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 growth chambers (Controlled Environments Ltd., Manitoba, Canada) and then in Percival Scientific E36-HID chambers (Perry, Iowa) alongside sw:cbf2 , sw:cbf123, and it:cbf123 mutant lines, which were generated via CRISPR/Cas9 and plants used in this study were confirmed by Sanger sequencing to carry the expected mutations. For more information on the ecotypes, see (Oakley et al., 2014; Stewart et al., 2017). The following four growth conditions—based on a factorial design of contrasting light intensities and temperature regimes—were employed: low light, warm temperatures (LLW; 9-h photoperiods of 100 µmol photons m−2 s−1 and 25°C/20°C [light/dark] leaf temperatures), low light, cool temperatures (LLC; 9-h photoperiods of 100 µmol photons m−2s−1 and 16°C/12.5°C [light/dark] leaf temperatures), high light, warm temperatures (HLW; 9-h photoperiods of 1000 µmol photons m−2 s−1 and 25°C/20°C [light/dark] leaf temperatures), and high light, cool temperatures (HLC; 9-h photoperiods of 1000 µmol photons m−2 s−1 and 14°C/12.5°C [light/dark] leaf temperatures). All plants were grown from seeds that were kept in H2O at 4°C for 4 days and then germinated in six-pack seed-starting trays containing 50-mL soil (Fafard Growing Mix 2; Sun Gro Horticulture, Massachusetts, 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 regime 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. Following this transplant, seedlings to be grown under LLW were kept at the air temperature at which they germinated (i.e., 25°C/20°C [light/dark] and 9-h photoperiods of 100 µmol photons m−2 s−1), and seedlings to be grown in HLW were transferred to a constant air temperature of 20°C so that the leaf temperatures were 25°C during the photoperiod and 20°C during the dark period. Seedlings to be grown in LLC were transferred to 16°C/15°C (light/dark) for 7 days and then to 16°C/12.5°C (light/dark) air temperatures, and HLC plants were transferred to a constant air temperature of 15°C for 7 days and then to an 8°C/12.5°C (light/dark) air temperature regime so that the leaf temperatures were 14°C during the photoperiod and 12.5°C during the dark period. The leaves of LLW and LLC plants did not differ from the respective air temperatures. Plants were grown under their respective final growth conditions for several weeks and received water daily with nutrients added every other day as previously described (Stewart et al., 2015).