Interaction of growth environment with ecotype in shaping
photosynthetic characteristics, expression of CBF genes, and leaf
transcriptome
The highest photosynthetic capacity (Fig. 1a), leaf dry mass per area
(Fig. 1b), and chlorophyll a + b (Fig. 1c) were achieved
under HLC (high light, cool temperature growth conditions). While
photosynthetic capacity and leaf dry mass per area were also higher in
HLW (high light, warm temperature growth conditions), chlorophylla + b levels remained similar to those under LLW
conditions (low light, warm temperature growth conditions). Chlorophylla /b ratios responded similarly to growth light conditions,
with a strong ecotypic difference of higher chlorophylla /b in IT versus SW under HLW but not HLC (Fig. 1d).
Significant ecotype-specific differences were also observed in terms of
higher photosynthetic capacity in SW under HLC and HLW (Fig. 1a), higher
leaf dry mass per area in SW under HLC (Fig. 1b), and higher chlorophylla + b in SW under all conditions tested (Fig. 1c). In both
ecotypes, the strongest CBF1–3 transcript expression was also
achieved in HLC (Fig. 2). As observed for photosynthetic capacity and
leaf dry mass per area, CBF1 and CBF3 expression were
greater in the SW versus IT HLC plants, but this pattern was somewhat
different in LLC (low light, cool temperature growth conditions), under
which the IT moderately induced CBF1–3 while SW did not induceCBF1–3 (Fig. 2).
Growth under HLC relative to LLW also induced sweeping changes in the
leaf transcriptome in both ecotypes (Fig. 3). 2086 and 2176 genes were
induced under HLC in 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 versus
LLW, i.e., 2073 and 1992 genes for IT and SW, respectively (Table S3,
S4). All three biological replicates co-clustered for both ecotypes
grown under each of the four conditions upon hierarchical clustering,
and the transcriptomic response of IT and SW in HLC conditions
co-branched (Fig. 3a). This co-branching of HLC transcriptomes of the
two ecotypes was due at least in part to large blocks of co-clustering
genes that were specifically induced under HLC (HLC-specific genes) and
downregulated under HLC (HLC-downregulated genes) in both ecotypes (Fig.
3a, Table S5-S7). HLC-specific induced genes were strongly enriched for
a number of the Gene Ontology (GO) categories, of which the most
enriched three categories were starch catabolism (GO:0005983), cold
acclimation (GO:0009631), and protein refolding (GO:0042026) (Table S8).
Similarly, GO analysis revealed pathways repressed specifically in HLC.
For instance, the three most strongly over-represented pathways among
genes specifically downregulated in HLC were water transport
(GO:0006833), auxin polar transport (GO:0009926), and
cytokinin-activated signaling pathways (GO:0009736) (Table S9).
Photosynthesis-related genes tended to have unique expression patterns
in each ecotype under HLC. Photosynthesis-related genes tended to be
downregulated under HLC in IT and upregulated in SW. Genes specifically
downregulated in IT under HLC were in the categories of light harvesting
in photosystem I (GO:0009768), light reaction (GO:0019684), and
photosynthesis (GO:0015979) (Fig. 3b, Table S10-S12). In contrast, genes
induced specifically in SW under HLC were enriched for both
photosynthesis (GO:0015979), plastid organization (GO:0009657), and
antioxidant biosynthesis pathways (GO:0006766) GO categories (Table
S13-S15). The photosynthesis-related genes selectively upregulated in SW
under HLC included multiple genes involved in the Calvin-Benson-Bassham
cycle, cyclic electron flow around PSI, and chlorophyll and tocopherol
biosynthesis (Fig. 3c).
Genes induced under HLC were also enriched for genes constitutively
induced in CBF overexpression lines (Park et al., 2018), withp -values of 10-66 and 10-38for IT and SW, respectively (Fig. 3d, Table S16). IT in LLC was the only
other case where significant overlap with this set of ectopically
expressed CBF-dependent genes was seen (p -value = 3.0 x
10-14). Genes induced under HLC in both ecotypes, as
well as genes induced in IT in LLC, were also enriched for genes
downregulated in it:cbf123 and sw:cbf123 following sudden
transfer from warm growth conditions to 4°C for 24 h (Park et al.,
2018), with p -values of 10-53,
10-19 and 0.003, respectively (Table S17).
The response of chloroplast redox state to experimental exposure to
different light intensities was ascertained in leaves of plants
acclimated to HLC and LLW. QA reduction state was
similar in the two ecotypes under LLW across a range of light
intensities (Fig. 4a), with both ecotypes already exhibiting relatively
high QA reduction states under relatively low light
intensities. In addition, HLC plants of both ecotypes exhibited
consistently lower QA reduction states than the LLW
plants (Fig. 4a,b). The light response of QA reduction
state differed between HLC in IT and SW, with SW exhibiting a
significantly lower QA reduction state (more oxidized
QA) than IT at higher light intensities (Fig. 4b).