Genome-Wide Identification of the Double B-Box (DBB) Family in Three Cotton Species and Functional Analysis of GhDBB22 Under Salt Stress

Abstract

Salt stress causes harm to plants through multiple aspects, such as osmotic pressure, ion poisoning, nutrient imbalance, and oxidative damage. Zinc finger proteins harboring two B-box domains, known as double B-box (DBB) proteins, constitute the DBB family. While DBB genes have been implicated in regulating circadian rhythms and stress responses in various plant species, their functions in cotton remain largely unexplored. The present study characterized the DBB gene family across the genomes of Gossypium hirsutum L., Gossypium raimondii L., and Gossypium arboreum L., revealing a complement of 58 members. These DBB genes were assigned to three separate clades based on phylogenetic analysis. Members possessing close phylogenetic relationships have similar conserved protein motifs and gene structures. All DBB proteins were predicted to be nuclear-localized, consistent with their roles as transcription factors. Furthermore, the presence of multiple cis-acting elements related to light, hormone, and stress responses in the promoters implies that GhDBBs are integral to cotton's environmental stress adaptation. Expression pattern analysis indicated that the expression of GhDBB genes was associated with the plant's response to multiple abiotic stresses, such as salt, drought, heat (37 °C), and cold (4 °C). The reliability of the expression data was confirmed by qPCR analysis of eight selected GhDBBs. Under 200 mM NaCl, Arabidopsis plants overexpressing GhDBB22 displayed longer roots and healthier true leaves than the wild-type controls. Conversely, VIGS-mediated silencing of GhDBB22 in G. hirsutum led to significantly reduced salt tolerance, accompanied by exacerbated oxidative damage. Taken together, the findings from our integrated genomic and functional analyses provide a foundational understanding of the molecular mechanisms through which proteins encoded by DBB genes are involved in the plant's response to salt stress.

Keywords: DBBs; Gossypium; gene family; salt tolerance.

Figure 1

Physical location of DBBs on chromosomes of Gossypium hirsutum, Gossypium arboreum, and Gossypium raimondii. The distribution of color blocks on the chromosome represents gene density.

Figure 2

Phylogenetic trees of DBB amino acids in A. thaliana, G. hirsutum, G. arboreum, and G. raimondii. The four species are marked with graphics of different colors.

Figure 3

The gene structure and conserved motifs of DBBs in G. hirsutum, G. arboreum, and G. raimondii. (A) Evolutionary trees were constructed using DBB protein sequences from three cotton species. Groups I, II, and III are highlighted in purple, green, and yellow. (B) The motifs distribution of DBBs. (C) The genetic structure distribution of DBBs. The reference genome annotations for G. arboreum and G. hirsutum lacked UTR information.

Figure 4

Collinearity analysis of DBBs in G. hirsutum, G. arboreum, and G. raimondii. (A) Intraspecific collinearity of GhDBBs. Homologous GhDBB gene pairs are linked by red lines in the synteny plot. The outer circle shows the gene density, with red and blue corresponding to high and low gene abundance, respectively. (B) Interspecific collinearity of DBB genes among three cottons. Red lines denote collinear pairs between GhDBBs and GaDBBs; orange lines denote collinear pairs between GhDBBs and GrDBBs; blue lines denote collinear pairs between GaDBBs and GrDBBs.

Figure 5

Distribution of cis-acting elements in G. hirsutum, G. arboreum, and G. raimondii promoters. The heatmap depicts elements associated with stress response, hormone response, and growth and development. Color intensity in each cell corresponds to the relative abundance of each element, with red denotes a higher abundance. The number in each color block indicates the specific quantity of the component.

Figure 6

Expression profiles of GhDBBs in response to various stresses and across tissues. Expression patterns of GhDBBs under (A) 4 °C, (B) 37 °C, (C) 20% PEG6000 stress, and (D) 400 mM NaCl stress. (E) Transcriptional abundance of GhDBBs in different tissues. (F) Expression profiles of eight GhDBBs under 400 mM NaCl treatment. The sampling times were 0, 1, 3, 6, 12 and 24 h after processing. The results are the average of three repetitions. Vertical bars represent the standard deviation (±SD) from three biological replicates, and significant differences (p < 0.05, Student’s t-test) are denoted by different letters above the bars.

Figure 7

Heterologous expression of GhDBB22 confers enhanced salt stress tolerance in Arabidopsis. (A) Phenotypes of WT and OE plants grown for 20 days under 0, 100, and 150 mM NaCl. (B) Phenotypes of Arabidopsis after 7 days of 200 mM NaCl stress. (C) Root length of Arabidopsis under stress for 20 days. (D) Chlorophyll content, (E) relative electrical conductivity, (F) SOD activity, (G) POD activity, and (H) MDA content in Arabidopsis leaves after 7 days of 200 mM NaCl treatment. Significant differences are indicated by * (p < 0.05) and ** (p < 0.01).

Figure 8

Silencing of GhDBB22 compromises salt tolerance in cotton. (A) Cotton plant phenotypes after 14 days of 300 mmol/L NaCl immersion. (B) GhDBB22 gene silencing efficiency and the albino phenotype of the plants infected with TRV2:GhCLA1. Relative electrical conductivity (C), total chlorophyll content (D), SOD activity (E), POD activity (F) and MDA content (G) of leaves were changed after salt treatment. Data are presented as the mean ± SD of three biological replicates. * (p < 0.05) and ** (p < 0.01) showed significant differences.