2.3 ⎪ Freezing tolerance assays
Freezing tolerance of leaf tissue was determined via electrolyte leakage assays based on those described by Thalhammer, Hincha & Zuther 2014. Leaves (grown under LLW or HLC conditions) with fresh-cut petioles were placed in 300 ml of deionized H2O (petioles submerged) and subjected to subfreezing temperatures using an Arctic A25 refrigerated water bath (Thermo Fisher Scientific, Waltham, MA, USA) and a cooling rate of 4°C h−1. Electrical conductivity was measured using an Exstik II probe (Extech Instruments, Nashua, NH, USA). The data for each replicate were fitted to a four-parameter logistic model, and lethal freezing temperatures (LT50) values were determined as the inflection points from these models. Maximal intrinsic photosystem II efficiency in darkness was assessed in parallel with the electrolyte leakage assays after overnight incubation on ice (4°C) to thaw frozen leaves for measurements of chlorophyll fluorescence with an Imaging-PAM Maxi (Walz, Effeltrich, Germany). Minimal fluorescence levels (Fo) were recorded after a 20-min dark period at room temperature following the freezing treatments, and then maximal fluorescence levels (Fm) were recorded by applying a pulse of saturating light (2500 µmol photons m−2 s−1). Maximal intrinsic photosystem II efficiency was calculated as Fv/Fm = (Fm − Fo)/Fm, and false-colored images of Fv/Fm were generated using ImageJ (Schindelin et al. 2012).
Freezing tolerance of whole plants was determined via survival assays based on previously described protocols (Xin & Browse 1998; Sandersonet al. 2020). Seeds were germinated and transferred to LLW or HLC growth conditions as described above with the exception that seedlings were not transferred to individual pots and were instead thinned to prevent overcrowding. After ten days under LLW or HLC growth conditions, plants with six to eight leaves were transferred to ½ MS-agar plates, chilled to −1°C in the presence of ice chips for 8 h, and frozen overnight (16 h) at an average freezer temperature of −10°C. Plates were then transferred to 4°C for one day, and plant survival was assessed after another two days of recovery in LLW conditions. Surviving plants remained green and erect, whereas non-surviving plants were white and no longer erect.