Investigation of the EIL/EIN3 Transcription Factor Gene Family Members and Their Expression Levels in the Early Stage of Cotton Fiber Development

Abstract

The ethylene-insensitive3-like/ethylene-insensitive3 (EIL/EIN3) protein family can serve as a crucial factor for plant growth and development under diverse environmental conditions. EIL/EIN3 protein is a form of a localized nuclear protein with DNA-binding activity that potentially contributes to the intricate network of primary and secondary metabolic pathways of plants. In light of recent research advances, next-generation sequencing (NGS) and novel bioinformatics tools have provided significant breakthroughs in the study of the EIL/EIN3 protein family in cotton. In turn, this paved the way to identifying and characterizing the EIL/EIN3 protein family. Hence, the high-throughput, rapid, and cost-effective meta sequence analyses have led to a remarkable understanding of protein families in addition to the discovery of novel genes, enzymes, metabolites, and other biomolecules of the higher plants. Therefore, this work highlights the recent advance in the genomic-sequencing analysis of higher plants, which has provided a plethora of function profiles of the EIL/EIN3 protein family. The regulatory role and crosstalk of different metabolic pathways, which are apparently affected by these transcription factor proteins in one way or another, are also discussed. The ethylene hormone plays an important role in the regulation of reactive oxygen species in plants under various environmental stress circumstances. EIL/EIN3 proteins are the key ethylene-signaling regulators and play important roles in promoting cotton fiber developmental stages. However, the function of EIL/EIN3 during initiation and early elongation stages of cotton fiber development has not yet been fully understood. The results provided valuable information on cotton EIL/EIN3 proteins, as well as a new vision into the evolutionary relationships of this gene family in cotton species.

Keywords: Cis-element; EIL/EIN3; developmental stages; gene structure; identification; phylogeny.

Figure 1

Structural features of EIL/EIN3 transcription factor in cotton. (A) The exon–intron arrangement of EIL/EIN3 genes. The blue shapes represent upstream/downstream, yellow shapes indicate CDS (exons), and black lines represent introns. (B) Ten motifs identified by the MEME tool are represented by colored boxes, and their consensus sequences are shown in Figure S2.

Figure 2

Phylogenetic tree relationships between 18 G. hirsutum, 9 G. arboreum, 10 G. raimondii, 6 Arabidopsis, 7 P. trichocarpa, 6 maize, 3 sorghum, 3 T. cacao, and 12 soybean EIL/EIN3 proteins. The phylogenetic tree was created by the MEGA 6.0 program using the NJ (neighbor-joining) method. The bootstrap test was done with 1000 iterations. The three groups (A, B, and C) are shown in colors.

Figure 3

Functional analysis of EIL/EIN3 proteins in upland cotton. (A) Gene ontology (GO) annotation results of the EIL/EIN3 proteins. GO analysis of EIL/EIN3 proteins tested for their function in biological processes, molecular functions, and cellular components. (B) Protein–protein interaction networks of EIL/EIN3 transcription factors. Each node represents a protein and each edge represents an interaction between two proteins.

Figure 4

Expression patterns of EIL/EIN3 genes in different stages of cotton fiber development and leaf. (A) Hierarchical clustering of expression profiles of the EIL/EIN3 genes in cotton leaf and fiber development at 0, 3, and 8 days post anthesis (DPA) based on RNAseq data. (B) Hierarchical clustering of expression profiles of the EIL/EIN3 genes in cotton fiber development at 0, 3, 5, 8, and 10 DPA using qRT-PCR analysis. The fold change values were log2 transformed. The standardized expression data were used to create heatmap with hierarchical clustering according to the Manhattan correlation with average linkage using the MeV software package. The color scale above the heatmap shows the expression levels: red indicates up-regulated while green indicates down-regulated genes.