Comparative transcriptomics analysis reveals SaCBL1-Like positively regulates constitutive and cold-acclimated freezing tolerance in potato via CBF-regulon

Freezing stress is a major limiting factor in potato production. Deciphering the regulatory gene that controlling potato freezing tolerance is vital for breeding against hardness. Potato cultivars (Solanum tuberosum) are freezing-sensitive but, some well-known potato wild species are freezing tolerant including S. commersonii, S. malmeanum and S. acaule. However, the common freezing-tolerance regulatory genes of the wild species remain to be deciphered. In the present study, Comparative cold-responsive transcriptomics analysis was carried out and the result showed that, CBL1-like (calcineurin B-like protein 1-like), up-regulated in all of the freezing-tolerant genotypes but not in the freezing-sensitive genotypes. Result of qPCR evaluation was consistent with the transcriptomics analysis. 2) Transgenic lines were obtained of SaCBL1-like over-expressing and knocking-down. The three knocked-down transgenic lines showed decreased constitutive and cold-acclimated freezing tolerance. While the three over-expression transgenic lines showed improved freezing-tolerance. Moreover, main detected agricultural traits of over-expression transgenic lines were not significantly imparted. 3) Result of function mechanism analysis showed that SaCBL1-like improved expression of CBF-regulon (C-repeat binding factor) as well as prolonged higher expression of CBF1 after exposure to cold conditions. Furthermore, SaCBL1-like only interacted with SaCIPK3-1 (CBL-interacting protein kinase) among all of the apparently cold-responsive CIPK. Moreover, SaCBL1-like could recruit SaCIPK3-1 (CBL-interacting protein kinase) to the plasma membrane. Furthermore, co-transformation of both SaCBL1-like and SaCIPK3-1 in the tobacco increased the expression of CBF1 after exposure to cold condition. SaCBL1-like probably interacted with SaCIPK3-1 to confer potato freezing tolerance via CBF-regulon. Our study presents the role of SaCBL1-like in conferring potato freezing tolerance without negatively impacting main agricultural traits, which provides the basis for a targeted potato breeding against hardness.