Genome-wide analyses of member identification, expression pattern, and protein-protein interaction of EPF/EPFL gene family in Gossypium

Abstract

Background: Epidermal patterning factor / -like (EPF/EPFL) gene family encodes a class of cysteine-rich secretory peptides, which are widelyfound in terrestrial plants.Multiple studies has indicated that EPF/EPFLs might play significant roles in coordinating plant development and growth, especially as the morphogenesis processes of stoma, awn, stamen, and fruit skin. However, few research on EPF/EPFL gene family was reported in Gossypium.

Results: We separately identified 20 G. raimondii, 24 G. arboreum, 44 G. hirsutum, and 44 G. barbadense EPF/EPFL genes in the 4 representative cotton species, which were divided into four clades together with 11 Arabidopsis thaliana, 13 Oryza sativa, and 17 Selaginella moellendorffii ones based on their evolutionary relationships. The similar gene structure and common motifs indicated the high conservation among the EPF/EPFL members, while the uneven distribution in chromosomes implied the variability during the long-term evolutionary process. Hundreds of collinearity relationships were identified from the pairwise comparisons of intraspecifc and interspecific genomes, which illustrated gene duplication might contribute to the expansion of cotton EPF/EPFL gene family. A total of 15 kinds of cis-regulatory elements were predicted in the promoter regions, and divided into three major categories relevant to the biological processes of development and growth, plant hormone response, and abiotic stress response. Having performing the expression pattern analyses with the basic of the published RNA-seq data, we found most of GhEPF/EPFL and GbEPF/EPFL genes presented the relatively low expression levels among the 9 tissues or organs, while showed more dramatically different responses to high/low temperature and salt or drought stresses. Combined with transcriptome data of developing ovules and fibers and quantitative Real-time PCR results (qRT-PCR) of 15 highly expressed GhEPF/EPFL genes, it could be deduced that the cotton EPF/EPFL genes were closely related with fiber development. Additionally, the networks of protein-protein interacting among EPF/EPFLs concentrated on the cores of GhEPF1 and GhEPF7, and thosefunctional enrichment analyses indicated that most of EPF/EPFLs participate in the GO (Gene Ontology) terms of stomatal development and plant epidermis development, and the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways of DNA or base excision repair.

Conclusion: Totally, 132 EPF/EPFL genes were identified for the first time in cotton, whose bioinformatic analyses of cis-regulatory elements and expression patterns combined with qRT-PCR experiments to prove the potential functions in the biological processes of plant growth and responding to abiotic stresses, specifically in the fiber development. These results not only provide comprehensive and valuable information for cotton EPF/EPFL gene family, but also lay solid foundation for screening candidate EPF/EPFL genes in further cotton breeding.

Keywords: EPF/EPFL gene family; Cotton; Expression pattern; Protein–protein interaction; qRT-PCR verification.

Fig. 1

Phylogenetic analysis of EPF/EPFL proteins from Gossypium, Arabidopsis thaliana, Oryza sativa, and Selaginella moellendorffii. The blue checkmark presents the AtEPF/EPFL proteins, and the purple square presents the OsEPF/EPFL proteins. The red check mark presents the SmEPF/EPFL proteins, and the green triangle presents the GaEPF/EPFL proteins. The red square presents the GrEPF/EPFL proteins, and the yellow star and blue circle present the GhEPF/EPFL and GbEPF/EPFL proteins, respectively

Fig. 2

Chromosomal location of cotton EPF/EPFL genes

Fig. 3

Gene structure and conserved motif identification of cotton EPF/EPFLs. a represents the evolutionary relationships of cotton EPF/EPFL genes, and b and c separately represent the conserved motifs and gene structures of cotton EPF/EPFL genes

Fig. 4

Collinearity events of duplication gene pairs of EPF/EPFL genes in four cotton species. The different color rectangles represented the chromosomes derived from the different cotton species, and the different color lines represented the collinearity relationships between and among the different cotton species

Fig. 5

Cis-regulatory elements in the promoter regions of cotton EPF/EPFL genes

Fig. 6

The tissue-specific expression and expressed patterns responding to abiotic stresses. A presented the analyses of specific expression of GhEPF/EPFL and GbEPF/EPFL genes in 9 tissues, and B presented the expression patterns of GhEPF/EPFL genes in response to high-temperature (37℃ treatment), low-temperature (4℃ treatment), salt stress (NaCl treatment), and drought stress (PEG treatment) at 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h, respectively

Fig. 7

The expression patterns and qRT-PCR verification of cotton EPF/EPFL genes during fiber development. A presented the analyses of expression patterns of GhEPF/EPFL and GbEPF/EPFL genes on the developing ovule (0, 1, 3, 5, 10, and 20 DPA) and fiber (10, 20, and 25 DPA), and B presented the qRT-PCR verification of 15 highly expressed GhEPF/EPFL genes on the high-yield and wide-adaptability CCRI36 and superior fiber-quality and high VW-resistance Hai1 at 10, 20, and 25 DPA, respectively

Fig. 8

The protein–protein interaction and functional enrichment of GhEPF/EPFL genes. A presented the analysis of protein–protein interaction of GhEPF/EPFL genes based on the String database, and B and C separately presented the enriched GO categories and KEGG pathways of GhEPF/EPFL genes