Identification of SFR6, a key component in cold acclimation acting post-translationally on CBF function.

Abstract

The sfr6 mutant of Arabidopsis thaliana was identified previously, on the basis of its failure to undergo acclimation to freezing temperatures following exposure to low positive temperatures. This failure is attributed to a defect in the pathway leading to Cold On-Regulated (COR) gene expression via CBF (C-box binding factor) transcription factors. We identified a region of chromosome IV containing SFR6 by positional-mapping. Fine mapping of the sfr6-1 EMS mutation proved impossible as the locus resides very close to the centromere. Therefore, we screened 380 T-DNA lines with insertions in genes within the large region to which sfr6-1 mapped. This resulted in the identification of 2 further mutant alleles of SFR6 (sfr6-2 and sfr6-3); like the original sfr6-1 mutation, these both disrupt freezing tolerance and COR gene expression. To determine the protein sequence, we cloned an SFR6 cDNA based on the predicted coding sequence but this offered no indication as to the mechanism by which SFR6 acts. The SFR6 gene itself is not strongly regulated by cold thus discounting regulation of SFR6 activity at the transcriptional level. We show that overexpression of CBF1 or CBF2 transcription factors, which constitutively activate COR genes in wild type, cannot do so in sfr6-1. We demonstrate that CBF protein accumulates to wild-type levels in response to cold in sfr6-1. These results point to a role for the SFR6 protein affecting the CBF pathway downstream of CBF translation. Demonstration that SFR6 protein is targeted to the nucleus may suggest a direct role in modulating gene expression.