The hexokinase Gene Family in Cotton: Genome-Wide Characterization and Bioinformatics Analysis

Abstract

Hexokinase (HXK) is involved in hexose phosphorylation, sugar sensing, and signal transduction, all of which regulate plant growth and adaptation to stresses. Gossypium hirsutum L. is one of the most important fiber crops in the world, however, little is known about the HXKs gene family in G. hirsutum L. We identified 17 GhHXKs from the allotetraploid G. hirsutum L. genome (AADD). G. raimondii (DD) and G. arboreum (AA) are the diploid progenitors of G. hirsutum L. and contributed equally to the At_genome and Dt_genome GhHXKs genes. The chromosomal locations and exon-intron structures of GhHXK genes among cotton species are conservative. Phylogenetic analysis grouped the HXK proteins into four and three groups based on whether they were monocotyledons and dicotyledons, respectively. Duplication event analysis demonstrated that HXKs in G. hirsutum L. primarily originated from segmental duplication, which prior to diploid hybridization. Experiments of qRT-PCR, transcriptome and promoter cis-elements demonstrated that GhHXKs' promoters have auxin and GA responsive elements that are highly expressed in the fiber initiation and elongation stages, while the promoters contain ABA-, MeJA-, and SA-responsive elements that are highly expressed during the synthesis of the secondary cell wall. We performed a comprehensive analysis of the GhHXK gene family is a vital fiber crop, which lays the foundation for future studies assessing its role in fiber development.

Keywords: Gossypium hirsutum; expression pattern; hexokinase; sequence analysis; the evolutionary.

FIGURE 1

Chromosomal distribution of GhHXKs. The chromosome number is shown above each chromosome. The chromosomal location of each GhHXKs is shown from the top to the bottom of the corresponding chromosome. The scale bars beside the chromosome indicate the length of megabases (Mb). The arrows show the transcription directions of GhHXK genes.

FIGURE 2

Chromosome distribution diagram of HXK genes in G. hirsutum L., G. arboreum, and G. raimondii. Colorful triangles represent the HXK genes and their transcription direction.

FIGURE 3

The phylogenetic tree (A) and gene structure (B) of GhHXKs. The phylogenetic tree was constructed with the CDS sequences of GhHXKs by MEGA 7.0 software with default parameters. The exons, UTR, and introns are indicated by blue ovals, red rectangles, and black lines, respectively.

FIGURE 4

Phylogenetic analysis of GhHXK proteins in G. hirsutum L. An unrooted phylogenetic tree was constructed using HXK protein sequences from A. thaliana (AtHXK), O. sativa (OsHXK), P. edulis (PeHXK), M. esculenta (MeHXK), B. napus (BnHXK), G. hirsutum L. (GhHXK), G. raimondii (GrHXK), and G. arboreum (GaHXK), and are displayed in purple, green, yellow, red, light blue, dark blue, dark red, and orange, respectively.

FIGURE 5

Protein features of HXKs in G. hirsutum L. (A) The weblogo of GhHXK protein glucose-binding sites. (B) The conserved adenosine binding sites of GhHXKs. (C) The motifs of HXK sequences from G. hirsutum L.

FIGURE 6

Circos plot showing GhHXKs paralogous gene pairs and orthologous gene pairs. Black lines connect the paralogous GhHXKs gene pairs. Red lines connect the paralogous gene pairs between GhHXKs and GrHXKs. Blue lines connect the paralogous gene pairs between GhHXKs and GaHXKs.

FIGURE 7

Cis-elements in the promoters of GhHXK genes. Numbers in the box are the number of cis-elements.

FIGURE 8

RNA-seq analysis of GhHXKs genes during fiber development at 0, 5, 10, 15, 20, 25, 30 and 35 DPA (A), and plant hormone-related cis-elements in the GhHXK promoter regions (B). The transcriptome data were normalized by fragments per kilobase of transcript per million mapped reads (FPKM) and visualized using the pheatmap software (https://www.omicshare.com/tools/Home/Soft/heatmap). The colorful bars from green to red indicate the expression levels from low to high, respectively.

FIGURE 9

The expression levels of GhHXKs during fiber developmental stages (0, 5, 10, 15, 20, and 25 DPA) analyzed by qRT-PCR. Error bars represent means ± SE from three independent biological repetition. The relative expression level was calculated by using GhUBQ7 as the internal control.

FIGURE 10

GhHXKs expression levels are induced by GA (A) and auxin (B) analyzed by qRT-PCR. GA treatment induced expression of most GhHXKs with GARE-motif, P-box or TATC-box in their promoter regions (A). The relative expression levels of GhHXKs with AuxRR-core or TGA-element in their promoter regions (B). The error bars represent means ± SE from three independent biological repetition. The y-axis represents the relative expression level. The x-axis represents the 0 DPA ovules treated with GA or IAA for 0, 12, 24, and 36 h, respectively.

FIGURE 11

A hypothesis about the roles of GhHXKs in cotton fiber development. (A) During fiber developmental stages, from initiation to elongation, the sucrose content decreased, and the fructose and glucose content increased. (B) The phosphorylated hexose functions in energy supply and signal transduction in fiber development; Meanwhile, intracellular hexose levels are regulated by IAA, GA, and ABA, and GhHXK is the center of this pathway. The blue, red and green dots represent sucrose, glucose and fructose, respectively. The arrows and blunt arrows indicate positive and negative regulation of the specific processes, respectively.