Comprehensive analyses of ZFP gene family and characterization of expression profiles during plant hormone response in cotton

Abstract

Background: Zinc finger proteins (ZFPs) containing only a single zinc finger domain play important roles in the regulation of plant growth and development, as well as in biotic and abiotic stress responses. To date, the evolutionary history and functions of the ZFP gene family have not been identified in cotton.

Results: In this paper, we identified 29 ZFP genes in Gossypium hirsutum. This gene family was divided into seven subfamilies, 22 of which were distributed over 17 chromosomes. Bioinformatic analysis revealed that 20 GhZFP genes originated from whole genome duplications and two originated from dispersed duplication events, indicating that whole genome duplication is the main force in the expansion of the GhZFP gene family. Most GhZFP8 subfamily genes, except for GhZFP8-3, were highly expressed during fiber cell growth, and were induced by brassinosteroids in vitro. Furthermore, we found that a large number of GhZFP genes contained gibberellic acid responsive elements, auxin responsive elements, and E-box elements in their promoter regions. Exogenous application of these hormones significantly stimulated the expression of these genes.

Conclusions: Our findings reveal that GhZFP8 genes are involved in cotton fiber development and widely induced by auxin, gibberellin and BR, which provides a foundation for the identification of more downstream genes with potential roles in phytohormone stimuli, and a basis for breeding better cotton varieties in the future.

Keywords: Cotton; Expression patterns; Fiber development; Plant hormone; Zinc finger proteins.

Fig. 1

Phylogenetic analysis of the ZFP gene family. The phylogenetic tree was constructed using the full length ZFP protein amino acid sequences from G. arboreum, G. raimondii, G. hirsutum and A. thaliana. MEGA 6.0 software was used with the neighbor-joining method and bootstrapping with 1,000 iterations. At, Arabidopsis thaliana; Ga, Gossypium arboreum; Gr, Gossypium raimondii; Gh, Gossypium hirsutum

Fig. 2

Chromosomal location of GhZFP genes on 17 chromosomes. The genome visualization tool CIRCOS was used to display the chromosomal location of GhZFP genes. The chromosome number is shown on the top of each chromosome. The scale bar indicates the length in megabases (Mb)

Fig. 3

Phylogenetic relationships, gene structures and protein domain architecture of GhZFP genes. a Phylogenetic relationships between GhZFPs. A phylogenetic tree was generated using the maximum likelihood method with 1,000 bootstrap iterations in MEGA 6.0 software. b Gene structure (exon-intron organization) analysis of GhZFPs. Exons and introns are represented by purple boxes and black lines, respectively. The scale bar is shown at the bottom. c The protein domain architecture of GhZFP genes. The number and order of motifs in each GhZFP genes are shown. Motif 2 in (c) is the ZFP domain

Fig. 4

Expression profiles of GhZFP genes during fiber development. a RNA-seq data heat map of GhZFP gene expression levels during different stages of fiber growth. The differences in gene expression are shown in different colors. b Cis-element analysis of GhZFP8 promoters. E-box (CANNTG) motifs (red) were predicted using the online toolkit, PLACE. A total of 2,000 bp promoter sequence for each GhZFP8 were used for the analysis. c Exogenous BR promotes the transcription of GhZFP8. Statistical significance was determined using one-way analysis of variance combined with Tukey’s test. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 5

Expression profiles of 19 GhZFP genes under GA₃ treatment. Analysis of 19 GhZFP genes with GARE elements present in their promoter regions (left). GAREs (green) were predicted using the PLACE website (http://www.dna.affrc.go.jp/htdocs/PLACE/). Fold change of 19 GhZFP genes under GA₃ treatment (right). All treatments were performed with three biological and three technical replicates. The relative gene expression levels were determined using cotton GhUBQ7 as a control

Fig. 6

Expression profiles of 10 GhZFP genes under NAA treatment. a Analysis of 10 GhZFP with AuxRE elements in their promoter regions. AuxREs (blue) were predicted using the PLACE website. b NAA activated transcription of most GhZFP genes with AuxRE elements in their promoter regions. Relative expression levels of each gene were determined after normalizing to the expression level in CK (no chemical added) ovules, which was set to 1.0

Fig. 7

Heat map of expression levels of GhZFP genes with the BES1 element in their promoter regions under BR treatment. Differences in gene expression are shown in different colors indicated in the scale. All treatments were performed with three biological and three technical replicates