Leaf Excision and Exposure Duration Alter the Estimates of the Irreversible Photosynthetic Thermal Thresholds

Understanding plant heat tolerance requires assessing their thermal thresholds, but commonly used methods have rarely been compared. Moreover, whether the photosynthetic machinery is irreversibly damaged past these thresholds remains unclear. We determined the critical temperature (T_crit), the temperature causing a 50% reduction (T₅₀), and the maximum tolerable temperature (T_max) of photosystem II in Mediterranean cypress, Aleppo pine, and Scots pine saplings using 15- or 30-min heat exposure curves performed on living plants (in-vivo), excised needles (ex-vivo), and excised needles continuously exposed to each rising temperature (ex-vivo continuous). Dark-adapted fluorescence (F_v/F_m) and gas exchange were recorded for 4 days postheat stress to track recovery. Longer heat exposure (30 vs. 15 min) consistently led to lower F_v/F_m, T₅₀, and T_max. T₅₀ and T_max were reduced in both ex-vivo conditions compared to in-vivo ones. Conversely, T_crit remained consistent between species, exposure durations, and methods. Gas exchange and F_v/F_m recovery mainly occurred before reaching T₅₀ values (about 45°C). Our work highlights the importance of exposure duration and method selection when measuring and comparing thermal thresholds. Moreover, while T_crit appears to be a reversible threshold, the photosynthetic machinery of studied species appears irreparably damaged past their T50.