3.2 ⎪ CBF1–3 deficiency, photosynthesis, morphology, freezing tolerance, and gene expression
3.2.1 Photosynthesis and morphology
CBF1–3- deficiency significantly lessened the extent of upregulation of photosynthetic capacity and leaf dry mass under HLC relative to LLW and abolished upregulation of chlorophyll a +b content in it:cbf123 compared to IT but, remarkably, did not impede upregulation of these traits in HLC compared to LLW in sw:cbf123 compared to SW (Fig. 5a–c, Table 1). Despite the difference in chlorophyll a + b content, IT and it:cbf123 did not differ in chlorophyll a /b under either LLW or HLC (Fig. 5d).
Similar trends were observed for leaf morphology in that IT and it:cbf123 grown under HLC exhibited significant differences, whereas SW and sw:cbf123 did not (Figs. 6,7). Specifically, leaves were thinner (Fig. 6a–c) and rosettes were larger (had a larger diameter) in it:cbf123 compared to IT (Fig. 7a–c) in plants grown in HLC. In contrast, leaf thickness was the same (Fig. 6a,d,e) and rosette diameter was similar in HLC-grown plants of SW and sw:cbf123 (Fig. 7a,d,e).
3.2.2 Freezing tolerance
An initial assessment of leaf freezing tolerance was made using electrolyte leakage and chlorophyll fluorescence, where a sharp increase in leakage and/or decrease in intrinsic photosystem II indicates freezing damage to membranes (Fig. 8). While LLW-grown plants of all genotypes exhibited the same high susceptibility to freezing damage by these criteria (Fig. 8a), HLC-grown plants were shifted to greater tolerance that was also more pronounced in SW compared to IT and was substantially impaired by CBF1–3 deficiency in both backgrounds (Fig. 8b, Table 2). Figure 8c shows these same data transformed to mean lethal temperature (LT50) upon exposure to stress; onset of significant electrolyte leakage occurred with an LT50near -5.6°C for all genotypes grown in LLW but was shifted to lower sub-freezing temperatures in leaves grown in HLC compared to LLW to varying degrees depending on genotype. LT50 of freezing tolerance in sw:cbf123 was 3.5°C warmer than that of SW (Fig. 8b,c). Similarly, LT50 of it:cbf123 was 3.4°C warmer than that of IT (Fig. 8b,c). This greater electrolyte leakage in sw:cbf123 compared to SW and it:cbf123 compared to IT was accompanied by more pronounced freezing-induced depression of intrinsic PSII efficiency Fv/Fm (Fig. 8d). At the same time, the lesser electrolyte leakage in both it:cbf123 and sw:cbf123 lines grown under HLC compared to LLW indicated contributions from CBF1–3-independent freezing-tolerance mechanisms.
The results from excised leaves (Fig. 8) were complemented by tests of whole plant survival upon exposure to freezing temperatures (Fig. 9). Whole-plant survival was extremely low in LLW-grown plants of all genotypes and was much enhanced by growth under HLC (Fig. 9). The whole plants (Fig. 9) were even more sensitive to freezing stress than the leaves shown in Figure 8. The impairment of freezing tolerance of whole plants by CBF1–3-deficiency was much more pronounced than that of leaves in the IT background, i.e., in it:cbf123 compared to IT. In contrast, there was much less impairment of freezing tolerance in whole plants by CBF1–3- deficiency in sw:cbf123 compared to SW (Fig. 9).
3.2.3 CBF1–3-dependent gene expression
This section focuses on selected genes that exhibited response patterns reminiscent of the trends exhibited by photosynthesis and leaf/plant morphology (Fig. 10, Table 1) as well as selected genes known to be cold regulated (Fig. 11, Table 2). From among 31 genes that were identified as CBF1–3-target genes in prior work (Park et al. 2018) and showed considerable induction under HLC in IT (Fig. 4), nine were selected for validation by RT-qPCR with priority given to genes encoding proteins that can be linked to a role in photosynthetic or leaf-morphological acclimation phenotypes based on either previous studies on these proteins or the presence of a protein domain with an established role in acclimation phenotypes. Expression level of five of these nine genes (Fig. 10; Table 1) exhibited an impact of CBF1–3 deficiency mirroring that on leaf photosynthetic and morphological traits in the two ecotypes. Specifically, these five genes exhibited a strong reduction in the extent of upregulation under HLC compared to LLW in it:cbf123 compared to IT but no to little difference in sw:cbf123 compared to SW. These genes included cold- and salt-responsive protein RCI2A (AT3G05880; Fig. 10a), transmembrane protein AT5G44565 (Fig. 10b), sucrose synthase SUS1(AT5G20830; Fig. 10c), cysteine-rich, defensin-like protein LCR69(AT2G02100; Fig. 10d), and oleosin-B3-like stress protein AT1G13930 (Fig. 10e).
Moreover, expression of nine selected cold acclimation genes was affected by CBF1–3 activity in both IT and SW grown in HLC (Fig. 11). Under HLC, expression of galactinol synthase GolS3 (AT1G09350), the protein kinases CIPK25 (AT5G25110) and KIN2(AT5G15970), and the protein phosphatase EGR2 (AT5G27930) were higher in SW compared to IT and were also higher in both wildtype genotypes compared to their corresponding CBF1–3-deficient mutants (Fig. 11a–d). In contrast, expression of cold-regulated genesCOR78 (AT5G52310) and COR15A (AT2G42540) was higher in IT compared to SW (Fig. 11e,f), and expression of the dehydrin LTI30(AT3G50970), the cold-regulated gene (necessary for chloroplast membrane integrity in freezing) COR15B (AT2G42530), and lipid-sensing-domain-containing AT1G21790 was similar in SW and IT (Fig. 11g–i). Induction of the latter genes (expressed either more strongly, or similarly, in IT compared to SW) under HLC versus LLW was associated to some extent with CBF1–3 since it was partially inhibited in sw:cbf123 compared to SW (Fig. 11, Table 2) and partially (Fig. 11a–c,e–h) or completely (Fig. 11d,i) inhibited in it:cbf123compared to IT (Table 2).
While the focus of this work was the effect of complete CBF1–3 deficiency, it should be noted that there was no difference, or only a relatively small effect, for expression of selected CBF1–3-regulated genes in plants deficient only in CBF2 in the SW background (sw:cbf2 ) relative to SW in HLC (Table S19). Similarly, no difference was observed between HLC-grown sw:cbf2 and SW in leaf photosynthetic or morphological phenotypic traits (Figs. 5–7, Table S19) or in freezing tolerance of excised leaves (via electrolyte leakage or Fv/Fm; Fig. 8, Table S19). A small decrease in freezing tolerance of whole plants was observed for sw:cbf2 relative to SW seedlings grown in HLC conditions, but its survivorship was still higher than that of IT plants (Fig. 9, Table S19).