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).