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).