DREB1A/CBF3 Is Repressed by Transgene-Induced DNA Methylation in the Arabidopsis ice1 -1 Mutant

DREB1/CBFs are key transcription factors involved in plant cold stress adaptation. The expression of s triggers a cold-responsive transcriptional cascade, after which many stress tolerance genes are expressed. Thus, elucidating the mechanisms of cold stress-inducible expression is important to understand the molecular mechanisms of plant cold stress responses and tolerance. We analyzed the roles of a transcription factor, INDUCER OF CBF EXPRESSION1 (ICE1), that is well known as an important transcriptional activator in the cold-inducible expression of in Arabidopsis ( ). is a widely accepted mutant allele known to abolish cold-inducible expression, and this evidence has strongly supported ICE1- regulation for many years. However, in outcross descendants, we unexpectedly discovered that repression was genetically independent of the allele ICE1(R236H). Moreover, neither overexpression nor double loss-of-function mutation of and its homolog altered expression. Instead, a transgene locus harboring a reporter gene in the genome was responsible for altering expression. The promoter was hypermethylated due to the transgene. We showed that repression in results from transgene-induced silencing and not genetic regulation by ICE1. The ICE1(R236H) mutation has also been reported as , which confers constitutive stomatal differentiation. The phenotype and the expression of a stomatal differentiation marker gene were confirmed to be linked to the ICE1(R236H) mutation. We propose that the current ICE1- regulatory model should be revalidated without the previous assumptions.