Genome-Wide Identification and Evolutionary Analysis of Gossypium Tubby-Like Protein (TLP) Gene Family and Expression Analyses During Salt and Drought Stress

Abstract

Tubby-like proteins (TLPs) possess a highly conserved closed β barrel tubby domain at C-terminal and N-terminal F-box. The role of TLP gene family members has been widely discussed in numerous organisms; however, the detailed genome-wide study of this gene family in Gossypium species has not been reported till date. Here, we systematically identified 105 TLP gene family members in cotton (Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense) genomes and classified them into eight phylogenetic groups. Cotton TLP12 gene family members clustered into two groups, 4 and 8. They experienced higher evolutionary pressure in comparison to others, indicating the faster evolution in both diploid as well as in tetraploid cotton. Cotton TLP gene family members expanded mainly due to segmental duplication, while only one pair of tandem duplication was found in cotton TLPs paralogous gene pairs. Subsequent qRT-PCR validation of seven putative key candidate genes of GhTLPs indicated that GhTLP11A and GhTLP12A.1 genes were highly sensitive to salt and drought stress. The co-expression network, pathways, and cis-regulatory elements of GhTLP11A and GhTLP12A.1 genes confirmed their functional importance in salt and drought stress responses. This study proposes the significance of GhTLP11A and GhTLP12A.1 genes in exerting control over salt and drought stress responses in G. hirsutum and also provides a reference for future research, elaborating the biological roles of G. hirsutum TLPs in both stress responses.

Keywords: expression; genome-wide analysis; network; phylogenetic analysis; salt and drought stress responses; transcription factor.

Figure 1

Phylogenetic relationship of Gossypium tubby-like proteins (TLPs) from A. thaliana. The phylogenetic ML tree was built, using the amino acid sequence with 1,000 bootstrap value with MEGA 7.0 software. At, A. thaliana; Ga, Gossypium arboretum; Gr, Gossypium raimondii; Gh, Gossypium hirsutum; Gb, Gossypium barbadense.

Figure 2

Synteny analysis of cotton TLP genes with T. cacao (Malvaceae) and A. thaliana (Brassicaceae) genomes. A syntenic representation of the identified TLP genes between (A) G. arboreum (Chr01 to Chr13) vs. T. cacao (B) G. raimondii (Chr01 to Chr13) vs. T. cacao (C) G. hirsutum (At1 to At13 and Dt1 to Dt13) vs. T. cacao (1–10) (D) G. barbadense (At1 to At13 and Dt1 to Dt13) vs. T. cacao (1–10) (E) G. arboreum (Chr01 to Chr13) vs. A. thaliana (F) G. raimondii (Chr01 to Chr13) vs. A. thaliana (G) G. hirsutum (At1 to At13 and Dt1 to Dt13) vs. A. thaliana (H) G. barbadense (At1 to At13 and Dt1 to Dt13) vs. A. thaliana chromosomes. Red lines indicate duplicated orthologous TLP genes, and cyan and green lines show the paralogous TLPs genes in G. hirsutum, G. raimondii, G. arboreum, T. cacao, and A. thaliana.

Figure 3

Phylogenetic tree, gene structure, and conserved protein motifs analysis of TLPs in G. arboreum, G. raimondii, G. hirsutum, and G. barbadense. (A) Phylogenetic tree of G. arboreum, G. raimondii, G. hirsutum, and G. barbadense TLPs built with the ML (maximum likelihood) method and using 1,000 bootstrap values. Different colors of lines denoted the different species of Gossypium (Blue, G. arboreum; Red, G. raimondii; Black, G. hirsutum; and cyan, G. barbadense). Groups 1–7 distributed in green, yellow, red, royal, violet, orange, and sky blue colors, respectively. (B) Showing the exon-intron organization of TLPs genes in Gossypium (Pink boxes, exon, and black lines, introns). (C) Identified conserved protein motifs in the Gossypium TLPs, and each motif is indicated with a specific color. (D) The logos are given for functionally annotated motifs only, where the heights of logo depict the degree of conservation of amino acid within the motif. The motifs order corresponds to their positions in protein sequence. Motif descriptions are given in Supplementary Table 3.

Figure 4

Chromosomal arrangement and paralogous gene duplication of TLP genes on the four genomes of Gossypium. A physical map of chromosomes demonstrates the position of TLP genes on A, D, and AD genomes, respectively. The paralogous TLP genes (segmental gene duplication) linked with blue lines. The locations of each TLP genes are represented through the horizontal gray lines. The numbers of chromosomes are showed at the top. (A,B) Represent the distribution of TLP genes in G. arboreum and G. raimondii genomes, (C,D) showed the arrangement of TLP genes in G. hirsutum genome, and (E,F) represented the TLP genes in G. barbadense genome, respectively. The scale is in mega bases (Mb). (G) The orthologous TLP duplicated genes pairs among four cotton genomes (G. arboreum, G. raimondii, G. hirsutum, and G. barbadense) were also shown with blue lines, and CIRCOS was used to visualize this plot. The gene name and the chromosome of G. arboreum, G. raimondii, G. hirsutum, and G. barbadense were shown with green, violet, pink, and cyan colors, respectively.

Figure 5

The expression pattern of the TLP gene family members in the G. hirsutum (A–G,I–O) qRT-PCR expression pattern of seven putative genes in normal (0 h), 6, 12, 24, 48, and 72 h of salt (300 MM) and drought (20% PEG solutions PEG8000) stress in cotton. Ubiquitin was used as the loading control. Three replicates were used for each experiment. The statistical analysis was performed, using two-tailed Student's T-tests. The data are plotted as means ± s.d. The error bars represent standard deviations. The asterisks indicate significant differences: *P < 0 0.1; **P < 0 0.01; ***P < 0 0.001; ****P < 0.0001. (H,P) Gene expression profiles of TLP genes under salt and drought condition in RNA-Seq, using Log2 (fold change) values.

Figure 6

cis-regulatory elements analysis of G. hirsutum TLP genes. The micro-parts in diverse colors are the putative elements sequence.

Figure 7

The homology 3D model of the GhTLPs tubby domain. The GhTLPs were selected for three-dimensional structure prediction and display with confidence level >99%. The alpha helix is shown with red and beta fold is shown with violet color.