3.1.2 ⎪ Expression of CBF1–3 genes and leaf transcriptome
In both ecotypes, the strongest CBF1–3 transcript expression was
also seen in plants grown under HLC (Fig. 3). As was the case for
photosynthetic capacity and leaf dry mass per area, CBF1 andCBF3 expression were also greater in SW compared to IT plants
grown under HLC. In plants grown under LLC on the other hand, only IT
moderately induced CBF1–3 but SW did not (Fig. 3).
Growth under HLC compared to LLW also resulted in sweeping differences
in the leaf transcriptome in both ecotypes (Fig. 4). 1912 and 1415 genes
were induced in HLC-grown IT and SW, respectively, with an adjustedP -value of less than 0.01 and a minimum fold-change of 2 (Table
S1, S2). Similar numbers of genes were downregulated under HLC compared
to LLW, i.e., 1671 and 1531 genes, for IT and SW, respectively (Tables
S3, S4). For both ecotypes grown under each of the four conditions, all
three biological replicates co-clustered (after hierarchical clustering)
and the transcriptomic response of IT and SW in HLC conditions
co-branched (Fig. 4a). This co-branching of HLC transcriptomes of the
two ecotypes was due at least in part to large blocks of co-clustering
genes either induced specifically or downregulated under HLC in both
ecotypes (Fig. 4a, Tables S5–S7). Genes specifically induced under HLC
were strongly enriched for a number of gene ontology (GO) categories, of
which the most enriched unique categories were starch catabolism/starch
metabolism (GO:0005983/GO:0005982), sucrose biosynthesis (GO:0005986),
and cold acclimation (GO:0009631) (Table S8). GO analysis also revealed
pathways repressed specifically in HLC; the three categories most
strongly downregulated in HLC were water transport (GO:0006833),
brassinosteroid metabolism (GO:0016131), and auxin polar transport
(GO:0009926) (Table S9).
In addition to these shared responses, there were also substantial
differences between IT and SW in how gene expression responded to HLC
(Table S10-16). In SW compared to IT, growth under HLC conditions
enriched more strongly for induction of genes in the overlapping GO
categories of cold acclimation (GO:0009631), response to water
deprivation (GO:0009414), and response to abscisic acid (GO:0009737)
(Table S11). Conversely, the three most enriched unique GO categories
among genes with significantly higher induction in IT compared to SW in
HLC were protein refolding (GO:0042026), glucosinolate biosynthesis
(GO:0019761), and phosphate starvation response (GO:0016036) (Table
S14). Moreover, genes exhibiting greater down-regulation in HLC in IT
compared to SW were enriched for cytokinin-activated signaling pathway
(GO:0009736), auxin polar transport (GO:0009926), and response to auxin
(GO:0009733) (Table S16).
Genes in the photosynthesis GO category (GO:0015979) also exhibited
unique expression patterns in each ecotype under HLC (Fig. S2). With the
exception of the light-stress-induced light-harvesting complexLHCB4.3 (AT2G40100), genes involved in light harvesting were
downregulated in both ecotypes under HLC, but more so in IT relative to
SW. Conversely, genes more strongly induced in IT compared to SW (Fig.
S2) had in common that they are typically induced under abiotic and/or
oxidative stress (see discussion), such as chloroplast glucose-6
phosphate/phosphate translocator GPT2 (AT1G61800), chloroplast
envelope K+/H+ antiporterKEA2 (AT4G00630), phosphofructokinase (AT1G76550), cytosolic
fumarase (AT5G76550), ferritin (AT2G30400/AT3G56090), and pyridoxal
phosphate synthase protein (AT5G01410).
For both ecotypes, genes preferentially induced under HLC were enriched
for those that had also been induced by CBF1–3 overexpression in
the absence of either high light or cool temperatures (Park et
al. 2018) (P -values of 10−19 and
10−18 for IT and SW, respectively; Fig. 4b, Table
S17). Moreover, these genes preferentially induced under HLC were also
enriched for genes expressed at lower levels in it:cbf123 and
sw:cbf123 following sudden transfer from warm growth conditions
to 4°C for 24 h (Park et al. 2018) (P -values of
10−28 and 10−38, for IT and SW,
respectively; Table S18). Overall, while CBF1–3 target genes (Jiaet al. 2016; Park et al. 2018) were strongly induced in
both ecotypes in HLC, these genes tended to be more strongly induced in
SW compared to the IT ecotype in this condition. Examples for genes in
this previously defined CBF1–3-regulated group that were more strongly
induced in SW included the cold-acclimation-regulating protein kinaseCIPK25 (AT5G25110) and a suite of cold-induced dehydrin proteins,COR47 (AT1G20440), LTI30 (AT3G50970), and LTI29(AT1G20450) (Fig. 4b). In contrast, CBF1–3-regulated protein
phosphatase EGR2 (AT5G27930), a repressor of growth, was more
strongly induced in IT compared to SW under HLC (Fig. 4b).