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

## TL;DR

This study identifies and analyzes the DBB gene family in cotton, revealing their role in helping plants tolerate salt stress.

## Contribution

The study provides the first comprehensive analysis of the DBB gene family in cotton and demonstrates the function of GhDBB22 in salt stress tolerance.

## Key findings

- The DBB gene family in three cotton species comprises 58 members, grouped into three clades with conserved motifs.
- GhDBB22 overexpression in Arabidopsis improves salt tolerance, while its silencing in cotton reduces it and increases oxidative damage.
- GhDBB genes are associated with responses to multiple abiotic stresses, including salt, drought, heat, and cold.

## 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.