Genome-wide identification of VOZ gene family in ten cotton species and the function analysis of GhVOZ2 involved in heat stress response

Abstract

Background: Vascular plant one zinc-finger protein (VOZ) is a type of special transcription factor and plays important roles in regulating various biological processes in plants. However, a genome-wide analysis of VOZ gene family in cotton is still not clear, which greatly limits the further understanding of VOZ gene function and regulatory mechanism.

Results: We identified 44 VOZ family genes in ten cotton species including 3 in Gossypium herbaceum, G. arboretum, G. thurberi, and G. Raimondii, 6 in G. hirsutum, G. barbadense, G. tomentosum, and G. mustelinum, and 5 in G. darwinii. The phylogenetic tree analysis indicated that VOZ gene family was subdivided into four subgroups. Chromosome location and collinearity analysis revealed that this gene family had experienced segment duplication events. The exon-intron structure and conserved motif analysis showed high conservations in each branch of the cotton VOZ genes. Cis-acting element component analysis displayed that VOZ gene family may be involved in various stresses. Expression profile analysis of GhVOZ genes through both publicly available RNA-seq data from G. hirsutum and quantitative reverse transcription PCR (RT-qPCR) indicated that GhVOZ members are involved in tissue development and biotic and abiotic responses, especially GhVOZ2 showed continuous high expressions in different tissues and stress treatments, with the most significantly induced accumulation under heat stress. Suppressing GhVOZ2 expression in virus-induced silencing (VIGS) cotton lines significantly decreased the tolerance to heat stress. Further RNA-seq analysis of TRV::GhVOZ2 showed that the down-regulated differentially expressed genes (DEGs) were mainly enriched in hormone signal pathways (auxin and BRs), phenylpropanoid and flavonoid biosynthesis, fatty acid elongation, starch and sucrose metabolism pathways, suggesting their potential important roles in heat response mediated by GhVOZ2.

Conclusions: A total of 44 VOZ genes were obtained in ten cotton species, and GhVOZ2 might perform significant positive function in cotton in response to heat stress via regulating the pathways of hormone signal and metabolisms. Our results provide insights into the function and regulatory mechanism of VOZ gene family and the further utilization of excellent cotton material breeding.

Keywords: VOZ gene family; Cotton; Heat stress; RNA-seq; Virus-induced gene silencing.

Fig. 1

Phylogenetic tree of VOZ gene family of ten cotton species, O. sativa, Z. mays, T. cacao, P. tomentosa, V. vinifera, S. bicolor and A. thaliana. Four subfamilies (Clade A-D) were colored in different colors. The neighbor-joining phylogeny tree was created using MEGA11 with 1000 bootstrap. Ghe: G. herbaceum; Ga: G. arboretum; Gth: G. thurberi; Gr: G. raimondii; Gtu: G. turneri; Gh: G. hirsutum; Gb: G. barbadense; Gt: G. tomentosum; Gm: G. mustelinum; Gd: G. darwinii; Os: O. sativa; At: A. thaliana; Zm: Z. mays; Tc: T. cacao; Pt: P. tomentosa; Vv: V. vinifera; Sb: S. bicolor

Fig. 2

Chromosome locations of VOZ genes in ten cotton species. The scale on the left is in mega-bases. The gene ID on the right side of each chromosome corresponds to the location of each VOZ gene. A-J The chromosomes of G. herbaceum, G. arboretum, G. hirsutum, G. barbadense, G. tomentosum, G. mustelinum, G. darwinii, G. thurberi, G. raimondii, G. turneri

Fig. 3

The collinearity analysis of VOZ genes in ten cotton species. A The collinearity between ten cotton species. B-J The homologous gene pairs of G. arboretum, G. hirsutum, G. barbadense, G. tomentosum, G. mustelinum, G. darwinii, G. thurberi, G. raimondii, G. turneri, G. herbaceum

Fig. 4

Predicted cis-elements in the promoter regions of VOZ genes in G. hirsutum. A The distribution of cis-elements. B The number of different cis-elements. C The number of different cis-elements are presented in the form of bar graphs, and the same category of cis-elements is exhibited with the same colors

Fig. 5

Conserved motifs and exon-intron organizations of VOZ genes in ten cotton species. A Neighbor-joining phylogenetic tree of 48 VOZ proteins was generated using the MEGA program. B The conserved protein motifs of VOZ were indicated by different colored boxes. C Exon-intron structure analysis of VOZ genes. D The MSA of GhVOZ proteins, the important domains (Domain A and Domain B) have been marked

Fig. 6

Interaction network analyses. A Protein-protein interaction (PPI) network analysis of GhVOZ proteins interactions. Each circle represents a protein, and the two dots with black lines show that the two proteins interact. B miRNA interaction analyses. The GhVOZ genes are targeted by miRNA via cleavage inhibition. The figure was constructed by using the psRNAtarget webserver and Adobe Illustrator software

Fig. 7

Expression profiles of GhVOZ genes in G. hirsutum. A Expression pattern of GhVOZ genes in different issues. B Expression pattern of GhVOZ genes in ovule and fiber development. C Expression pattern of GhVOZ genes under different abiotic stresses. D Expression pattern of GhVOZ genes under Verticillium dahlia stress. The heatmap was drawn based on RNA-seq data using TBtools. Scale bar represents log₂(FPKM). E. RT-qPCR analysis of GhVOZ genes in different abiotic stresses. The symbols *, **, and ***, represent significant differences of p<0.05, p<0.01, p<0.001. Two-way anova was used to compare the expression differences of GhVOZ gene family between control and treatments

Fig. 8

A The phenotype of positive control TRV::PDS. B RT-qPCR analysis of the change in the expression level of GhVOZ2 gene on the leaf of the cotton plants. The symbols *** represent significant differences of p<0.001. C The phenotypes of silencing of the GhVOZ2 before and heat stress treatment in G. hirsutum. Scale bar = 2 cm

Fig. 9

Transcriptome analysis of silence of GhVOZ2 gene in G. hirsutum. A The number of DEGs. B KEGG enrichment of DEGs. C The heatmap of key genes in auxin signal transduction. D The heatmap of key genes in brassinosteroids signal transduction. E Flavonoid biosynthesis (map00941). F Phenylpropanoid biosynthesis (map00940). G Starch and sucrose metabolism (map00500). H Fatty acid elongation (map00062)

Fig. 10

TFs analysis of DEGs after silence of GhVOZ2 gene in G. hirsutum (A) Up-regulated TFs. B Go enrichment analysis of up-regulated TFs. C KEGG enrichment analysis of up-regulated TFs. D Down-regulated TFs. E Go enrichment analysis of down-regulated TFs. F KEGG enrichment analysis of down-regulated TFs