Molecular and physiological indicators of heat tolerance in upland cotton ( <i>Gossypium hirsutum</i> L.)

Heat stress is a major abiotic constraint limiting cotton (Gossypium hirsutum L.) productivity under climate change. This study evaluated the integrated molecular and physiological responses of fifteen upland cotton cultivars exposed to acute heat stress (45 °C for 6 h) followed by recovery under controlled phytotron conditions. The relative expression of antioxidant genes encoding catalase (GhCAT), peroxidase (GhPOD), and superoxide dismutase (GhSOD) was quantified using RT–qPCR with UBQ as a reference. The net photosynthetic rate (Pn), transpiration rate (E), stomatal conductance (W), leaf–air temperature difference (ΔT), and vapor pressure deficit (VPD) were assessed. Heat stress significantly altered gene expression and gas exchange traits (ANOVA, p < 0.05). The heat-tolerant cultivars showed strong induction of GhCAT (18.4-fold) and GhPOD (17.1-fold), whereas the susceptible genotypes exhibited limited activation and extreme GhSOD overexpression (up to 133-fold). The tolerant cultivars maintained higher Pn (7–8 µmol m−2 s−1), greater transpirational cooling (ΔT to −7.27 °C), and lower VPD (2.6–3.2 kPa), while sensitive cultivars showed severe photosynthetic decline (<3 µmol m−2 s−1) and elevated VPD (>4.5 kPa). Principal component analysis separated tolerant and sensitive genotypes. Coordinated GhCAT and GhPOD activation with stable gas exchange underpins heat tolerance and supports integrated molecular–physiological screening for breeding programs.