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.