Figure Legends
FIGURE 1 (a) Photosynthetic capacity (i.e., maximal light- and
CO2-saturated rate of oxygen evolution) per leaf area,
(b) leaf dry mass per area, (c) level of chlorophyll a + bper leaf area, and (d) chlorophyll a/b ratio in leaves of IT (red
columns) and SW (blue columns) plants that were grown in low light/warm
temperature growth conditions (LLW), low light/cool temperature growth
conditions (LLC), high light/warm temperature growth conditions (HLW),
or high light/cool temperature growth conditions (HLC). Mean values ±
standard deviations (n = 3 or 4); groups that share the same
letters are not considered statistically different, and groups that do
not share the same letters are considered statistically different based
on one-way ANOVA and post hoc Tukey–Kramer HSD tests.
FIGURE 2 Light response of reduction state of the primary
electron acceptor of photosystem II (QA, quantified by
chlorophyll fluorescence using the equation 1 − qL) of
leaves from IT (red circles) and SW (blue squares) plants grown under
(a) LLW and (b) HLC. Mean values ± standard deviations (n = 3);
statistically significant differences between ecotypes based on
Student’s t -tests are indicated with asterisks (* =P < 0.05, ** = P < 0.01, *** =P < 0.001); n.s . = not significantly different.
FIGURE 3 Relative transcript abundance (via RT-qPCR) of (a)CBF1 , (b) CBF2 , and (c) CBF3 in leaves of IT (red
columns) and SW (blue columns) plants that were grown in LLW, LLC, HLW,
or HLC conditions. Values are presented relative to the expression level
for each respective gene in the IT ecotype grown under LLW. Mean values
± standard deviations (n = 3); groups that share the same letters
are not considered statistically different, and groups that do not share
the same letters are considered statistically different based on one-way
ANOVA and post hoc Tukey–Kramer HSD tests.
FIGURE 4 (a) Hierarchical clustering of the
log2 expression data (via RNAseq) for 7,933 genes with
an adjusted P- value below 0.01 in one of the pairwise comparisons
for differential expression between ecotypes and growth conditions. The
three biological replicates for each growth condition/ecotype set are
shown as separate columns. (b) Log2 expression data (via
RNAseq) for IT and SW in HLC relative to LLW for genes previously
characterized as regulated by CBF1–3 in Col-0 (Jia et al. 2016;
Park et al. 2018). CIPK25 and KIN2 are protein kinases and EGR2
is a protein phosphatase, all participating in cold acclimation
signaling (Thomashow 1999; Ding et al. 2019). COR47, LTI30, and
LTI29 each are cold and drought-induced dehydrin proteins (Puhakainenet al. 2004). SUS1 and GOLS3 are stress-induced sucrose synthase
and galactinol synthase enzymes, respectively (Maruyama et al.2009). COR15B is essential for chloroplast membrane integrity during
freezing (Thalhammer, Hundertmark, Popova, Seckler & Hincha 2010).
FIGURE 5 (a) Photosynthetic capacity (light- and
CO2-saturated rate of oxygen evolution) per leaf area,
(b) leaf dry mass per area, (c) level of chlorophyll a + bper leaf area, and (d) chlorophyll a/b ratio in leaves of IT (red
columns), it:cbf123 (light red columns), SW (blue columns), and
sw:cbf123 (light blue columns) plants that were grown in LLW or
HLC. Mean values ± standard deviations (n = 6); Groups that share
the same letters are not considered statistically different, and groups
that do not share the same letters are considered statistically
different based on one-way ANOVA and post-hoc Tukey–Kramer HSD tests.
FIGURE 6 (a) Leaf thickness of IT (red column),
it:cbf123 (light red column), SW (blue column), and
sw:cbf123 (light blue column) plants that were grown in HLC, as
well as representative images of leaf cross-sections for (b) IT, (c)
it:cbf123 , (d) SW, and (e) sw:cbf123 . For (a), mean values
± standard deviations (n = 3); groups that share the same letters
are not considered statistically different, and groups that do not share
the same letters are considered statistically different based on one-way
ANOVA and post-hoc Tukey–Kramer HSD tests.
FIGURE 7 (a) Rosette diameter of IT (red column),
it:cbf123 (light red column), SW (blue column), and
sw:cbf123 (light blue column) after 40 days of growth in HLC, as
well as images of representative (b) IT, (c) it:cbf123 , (d) SW,
and (e) sw:cbf123 plants. For (a), mean values ± standard
deviations (n = 5); groups that share the same letters are not
considered statistically different, and groups that do not share the
same letters are considered statistically different based on one-way
ANOVA and post-hoc Tukey–Kramer HSD tests.
FIGURE 8 (a–c) Cellular electrolyte leakage from mature
leaves, and (d) images of mature leaves with photosystem II
photochemical efficiency (visualized via false colors based on
Fv/Fm values) from IT (red circles),
it:cbf123 (light red circles), SW (blue squares), and
sw:cbf123 (light blue squares) plants grown under LLW or HLC. For
(a,b) mean values (n = 3). For (c), mean values ± standard
deviations (n = 3); groups that share the same letters are not
considered statistically different, and groups that do not share the
same letters are considered statistically different based on one-way
ANOVA and post-hoc Tukey–Kramer HSD tests.
FIGURE 9 Percent survival after an overnight freezing treatment
(16 h at −10°C) of IT (red columns), it:cbf123 (light red
columns), SW (blue columns), and sw:cbf123 (light blue columns)
plants grown under LLW or HLC. Mean values ± standard deviations
(n = 3 plates, each of which contained 40 plants); groups that
share the same letters are not considered statistically different, and
groups that do not share the same letters are considered statistically
different based on one-way ANOVA and post-hoc Tukey–Kramer HSD tests.
FIGURE 10 Relative transcript abundance (via RT-qPCR) for (a)
AT5G44565, (b) SUS1, (c) LCR69, (d) AT1G13930, and
(e) RCI2A in leaves of IT (red columns), it:cbf123 (light
red columns), SW (blue columns), and sw:cbf123 (light blue
columns) plants grown in LLW or HLC. All values are normalized based on
the expression levels of IT in LLW. Mean values ± standard deviations
(n = 3); groups that share the same letters are not considered
statistically different, and groups that do not share the same letters
are considered statistically different based on one-way ANOVA and
post-hoc Tukey–Kramer HSD tests. SUS1 is a stress induced sucrose
synthase (Barratt et al. 2009).
FIGURE 11 Relative transcript abundance (via RT-qPCR) for (a)GolS3 , (b) CIPK25, (c) KIN2 , (d) EGR2 , (e)COR78, (f) COR15A , (g) LTI30 , (h) COR15B ,
and (i) AT1G21790 in leaves of IT (red columns), it:cbf123 (light
red columns), SW (blue columns), and sw:cbf123 (light blue
columns) plants grown under LLW or HLC. CIPK25 and KIN2 are protein
kinases and EGR2 is a protein phosphatase, all participating in cold
acclimation signaling (Thomashow 1999; Ding et al. 2019). LTI30
is a drought-induced dehydrin protein (Puhakainen et al. 2004).
GOLS3 is a galactinol synthase enzyme (Fowler & Thomashow 2002). COR15B
is essential for chloroplast membrane integrity during freezing
(Thalhammer et al. 2010). All values are normalized based on the
expression levels of IT under LLW. Mean values ± standard deviations
(n = 3); groups that share the same letters are not considered
statistically different, and groups that do not share the same letters
are considered statistically different based on one-way ANOVA and
post-hoc Tukey–Kramer HSD tests.