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).