When it gets too hot: Chronology of critical heat thresholds stomatal response, PS II dysfunction, tissue heat damage, molecular denaturation and increased cuticle conductance

• Molecular denaturation via DSC primarily occurs at temperatures above heat damage. • Stomata responded to heat 10K below initial leaf damage, enabling leaf cooling. • Photosystem II is confirmed as early indicator of heat stress just before damage. • A significant increase of cuticle permeability occurred only 10K above heat damage. • The sequence of heat stress symptoms observed was species-specific. Climate change is intensifying the frequency and severity of heat waves, posing significant challenges to plant survival, particularly in alpine ecosystems. Elevated leaf temperatures combined with reduced water availability disrupt physiological processes, but the chronology of these events remains unclear. This study investigates the sequence of critical heat thresholds in three alpine plant species from contrasting habitats — Kalmia procumbens, Rhododendron ferrugineum, and Ranunculus glacialis . We assessed stomatal responses (g s ), increased cuticular water permeability (g min , via gas exchange), molecular aggregation and denaturation (via differential scanning calorimetry, DSC), PSII thermotolerance (chlorophyll fluorescence), and tissue heat damage (LT 50 ). Among the investigated species, the earliest detectable physiological response of heat stress was stomatal opening at around 31.9°C, which was followed by functional disturbances of PSII (T c ). With further increase of temperature heat damage progressively developed. Strikingly, an increase of cuticle permeability (g min ) and bulk molecular denaturation via DSC only occurred at significantly higher temperatures than those at which heat damage occurred. Critical heat thresholds were species-specific: R. glacialis showed the lowest heat thresholds, followed by R. ferrugineum and K. procumbens , which corresponds to increasing heat loads in their natural habitat. This study emphasizes the importance of understanding heat stress responses in alpine plants, which face disproportionate impacts from climate change. The findings offer key insights into alpine species’ adaptive strategies and suggest that stomatal and molecular heat thresholds may serve as indicators of heat resilience. These insights are critical for improving models of plant responses to global warming, particularly in vulnerable high-altitude ecosystems.