Transcriptomic analysis of response to high-temperature stress in cotton.

High temperature stress (HT) is a major abiotic factor limiting global crop production. Cotton (Gossypium hirsutum L.) is particularly susceptible to heat stress, yet the underlying molecular mechanisms remain poorly understood. Here, we integrated physiological, transcriptomic, and functional analyses to elucidate the response of cotton material ZhongMianSuo087 (ZMS087) to HT. Field-based assays revealed that heat stress compromised cotton productivity by impairing pollen viability, reducing chlorophyll biosynthesis and leaf area, and altering biomass partitioning, leading to significant yield losses. Controlled-environment experiments demonstrated that heat stress disrupted oxidative homeostasis and photosynthetic capacity, as evidenced by increased lipid peroxidation and osmoregulatory accumulation, reduced antioxidant enzyme activities, and decreased chlorophyll content. Transcriptome sequencing identified 5211 differentially expressed genes, with significant activation of the raffinose metabolic process. GhRS2 and GhGOLS1 emerged as key upregulated genes, corresponding with increased raffinose content. Functional validation via virus-induced gene silencing demonstrated that silencing either gene compromised thermotolerance by reducing raffinose accumulation, impairing antioxidant enzyme activities, and exacerbating oxidative damage. Furthermore, exogenous application of raffinose (20-200 mg/L) alleviated heat-induced damage by improving leaf water status, enhancing survival rates, and promoting biomass accumulation. Collectively, This study provides species-specific functional evidence in cotton that the GhRS2/GhGOLS1-mediated raffinose biosynthesis pathway positively regulates thermotolerance, and reveals a mechanistic link between raffinose metabolism and antioxidant responses under heat stress.