Leaf phenotypic traits
Leaf photosynthetic capacity was determined as light- and CO2-saturated oxygen evolution with leaf disc oxygen electrodes (Hansatech Instruments Ltd., Norfolk, UK) as previously described (Delieu & Walker, 1981). The reduction state of the primary electron acceptor of photosystem II, QA, was assessed via measurements of chlorophyll fluorescence using a pulse-amplitude-modulated (PAM) chlorophyll fluorometer (FMS2; Hansatech Instruments Ltd., Norfolk, UK). Leaves were darkened for 20 min, exposed to a far-red light of 0.6 µmol photons m−2s−1 for 5 min, and then subjected to 5-min exposures of increasing light intensities. At the end of each 5-min exposure, steady-state fluorescence (Strand et al., 1999) were recorded, maximum fluorescence levels (Fm´) were obtained by applying a saturating pulse of light (0.8 s of 3000 µmol photons m−2 s−1), and then minimum fluorescence levels (Fo´) were recorded by briefly darkening the leaf. QA reduction state was calculated as 1 − qL = (1/Fs − 1/Fm´)/(1/Fo´ − 1/Fm´). Measurements on LLW plants were conducted in the laboratory at ambient temperature (approximately 22°C), and measurements on HLC plants were conducted inside the growth chamber in which they were grown (with an air temperature of 8°C). Chlorophyll a and b content was determined via high-performance liquid chromatography as previously described (Stewart et al., 2015) or via spectrophotometry as previously described (Arnon, 1949) from leaf discs (0.30 cm2) collected at the end of the 15-h dark period.
Leaf dry mass was measured with an A-160 balance (Denver Instruments Company, Denver, CO, USA) from leaf discs that were dried at 70°C for 7 d. For leaf-thickness measurements, leaves were embedded in 7% (w/v) agarose and sectioned into 80–100 µm thick sections using a 752/M vibroslice tissue cutter (Campden Instruments Limited, Loughborough, England). Sections were stained with 0.02% toluidine blue O for 30 s, and images were taken approximately 150 μm away from the mid-vein (where no major veins or trichomes were present) with an AxioImager (Zeiss, Oberkochen, Germany) coupled with a MicroPublisher color camera (QImaging, Surrey, Canada). Leaf thickness was quantified for 10 representative sections of each plant (i.e., 10 technical replicates for each biological replicate) using ImageJ (Schneider et al., 2012).
Freezing-tolerance assays were performed as previously described (Thalhammer, Hincha, & Zuther, 2014). Leaves in 300 µl of deionized H2O where subjected to subfreezing temperatures using an Arctic A25 refrigerated water bath (Thermo Fisher Scientific, Waltham, MA) and a cooling rate of 4°C h−1. Electrical conductivity was measured using an Exstik II probe (Extech Instruments, Waltham, MA). 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 photosystem II efficiency in darkness was assessed via 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 (1800 µmol photons m−2s−1). Maximal photosystem II efficiency was calculated as Fv/Fm = (Fm − Fo)/Fm, and false-colored images of Fv/Fm were generated using ImageJ (Schneider, Rasband, & Eliceiri, 2012).