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

Premise Despite myriad examples of local adaptation, the phenotypes and genetic variants underlying such adaptive differentiation are seldom known. Recent work on freezing tolerance and local adaptation in ecotypes of Arabidopsis thaliana from Italy and Sweden provides an essential foundation for uncovering the genotype-phenotype-fitness map for an adaptive response to a key environmental stress. Methods We examined the consequences of a naturally occurring loss-of-function (LOF) mutation in an Italian allele of the gene that encodes the transcription factor CBF2, which underlies a major freezing-tolerance locus. We used four lines with a Swedish genetic background, each containing a LOF CBF2 allele. Two lines had introgression segments containing the Italian CBF2 allele, and two contained deletions created using CRISPR-Cas9. We used a growth chamber experiment to quantify freezing tolerance and gene expression before and after cold acclimation. Results Freezing tolerance was lower in the Italian (11%) compared to the Swedish (72%) ecotype, and all four experimental CBF2 LOF lines had reduced freezing tolerance compared to the Swedish ecotype. Differential expression analyses identified 10 genes for which all CBF2 LOF lines, and the IT ecotype had similar patterns of reduced cold responsive expression compared to the SW ecotype. Conclusions We identified 10 genes that are at least partially regulated by CBF2 that may contribute to the differences in cold-acclimated freezing tolerance between the Italian and Swedish ecotypes. These results provide novel insight into the molecular and physiological mechanisms connecting a naturally occurring sequence polymorphism to an adaptive response to freezing conditions.