Two class I family teosinte branched1/cycloidea/proliferating cell factor1 (TCP) proteins from allotetraploid cotton are involved in cotton fiber cell differentiation and elongation and root hair development. However, the biological function of most class II TCP proteins is unclear. This study sought to reveal the characteristics and functions of the sea-island cotton class II TCP gene GbTCP4 by biochemical, genetic, and molecular biology methods. GbTCP4 protein localizes to nuclei, binding two types of TCP-binding cis-acting elements, including the one in its promoter. Expression pattern analysis revealed that GbTCP4 is widely expressed in tissues, with the highest level in flowers. GbTCP4 is expressed at different fiber development stages and has high transcription in fibers beginning at 5 days post anthesis (DPA). GbTCP4 overexpression increases primary root hair length and density and leaf and stem trichomes in transgenic Arabidopsis relative to wild-type plants (WT). GbTCP4 binds directly to the CAPRICE (CPC) promoter, increasing CPC transcript levels in roots and reducing them in leaves. Compared with WT plants, lignin content in the stems of transgenic Arabidopsis overexpressing GbTCP4 increased, and AtCAD5 gene transcript levels increased. These results suggest that GbTCP4 regulates trichome formation and root hair development in Arabidopsis and may be a candidate gene for regulating cotton fiber elongation.

The SEVEN IN ABSENTIA (SINA) E3 ubiquitin ligase is widely involved in drought and salt stress in plants. However, the biological function of the SINA proteins in cotton is still unknown. This study aimed to reveal the function of GhSINAT5 through biochemical, genetic and molecular approaches. GhSINAT5 is expressed in several tissues of cotton plants, including roots, stems, leaves and cotyledons, and its expression levels are significantly affected by polyethylene glycol, abscisic acid and sodium chloride. When GhSINAT5 was silenced in cotton plants, drought and salinity stress occurred, and the length, area and volume of the roots significantly decreased. Under drought stress, the levels of proline, superoxide dismutase, peroxidase and catalase in the GhSINAT5-silenced cotton plants were significantly lower than those in the non-silenced control plants, whereas the levels of hydrogen peroxide and malondialdehyde were greater. Moreover, the expression of stress-related genes in silenced plants under drought stress suggested that GhSINAT5 may play a positive role in the plant response to drought and salt stress by regulating these stress response-related genes. These findings not only deepen our understanding of the mechanisms of drought resistance in cotton but also provide potential targets for future improvements in crop stress resistance through genetic engineering.

Cotton is an economically critical crop worldwide, and drought stress strongly affects its growth and development. Ubiquitination modifies protein activity and is crucial in numerous biological processes. Ubiquitin-conjugating enzymes serve as intermediaries in the protein ubiquitination process and play important roles in plant responses to abiotic stress. However, the impact of ubiquitination on the response of cotton to abiotic stress is not fully understood. Bioinformatic methods were employed in this study to analyze the physiochemical characteristics, gene structure, collinearity, expression patterns, and evolutionary relationships of GbUBC gene family members in sea-island cotton. In sea-island cotton, a minimum of 125 GbUBC genes are irregularly distributed across the 26 chromosomes, with multiple instances of gene duplication observed among the members. Phylogenetic analysis categorized the GbUBC gene family into 15 ubiquitin-conjugating enzyme (E2) subgroups, one ubiquitin E2 enzyme variant (UEV) subgroup, and one COP10 subgroup. GbUBC gene expression pattern analyses revealed that most GbUBC genes responded differently to cold, heat, NaCl, and polyethylene glycol (PEG) treatments, with certain GbUBC genes exhibiting high expression levels in specific fiber development period and organs. Furthermore, molecular biology methods were employed to elucidate the biological functions of GbUBC23. The GbUBC23 gene was highly expressed in the cotyledons of sea-island cotton and was activated by PEG treatment. GbUBC23 is localized to the nucleus and cytomembrane. The silencing of the GbUBC23 gene under drought conditions led to decreased drought tolerance and survival rates in sea-island cotton. Compared with those in the control plants, the activity of proline and superoxide dismutase and the expression levels of the drought-induced genes GbNCED3, GbRD22, GbRD26 were significantly lower, but the levels of malondialdehyde and hydrogen peroxide were significantly higher. Our findings revealed 125 members of the GbUBC gene family in sea-island cotton, with the GbUBC23 gene critically contributing to the abiotic stress response. These findings indicate that the GbUBC gene family may play a crucial role in the drought stress response in sea-island cotton.

RNA polymerase II (Pol II) has been shown to participate in various biological processes in plants, but its function in response to abiotic stress in cotton remains unclear. This study aimed to elucidate the role of the third-largest subunit of Pol II (NRPB3) in the response of cotton to drought and salt stress through molecular biology and physiological methods. Real-time fluorescence quantitative PCR was used to analyze the expression pattern of GhNRPB3 in roots, stems, leaves, and cotyledons and to detect changes in its expression under drought, NaCl, and ABA treatments. Using virus-induced gene silencing (VIGS) technology, GhNRPB3-silenced plants were obtained, and their physiological indicators under drought and salt stress, as well as the expression levels of the drought stress-related genes GhRD22 and GhRD26, were measured. This study revealed that GhNRPB3 is widely expressed in roots, stems, leaves, and cotyledons and that its expression is significantly induced by drought, NaCl, and ABA treatments. Compared to wild-type plants, the drought resistance, survival rate, and peroxidase activity of the GhNRPB3-silenced plants significantly increased, whereas the malondialdehyde content significantly decreased. Moreover, the expression levels of the drought-responsive genes GhRD22 and GhRD26 significantly increased. The salt tolerance of the GhNRPB3-silenced plants also increased, as reflected by decreased leaf wilting and significant increases in root growth parameters (including root length, root area, and root volume). These results indicate that GhNRPB3 plays a crucial role in mediating the adaptation of cotton to drought and salt stress by regulating the expression of stress-related genes.