Posttranslational regulation of multiple clock-related transcription factors triggers cold-inducible gene expression in Arabidopsis.

Cold stress is an adverse environmental condition that affects plant growth, development, and crop productivity. Under cold stress conditions, the expression of numerous genes that function in the stress response and tolerance is induced in various plant species, and the dehydration-responsive element (DRE) binding protein 1/C-repeat binding factor (DREB1/CBF) transcription factors function as master switches for cold-inducible gene expression. Cold stress strongly induces these DREB1 genes. Therefore, it is important to elucidate the mechanisms of DREB1 expression in response to cold stress to clarify the perception and response of cold stress in plants. Previous studies indicated that the central oscillator components of the circadian clock, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), are involved in cold-inducible DREB1 expression, but the underlying mechanisms are not clear. We revealed that the clock-related MYB proteins REVEILLE4/LHY-CCA1-Like1 (RVE4/LCL1) and RVE8/LCL5 are quickly and reversibly transferred from the cytoplasm to the nucleus under cold stress conditions and function as direct transcriptional activators of DREB1 expression. We found that CCA1 and LHY suppressed the expression of DREB1s under unstressed conditions and were rapidly degraded specifically in response to cold stress, which suggests that they act as transcriptional repressors and indirectly regulate the cold-inducible expression of DREB1s We concluded that posttranslational regulation of multiple clock-related transcription factors triggers cold-inducible gene expression. Our findings clarify the complex relationship between the plant circadian clock and the regulatory mechanisms of cold-inducible gene expression.

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 DREB1/CBFs triggers a cold-responsive transcriptional cascade, after which many stress tolerance genes are expressed. Thus, elucidating the mechanisms of cold stress-inducible DREB1/CBF 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 DREB1A/CBF3 in Arabidopsis (Arabidopsis thaliana). ice1-1 is a widely accepted mutant allele known to abolish cold-inducible DREB1A expression, and this evidence has strongly supported ICE1-DREB1A regulation for many years. However, in ice1-1 outcross descendants, we unexpectedly discovered that ice1-1 DREB1A repression was genetically independent of the ice1-1 allele ICE1(R236H). Moreover, neither ICE1 overexpression nor double loss-of-function mutation of ICE1 and its homolog SCRM2 altered DREB1A expression. Instead, a transgene locus harboring a reporter gene in the ice1-1 genome was responsible for altering DREB1A expression. The DREB1A promoter was hypermethylated due to the transgene. We showed that DREB1A repression in ice1-1 results from transgene-induced silencing and not genetic regulation by ICE1. The ICE1(R236H) mutation has also been reported as scrm-D, which confers constitutive stomatal differentiation. The scrm-D 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-DREB1 regulatory model should be revalidated without the previous assumptions.