Heat tolerance assay

Following the thermal tolerance measurement protocols adapted from G. Heinrich Krause et al. (2010), heat treatment assay was conducted on fully mature healthy leaves were transferred into a water trough in which leaf discs of ∅ 20.5 mm were cut underwater using a cork borer, consistently choosing the central part of the lamina, and avoiding the midrib area. One disc was cut from a single leaf except for a small number of cases where leaf material for analysis was limited when two discs per leaf were excised. Leaf discs were then randomly selected and wrapped in two layers of moist tissue paper on both sides covering the cut edge of the leaf discs. Care was taken not to touch the leaves, and the leaf discs always remained in water or covered in damp tissue paper to avoid anaerobiosis (Harris & Heber, 1993). The discs were transferred into separate plastic bags (30 × 100 mm) keeping them flat and covered in a thin film of water. The bags were subjected to heat treatment in sets of at least three discs per temperature point. Heat treatment was conducted in temperature-controlled thermos flasks with pre-heated water. The water temperature was recorded in the central area of the flask using an HI 9063 K-type thermocouple probe. Separate sets of leaf discs were treated to one of the following temperatures points: 30, 35, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, and 70 °C and a set of untreated discs were used as controls (about 25°C). Hence, one assay run, in most cases, includes measurements from at least 45 leaves. After the heat treatment, the leaf discs were dark adapted for at least 30 minutes before measuring Fv/Fm with a FluorPen FP100 (Photon System Instruments, Czech Republic). FP100 uses a 455nm light source at 1 kHz modulation frequency to irradiate a ∅ 5mm surface aperture. F 0 is read after 40 µs exposure to a 0.09 µ mol.m-2s-1 light per pulse and F mafter a 1s exposure to 3,000 µ mol.m-2s-1 light pulse. The response was measured on a PIN photodiode with a 667 to 750 nm bandpass filter for 10 µs. The leaf discs were incubated in lab conditions (25°C, ~ 20 µmol.m-2.s-1 light) in Petri plates with a thin film of water.F v/F m recovery after 24h was measured following a 30-minute period of dark adaptation.