima plants subjected to temperature stress treatment, 6∼9: C. mollissima plants were subjected to high-temperature stress for 0, 4, 8, and 12 h, respectively. 10∼13: C. mollissima plants were subjected to low-temperature stress for 0, 4, 8, and 12 h, respectively. (C) RT-qPCR of CmPEBP genes in C. mollissima leaves under high- and low-temperature stresses. Lowercase letter(s) above the bars indicate significant differences (α = 0.05, LSD) among the treatments.