Ecotype-specific responses to HLC growth for CBFs and
CBF-regulated genes
The fact that the strongest induction of CBF1-3 was achieved in
both ecotypes under HLC indicates a synergistic effect of cool
temperature and high light on CBF1-3 induction. Elevated
excitation pressure in the chloroplast may contribute to maintenance of
elevated CBF1-3 expression during long-term growth in HLC (Hüner
et al., 2012; Hüner et al., 2016). Other possible contributing signals
include tetrapyrrole Mg-ProtoIX-mediated retrograde signaling that
impacts CBF1-3 expression levels in specific mutant backgrounds
(Lee & Thomashow, 2012; Noren et al., 2016). Ecotypic differences
presumably also shape how HL-dependent signals are translated toCBF1-3 induction. For instance, the finding that SW in HLC
maintained a more oxidized QA state under experimental
exposure to high light relative to IT in HLC is consistent with other
phenotypic measures that indicate superior adaptation of SW to either
high light or cold temperature than IT (Adams et al., 2014, 2016, 2018;
Ågren & Schemske, 2012; Agrena, Oakley, McKay, Lovell, & Schemske,
2013; Cohu et al. 2013a,b; Oakley et al., 2014; Park et al., 2018;
Stewart et al., 2015, 2016, 2017).
CBF1-3 are essential for long term maintenance of freezing
tolerance in both ecotypes
The present finding that CBF1-3 are essential to the full
induction of freezing tolerance in the SW and IT ecotypes under HLC
confirms their importance in A. thaliana as a key survival trait
for overwintering plants. Previous studies showed that CBFs are
essential for full induction of freezing tolerance in mature A.
thaliana plants grown under warm conditions and transferred in one step
to chilling conditions (Jia et al., 2016; Park et al., 2018; Zhao et
al., 2016). The present study on plants grown from seed under differing
temperature regimes demonstrates that CBFs also have an essential role
in the long-term maintenance of elevated freezing tolerance. Moreover,
just as was concluded from short-term studies on warm-grown cbfmutants abruptly transferred to cold conditions (Jia et al., 2016; Park
et al., 2018; Zhao et al., 2016), both CBF-dependent and CBF-independent
pathways also appear to be required for long-term freezing tolerance in
HLC plants – as illustrated here by the moderately enhanced freezing
tolerance in both it:cbf123 and sw:cbf123 under HLC versus
LLW. This finding is also consistent with the observation that gene
expression of CBF-target genes under HLC was strongly reduced, but not
fully blocked, in cbf123 triple mutants in the present study.
Furthermore, the contribution of the CBF-dependent pathway to freezing
tolerance after long-term growth under HLC was similar in both ecotypes
(reduction of LT50 of freezing tolerance by 3.5 ºC and
3.4ºC under HLC in it:cbf123 and sw:cbf123 versus IT and
SW, respectively). These findings indicate that the greater freezing
tolerance of SW versus IT in HLC is due to CBF-independent pathways
contributing to freezing tolerance (see also Park et al., 2018).
The absence of a clear effect under HLC in the sw:cbf2 mutant is
best explained by the impact of paralog compensation (Gilmour et al.,
2004; Jia et al., 2016; Park et al., 2018; Zhao et al., 2016), i.e.,
functionally overlapping components that can partly compensate for each
other’s loss. The observation in the present study of stronger induction
of many CBF-target genes under HLC in IT with its pre-existingcbf2 mutation relative to SW could also be interpreted in the
context of paralog compensation by CBF1 and CBF3 in acbf2 mutant background. Several independent A. thalianalineages evolved loss-of-function mutations in individual CBFgenes without apparent severe adverse effects on survival in regions
with mild winters (Kang et al., 2013; Monroe et al., 2016). The full
suite of CBF1–3 may thus only be required for tolerance to
colder conditions than used here (daytime air temperature of 8ºC and
leaf temperature of ~14 ºC).
However, paralog compensation among CBF1–3 does not explain the
observed significant induction of CBF-target genes and moderately
elevated freezing tolerance of cbf123 mutants in HLC, i.e.,
completely CBF-independent induction of some level of freezing
tolerance. The concept of paralog compensation can also apply to entire
gene families that may functionally overlap and compensate for each
other’s loss. CBF1–3 belong to the ERF/AP2 A-1 subfamily that
includes three additional members located outside the CBF1–3gene locus in A. thaliana (Mizoi, Shinozaki, &
Yamaguchi-Shinozaki, 2012). However, these three other ERF/AP2 A-1
subfamily members (DDF1, AT1G12610; DDF2 , AT1G63030;CBF4 , AT5G51990) are not expressed at detectable levels in leaf
tissue of IT or SW under any of the four growth regimes. Additionally,
Park et al. (2018) observed no induction of these three genes in the
it:cbf123 and sw:cbf123 in their various cold treatments.
It is thus unlikely that paralog compensation accounts for the
CBF-independent induction of freezing observed in cbf123 mutants.
Instead, the present findings suggest involvement of unrelated signaling
networks.