Ecotype-specific role for CBF1–3 in photosynthetic upregulation under HLC
The finding that, in contrast to freezing tolerance, photosynthetic upregulation under HLC was not inhibited in sw:cbf2 or sw:cbf123 , and was only modestly reduced in it:cbf123needs to be examined in the context of the strong constitutive photosynthetic upregulation observed upon over-expression of CBFparalogs (Hüner et al., 2014; Savitch et al., 2005). Photosynthetic upregulation, a developmental process involving changes at the organelle, cell, tissue, and whole plant levels (Hoshino et al., 2019; Yano & Terashima, 2004), is likely to involve multiple regulatory pathways. For example, blue-light photoreceptor signaling and foliar sucrose levels (Hoshino et al., 2019; Katagiri et al., 2016; Kozuka, Kong, Doi, Shimazaki, & Nagatani, 2011; Lopez-Juez, Bowyer, & Sakai, 2007) make contributions to increases in leaf thickness in HL-grown plants of a similar magnitude as those observed for CBF-dependent leaf thickening in the it:cbf123 mutant under HLC. The present findings indicate that light-responsive signaling pathways with overlapping functions can fully compensate for the loss ofCBF1–3 in the sw:cbf2 and sw:cbf123 . Such alternative signaling pathways could thus include photoreceptors, photosynthetic sugar and redox signals, and phytohormone signals. Since loss of CBF activity in the it:cbf123 mutant under HLC resulted in a modest increase in the temperature at which electrolyte leakage occurred as well as a significantly lower capacity for photosynthesis, both photosynthetic upregulation and long-term freezing tolerance in IT appear to be more dependent on CBF transcriptional activity than in SW. Taking the present results from long-term HLC acclimation and previous results on short-term transfer to cold conditions together, we suggest an obligatory role of CBF1–3 as first-wave responders to abrupt chilling conditions (Fowler & Thomashow, 2002), and an apparent continuous engagement of IT in early-phase acclimation even after weeks of growth under HLC. In contrast, SW may achieve a state of complete cold acclimation, where CBF1–3 transcriptional activity becomes entirely dispensable to maintaining photosynthetic upregulation – and partly dispensable to the maintenance of elevated freezing tolerance. This novel hypothesis of a more complete acclimation to HLC in SW (but not IT) is also consistent with the stronger photosynthetic upregulation and less elevated QA reduction state of SW in HLC.
The present comparative transcriptomic analysis of two ecotypes thus lends further support to a greater relative importance of CBFgenes for long-term growth under HLC in IT versus SW, as had also been suggested for sudden transfer of warm-grown plants to chilling conditions but lower light intensity than used in the present study (Park et al., 2018; Sanderson et al., 2020). This is also supported by the finding that, among all pairings of ecotype x growth conditions in the present study, the largest percentage of CBF-target genes was induced in HLC-grown IT (Supplemental Tables). For example, 78.9% of CBF-target genes defined as genes constitutively induced in CBFoverexpression lines were induced in HLC-grown IT, compared to only 49.3% in SW. While a hypothetical alternative explanation for this result would be transcriptional network divergence of CBF-regulated genes between SW and IT resulting in underestimation of CBF target genes in SW HLC plants, this explanation can be ruled out since Park et al., (2018) defined CBF regulons specific to IT and SW and all cross-comparisons to these gene lists consistently showed a greater percentage of CBF-target genes induced in HLC-grown IT (c.f., supplemental tables on CBF target genes). In summary, our evidence at the transcriptomic and physiological levels points towards a consistent trend of the CBF-dependent pathway having a greater ongoing role during long-term growth under HLC in IT versus SW, which might have been unexpected given the naturally occurring cbf2 mutation in the IT background.