Combined Day-Night Heat Damage in Soybean Is Driven by Nighttime Warming

Asymmetric warming, characterized by faster increases in nighttime than daytime temperatures, is an emerging threat to crop performance. However, the interactive physiological effects of day versus night heat remain insufficiently resolved in soybean (Glycine max L. Merr.). Here, we evaluated soybean responses to 4 day/night thermal regimes (28°C/22°C, 40°C/22°C, 28°C/28°C, and 40°C/28°C) to test whether nighttime warming is the dominant driver of stress disruption. Isolated daytime heat (40°C/22°C) enhanced photosynthetic performance and maintained biomass, indicating short-term functional plasticity. In contrast, treatments including elevated nighttime temperature impaired pre-dawn rehydration, reduced leaf water status, and intensified membrane damage, with the strongest effects under combined heat (40°C/28°C). Combined stress also caused severe stomatal limitation of CO₂ assimilation, depletion of soluble sugars, accumulation of proline and glycine betaine, and strong activation of antioxidant enzymes accompanied by high H₂O₂ and TBARS levels, indicating that oxidative load exceeded detoxification capacity. Multivariate integration reinforced this pattern by clearly separating combined heat from all other treatments and placing nighttime warming as the central axis of physiological disruption. Together, our findings demonstrate that restricted nocturnal recovery is a key mechanism underlying synergistic damage under sustained day-night heat, highlighting nighttime metabolism and overnight hydraulic recovery as priority targets for improving soybean heat resilience.