4.3.1 ⎪ Extent of CBF1–3 involvement in SW relative to IT
The present finding that CBF1–3 are necessary for full induction of freezing tolerance in SW and IT demonstrates their involvement inA. thaliana grown from seedling stage in HLC conditions as done here. Previous studies had shown that CBF1–3 are required for full induction of freezing tolerance in mature plants grown under warm conditions and transferred in one step to chilling conditions (Zhaoet al. 2016; Jia et al. 2016; Park et al. 2018). However, as was also concluded from studies on warm-grown CBF1–3-deficient mutants abruptly transferred to cold conditions (Zhaoet al. 2016; Jia et al. 2016; Park et al. 2018), both CBF1–3-dependent and CBF1–3-independent pathways contribute to freezing tolerance in plants grown from seedling stage under HLC – as illustrated here by the fact that freezing tolerance of both it:cbf123 and sw:cbf123 was greater in HLC compared to LLW and that the induction of genes previously defined as CBF1–3-target genes was reduced to varying degrees, but was not fully blocked in CBF1–3-deficient lines grown under HLC.
The striking difference in the extent to which CBF1–3- deficiency differentially impairs aspects of the acclimation process to HLC conditions in IT compared to SW is a key finding of the present study. While many genes previously defined as CBF1–3-responsive genes did exhibit strongly reduced expression in both it:cbf123 compared to IT and sw:cbf123 compared to SW, and may be associated with functions we did not characterize in this study, some genes instead exhibited trends matching those of photosynthetic acclimation and freezing tolerance of whole plants. For the latter genes, sw:cbf123 compared to SW exhibited little or no difference as the result of CBF1–3- deficiency, whereas it:cbf123 exhibited strongly reduced expression compared to IT. The central features of the acclimation of plant form and function to HLC, i.e., photosynthetic upregulation (and its associated morphological traits) as well as freezing tolerance, were only modestly impacted in sw:cbf123 but were strongly impacted (especially in whole plants for the case of freezing tolerance) in it:cbf123 compared to IT. These findings provide further indication for a role of CBF1–3-independent pathways in HLC acclimation of photosynthesis and freezing tolerance and suggest a greater contribution of such pathways in SW.
Growth is yet another trait exhibiting differential regulation between SW and IT in the context of CBF1–3-deficiency. The fact that rosettes of it:cbf123 were larger relative to IT, but those of sw:cbf123 were similarly large as those of SW under HLC is consistent with an obligatory role of CBF1–3 in growth depression under HLC conditions in IT but not in SW. Ding et al. (2019) reported a regulatory link between CBF1–3 induction by chilling stress, post-translational modification of EGR2, and whole-plant changes in rosette growth. As noted, under HLC conditions EGR2 was induced in both ecotypes (more strongly so in SW) and preferentially attenuated in it:cbf123 . Given EGR2 ’s role in repressing leaf elongation (Bhaskara et al. 2017), this gene may contribute to the larger rosette size of it:cbf123 relative to IT in HLC growth conditions. Future research should further clarify the role of CBF1–3 (in IT) and/other regulators (in SW) in inducing EGR2-dependent growth depression under HLC.