Physiological assessment

Arabidopsis thaliana chlorophyll content and stomatal conductance were measured of the 5th youngest leaf when plants reached the stage of 10 true leaves. Chlorophyll content was measured using a Chlorophyll Conductance Meter (Eijkelkamp CCM-300). Three measurements of the same leaf were taken and averaged. Stomatal conductance was measured using a Leaf Porometer (METER SC-1, Decagon Devices, Inc., Pullman, USA) on the same leaf.

For lettuce chlorophyll content, plants were grown in long day conditions (16-h light/8-h darkness) on a gradient set from 5 °C to 27 °C. Chlorophyll content was measured of plants with 6 true leaves. The measurement was taken on the second youngest leaf. Three measurements of the same leaf were taken and averaged.

Col-0 wild type plants were grown on soil as described above on a gradient set from 5 °C to 27 °C in short day conditions (8-h light/16-h dark). Plants were grown in each temperature condition until they were 3 weeks old. To determine ion leakage, the plants were cut at the base of the rosette. The whole rosettes were rinsed in deionized water and transferred to a 15 ml Griner tube (CELLSTAR, Greiner Bio-One) containing 10 ml deionized water. The tubes were shaken at room temperature for 1 h. Thereafter, 100 µl of solution was pipetted onto an Horiba LAQUAtwin-EC-33 conductivity meter to determine the initial conductivity. After measuring, the remaining solution was incubated in a water bath at 95 °C for 30 min. After cooling down to room temperature, 100 µl of the solution was pipetted onto the conductivity meter to determine the final conductivity. Ion leakage described above on a set gradient fromwas calculated as: Initial Conductivity / Final Conductivity * 100%. For each temperature conditions six biological replicates were included.

Col-0 wild type plants were grown on soil as described above on a set gradient from 5 °C to 27 °C in short day conditions (8-h light/ 16-h darkness). Plants were grown in each temperature condition until they were 3 weeks old. The plants were cut at the base of the rosette and placed into 6 well multi-well plates (Greiner Bio-One) and submerged in 1 mg/ml 3,3’-diaminobenzidine (DAB) solution, in 1x phosphate-buffer saline (PBS). The plates were covered by aluminium foil and placed in a vacuum desiccator for 15 min in darkness. After being exposed to a vacuum, the DAB solution was replaced by fresh DAB solution and again placed in a vacuum for 15 min. Thereafter, the plates were transferred to a shaking table and left shaking overnight at room temperature. The next day the DAB solution was removed and replaced by de-staining solution (acetic acid, glycerol, 96% ethanol, 1:1:3) and placed in an oven at 60 °C. After 60 min, the whole rosettes were transferred to a new plate containing 6 ml 90% lactic acid. Thereafter, the plates were scanned using a flatbed scanner. Acquired thermal images were analysed using ImageJ software (https://imagej.nih.gov/ij/). For each temperature six biological replicates were included.