Evolutionary Relationships and Divergence of KNOTTED1-Like Family Genes Involved in Salt Tolerance and Development in Cotton (Gossypium hirsutum L.)

Abstract

The KNOX (KNOTTED1-like homeobox) transcription factors play an important role in leaf, shoot apical meristem and seed development and respond to biotic and abiotic stresses. In this study, we analyzed the diversity and evolutionary history of the KNOX gene family in the genome of tetraploid cotton (Gossypium hirsutum). Forty-four putative KNOX genes were identified. All KNOX genes from seven higher plant species were classified into KNOXI, KNOXII, and KNATM clades based on a phylogenetic analysis. Chromosomal localization and collinearity analysis suggested that whole-genome duplication and a polyploidization event contributed to the expansion of the cotton KNOX gene family. Analyses of expression profiles revealed that the GhKNOX genes likely responded to diverse stresses and were involved in cotton growth developmental processes. Silencing of GhKNOX2 enhanced the salt tolerance of cotton seedlings, whereas silencing of GhKNOX10 and GhKNOX14 reduced seedling tolerance to salt stress. Silencing of GhSTM3 influenced the cotton flowering time and plant development. These findings clarify the evolution of the cotton KNOX gene family and provide a foundation for future functional studies of KNOX proteins in cotton growth and development and response to abiotic stresses.

Keywords: KNOX; artificial selection; cotton; development; evolutionary; stress response.

FIGURE 1

Phylogenetic relationship of KNOX family proteins of seven species. The phylogenetic tree was constructed from KNOX amino acid sequences using the neighbor-joining method with 1000 bootstrap replicates. The inner circle is marked in purple, orange, and green representing the KNOXI, KNOXII, and KNATM clades, respectively.

FIGURE 2

Phylogenetic relationships and genomic structure of GhKNOX genes. (A) Neighbor-joining tree of GhKNOXs. The GhKNOX genes were classified into four subclades (I, II, III, and IV). The subclades I and II were clustered in the KNOXI clade. The subclades III and IV belonged to the KNOXII and KNATM clades, respectively. (B) Exon–intron structural features of GhKNOX genes. Black boxes and lines indicate exons and introns, respectively.

FIGURE 3

Physical locations of KNOX genes on G. hirsutum chromosomes. The upper and lower panels represent A subgenome chromosomes and D subgenome chromosomes, respectively. The scale is provided in megabases.

FIGURE 4

Collinearity analysis of KNOX genes on G. hirsutum chromosomes. Green lines link two homoeologous genes located in the A and D subgenome chromosomes. Red and blue lines link the two homologs formed by segmental duplication within the D subgenome and A subgenome, respectively. A01–A13 indicate the chromosomes in the A subgenome and D01–D13 indicate the chromosomes in the D subgenome.

FIGURE 5

Expression patterns of GhKNOX genes in different tissues. The expression patterns were analyzed using hierarchical clustering. The FPKM values were calculated from RNA-seq data and are shown as a heatmap. The colored scale indicates the relative expression level.

FIGURE 6

Expression profiles of GhKNOX genes in response to four abiotic stress treatments. The ratios of FPKM between the treatments (at 1, 3, 6, 12, and 24 h) and the control (at 0 h) were calculated from RNA-seq data and are shown as a heat map. The colored scale indicates the relative expression level. An asterisk indicates that expression of these genes requires verification by qRT-PCR.

FIGURE 7

Expression patterns of selected GhKNOX genes in response to PEG or NaCl stress treatment. (A) Relative expression levels of GhKNOX genes between the control (0 h) and different time points (1, 3, 6, 12, and 24 h) under 20% PEG treatments. (B) Relative expression levels of GhKNOX genes between the control (0 h) and different time points (1, 3, 6, 12, and 24 h) under 200 mM NaCl treatment. GhACTIN (AY305733) was used as an internal control. Error bars indicate the standard deviation of three independent experiments. Relative expression was calculated using the 2^–△△Ct method. The significance of differences between means was determined using analysis of variance (*P < 0.05, **P < 0.01).

FIGURE 8

VIGS analysis of five G. hirsutum KNOX genes. (A–E) The left-hand plant is the control cotton transformed with the empty vector. In the right-hand plants, GhKNOX2-A, GhKNOX10-A, GhKNOX14-A, GhSTM2-A, and GhSTM3-A/D were silenced, respectively. (F) The control plant and GhSTM3-silenced plant of ‘CCRI50’ from left to right. (G) Malondialdehyde (MDA) content in TRV:00 and plants silenced for five G. hirsutum KNOX genes. (H) Peroxidase (POD) activity in TRV:00 and plants silenced for five G. hirsutum KNOX genes. (I) Relative expression levels of GhSOC1, GhFT, GhLFY, and GhAP1. The significance of differences was determined using Student’s t-test (*P < 0.05, **P < 0.01).

FIGURE 9

Genetic variation in G. hirsutum KNOX genes. (A) Density of SNPs in 19 GhKNOX genes. (B) The F_st values of the SNP loci in 19 GhKNOX genes. A F_st value for a SNP locus higher than 0.45 was considered to indicate putative sites under selection during domestication.