CBF-dependent and CBF-independent regulatory pathways contribute to the differences in freezing tolerance and cold-regulated gene expression of two Arabidopsis ecotypes locally adapted to sites in Sweden and Italy

Abstract

Arabidopsis thaliana (Arabidopsis) increases in freezing tolerance in response to low nonfreezing temperatures, a phenomenon known as cold acclimation. The CBF regulatory pathway, which contributes to cold acclimation, includes three genes—CBF1, CBF2 and CBF3—encoding closely-related transcription factors that regulate the expression of more than 100 genes—the CBF regulon—that impart freezing tolerance. Here we compare the CBF pathways of two Arabidopsis ecotypes collected from sites in Sweden (SW) and Italy (IT). Previous studies showed that the SW ecotype was more freezing tolerant than the IT ecotype and that the IT ecotype had a nonfunctional CBF2 gene. Here we present results establishing that the difference in CBF2 alleles contributes to the difference in freezing tolerance between the two ecotypes. However, other differences in the CBF pathway as well as CBF-independent pathways contribute the large majority of the difference in freezing tolerance between the two ecotypes. The results also provided evidence that most cold-induced CBF regulon genes in both the SW and IT ecotypes are coregulated by CBF-independent pathways. Additional analysis comparing our results with those published by others examining the Col-0 accession resulted in the identification of 44 CBF regulon genes that were conserved among the three accessions suggesting that they likely have important functions in life at low temperature. The comparison further supported the conclusion that the CBF pathway can account for a large portion of the increase in freezing tolerance that occurs with cold acclimation in a given accession, but that CBF-independent pathways can also make a major contribution.

Genetic and physiological mechanisms of freezing tolerance in locally adapted populations of a winter annual

“…Our work builds upon the recent study by Park et al. (), who used CRISPR‐Cas9 to produce mutations in CBF genes in the SW genetic background. Here we used a CBF2 null mutant from Park et al.…”

“…Here we used a CBF2 null mutant from Park et al. (), an additional independent CBF2 null mutant, and two NILs with IT loss‐of‐function mutations in a SW genetic background to hone in on the downstream targets of CBF2 that are in part responsible for differences in cold‐acclimated freezing tolerance between IT and SW.…”

“…The other very highly cold‐responsive gene in SW was AT4G30830 , but the expression differences between SW and IT explained by CBF2 for this gene were more modest (~44%). AT4G30830, which is a myosin‐like protein of unknown function (Krishnakumar et al., ) that has been described as cold responsive in other studies (Gehan et al., ; Park et al., ). Future studies to identify what role AT4G30830 plays in cold‐acclimated freezing tolerance would be worthwhile.…”

“…Two lines were produced (Appendix S1): SW:cbf2 a, which is the same line with a 19‐bp deletion in the coding region of CBF2 of Park et al. (), and SW:cbf2 b, with a 13‐bp deletion in the coding region of CBF2 .…”

“…Recent studies have primarily focused on the effects of loss‐of‐function mutations of all three CBF genes on freezing tolerance and global gene expression and typically identify over a hundred genes with differential expression after cold acclimation (Park et al., , ; Jia et al., ; Zhao et al., ). Here we are specifically interested in the effects of CBF2 on differential cold‐acclimated gene expression and survival through freezing between the IT and SW ecotypes because of direct implications of loss of function of CBF2 for local adaptation.…”