The cotton 70-kDa heat shock protein GhHSP70-26 plays a positive role in the drought stress response

• The cotton cytoplasmic subgroup gene GhHSP70-26 is transcribed in response to polyethylene glycol and abscisic acid treatment and salt, cold, and heat stress. • Overexpression of the GhHSP70-26 gene in tobacco and silencing of the GhHSP70-26 gene in cotton showed that the GhHSP70-26 gene can improve plant tolerance to drought stress. • GhbZIP43 and GhHSF8 bind to cis -acting ABREs and HSEs, respectively, to regulate the transcription of GhHSP70-26 , and the GhHSP70-26 protein interacts with the GhAPX2 protein. The 70-kDa heat shock protein (HSP70) is a molecular chaperone that plays an important role in the response of plants to abiotic stress, but the function and molecular mechanism of HSP70 in the cotton drought stress response are unclear. In this study, the HSP70-encoding gene GhHSP70-26 , which belongs to the cytoplasmic HSP70 subgroup, was cloned from upland cotton ( Gossypium hirsutum L.). The transcript level of GhHSP70-26 in cotton leaves was higher than that in cotton stems and roots. GhHSP70-26 was found to respond at the transcriptional level to polyethylene glycol (PEG) and abscisic acid (ABA) treatment and to cold, salt, and heat stress, and its transcript level was positively correlated with the drought resistance of different cotton varieties. Heterologous overexpression of the GhHSP70-26 gene improved the drought resistance of transgenic tobacco compared with that of wild-type (WT) tobacco, and the transgenic tobacco plants were characterized by low leaf wilting, high survival, low leaf water loss, increased root length and high chlorophyll content. Moreover, the transgenic tobacco plants overexpressing GhHSP70-26 had higher proline contents, higher superoxide dismutase (SOD) and peroxidase (POD) enzyme activities, and lower malondialdehyde (MDA) and reactive oxygen species (ROS) contents than the WT tobacco plants. The transcript levels of the stress response-related genes NtLEA , NtERD10D , NtPOD , NtSOD , and NtNCED3-1 in transgenic tobacco overexpressing GhHSP70-26 were significantly higher than those in WT tobacco. In contrast, under drought stress, unlike the results obtained with the pTRV2 empty vector (EV)-transformed and WT cotton plants, the use of virus-induced gene silencing (VIGS) technology to silence the GhHSP70-26 gene in cotton resulted in plants with severely wilted leaves, an increased water loss rate, a higher MDA content, and higher relative electrical conductivity (REC). Furthermore, yeast one-hybrid assays showed that GhbZIP43 and GhHSF8 bind to ABA-responsive elements (ABREs) and heat shock elements (HSEs) in the GhHSP70-26 promoter, respectively. Moreover, yeast two-hybrid, bimolecular fluorescence complementation and luciferase complementation assays showed that the GhHSP70-26 protein interacts with the ascorbate peroxidase (APX) GhAPX2 protein. These results indicate that by reducing the degrees of cell membrane damage and cellular damage caused by ROS stress, the GhHSP70-26 protein plays a positive role in the response of plants to drought stress.