2.1 ⎪ Plant material and growth conditions
Arabidopsis thaliana ecotypes IT (Castelnuovo-12 [ABRC stock
number: CS98761], sub-line 24) and SW (Rodasen-47 [ABRC stock
number: CS98762], sub-line 29) were grown from seed in Conviron E15
(Controlled Environments Ltd., Manitoba, Canada) and then in E36-HID
(Percival Scientific, Perry, IA, USA) growth chambers alongside the
corresponding CBF1–3-deficient lines it:cbf123 and
sw:cbf123 that had been generated by Park, Gilmour, Grumet, &
Thomashow (2018) via CRISPR/Cas9 (for more information on the ecotypes,
see Ågren & Schemske 2012; Adams et al. 2016). For selected
experiments, sw:cbf2 , a CBF2-deficient line created in SW by the
same group (Park, Gilmour, Grumet, & Thomashow 2018), was included as
well. CBF1–3 genotypes of it:cbf123 , sw:cbf123 ,
and sw:cbf2 plants used in this study were confirmed by Sanger
sequencing.
The following four growth conditions—based on a factorial design of
contrasting light intensities and leaf temperatures—were employed: low
light, warm temperature (LLW; 9-h photoperiod of 100 µmol photons
m−2 s−1 and leaf temperature
25°C/20°C [light/dark]), low light, cool temperature (LLC; 9-h
photoperiod of 100 µmol photons m−2s−1 and leaf temperature maximum of 16°C/12.5°C
[light/dark]), high light, warm temperature (HLW; 9-h photoperiod of
1000 µmol photons m−2 s−1 and leaf
temperature 25°C/20°C [light/dark]), and high light, cool
temperature (HLC; 9-h photoperiod of 1000 µmol photons
m−2 s−1 and leaf temperature maximum
of 16°C/12.5°C [light/dark]), the last of which was chosen as the
key condition for the present study based on previously reported
phenotypic differences between IT and SW (Cohu et al. 2013b;
Cohu, Muller, Demmig-Adams & Adams 2013a). The controlled conditions
used here do not represent the full extremes these plants experience in
natural settings; high/low light (HL/LL) and cool/warm (C/W) temperature
are used as descriptive terms to characterize the factorial growth
regime. All plants were grown from seeds soaked in water at 4°C for 4
days and then germinated in six-pack seed-starting trays containing 50
mL of Fafard Growing Mix 2 (Sun Gro Horticulture, Agawam, MA, USA) under
9-h photoperiods of either 100 (LLW and LLC) or 1000 (HLW and HLC) µmol
photons m−2 s−1 and a common air
temperature of 25°C during the photoperiod and 20°C during the dark
period. Following germination, individual seedlings were transplanted
with 50-mL soil from their respective cells into larger (2.9-L) pots,
and then transitioned to their final growth conditions (for details, see
Fig. S1; see also (Cohu et al. 2013b)). Plants received water
daily with nutrients added every other day as previously described
(Stewart et al. 2017a).