Cotton (Gossypium hirsutum L.), the most important textile crop worldwide, often encounters abiotic stress during its growing season and its productivity is significantly limited by adverse factors. Trihelix transcription factors (also known as GT factors) are important proteins involved in the morphological development and responses to abiotic stress in plants. However, their functions and molecular mechanisms in the cotton toward abiotic stress response remain unclear. In this study, a member (GhGT26) of the cotton Trihelix family was functionally characterized in the model plant Arabidopsis. This protein containing a SANT domain belongs to the GT-1 subgroup of trihelix proteins. GhGT26 was widely expressed in tissues (with the highest level in flower) and responded to high salt and ABA treatments at the transcriptional level. Using the Arabidopsis protoplast assay system, we found that the GhGT26 protein was located in the cell nuclei. The EMSA assay revealed that the GhGT26 protein could bind to the Site1-type GT cis elements (GT-3a) and MYB elements MRE3 and MRE4. The overexpression of GhGT26 improved plant tolerance to salt stress in transgenic Arabidopsis plants. Although ABA inhibits root elongation, the statistical analysis revealed that the root lengths of GhGT26-overexpressing Arabidopsis were the same as the wild plants after ABA treatment. Our results demonstrate that GhGT26 positively regulates salt stress via ABA-independent pathways. This evidence suggests that the GhGT26 may participate in the regulation of stress tolerance in cotton.

As a plant-specific Rho-like small G protein, the ROP (Rho-related GTPase of plants) protein regulates the growth and development of plants and various stress responses in the form of molecular switches. Drought is a major abiotic stress that limits cotton yield and fiber quality. In this study, virus-induced gene silencing (VIGS) technology was used to analyze the biological function of GhROP3 in cotton drought stress tolerance. Meanwhile, we used yeast two-hybrid and bimolecular fluorescence complementation assays to examine the interaction between GhROP3 and GhGGB. GhROP3 has a high expression level in cotton true leaves and roots, and responds to drought, high salt, cold, heat stress, and exogenous abscisic acid (ABA) and auxin (IAA) treatments. Silencing GhROP3 improved the drought tolerance of cotton. The water loss rates (WLR) of detached leaves significantly reduced in silenced plants. Also, the relative water content (RWC) and total contents of chlorophyll (Chl) and proline (Pro) of leaves after drought stress and the activities of three antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) significantly increased, whereas the contents of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) significantly reduced. In the leaves of silenced plants, the expression of genes related to ABA synthesis and its related pathway was significantly upregulated, and the expression of decomposition-related GhCYP707A gene and genes related to IAA synthesis and its related pathways was significantly downregulated. It indicated that GhROP3 was a negative regulator of cotton response to drought by participating in the negative regulation of the ABA signaling pathway and the positive regulation of the IAA signaling pathway. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that the GhROP3 protein interacted with the GhGGB protein in vivo and in vitro. This study provided a theoretical basis for the in-depth investigation of the drought resistance–related molecular mechanism of the GhROP3 gene and the biological function of the GhGGB gene.