Impact of CBFs on freezing tolerance and gene expression under LLW versus HLC
Freezing tolerance was significantly lower in plants acclimated to LLW versus HLC (Fig. 5a,b), with an LT50 near -5.6ºC for all five genotypes in LLW (Fig. 5a). For HLC plants, LT50 of freezing tolerance in sw:cbf123 was lower by 3.5ºC relative to the SW and sw:cbf2 lines (Fig. 5b). Similarly, LT50 of HLC plants was lower by 3.4ºC in it:cbf123 relative to IT. This reduced freezing tolerance in the sw:cbf123 and it:cbf123 lines was accompanied by more pronounced freezing-induced depression of PSII efficiency Fv/Fm (Fig. 5c). At the same time, the greater freezing tolerance in HLC versus LLW for it:cbf123 and sw:cbf123 lines indicated some contribution fromCBF1–3 -independent freezing-tolerance mechanisms.
At the molecular level, induction of selected CBF- regulated genes was strongly inhibited in it:cbf123 and sw:cbf123 under HLC (Fig. 6). Five genes were chosen for assaying by RT-qPCR based(i) on prior demonstration of their regulation by CBF1–3using short-term cold shifts and (ii) demonstrated function in cold acclimation or cold-induced signaling. These five genes were Ser/Thr kinase CIPK25 (AT5G25110), freezing-tolerance-related proteins COR78 (AT5G52310), LTI30 (AT3G50970),COR15A (AT2G42540), and GOLS3 (AT1G09350). All five genes were induced under HLC in both parental ecotypes and exhibited strongly reduced induction under HLC in the it:cbf123 and sw:cbf123lines (Fig. 6a–e). Two of the five genes, COR15A andGolS3 , also had weakly attenuated induction in sw:cbf2relative to SW under HLC (Fig. 6d,e).