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).