The C-REPEAT BINDING FACTOR (CBF) cold-response pathway has a prominent role in cold acclimation, the process whereby certain plants increase tolerance to freezing in response to low nonfreezing temperatures. In Arabidopsis, the CBF pathway is characterized by rapid induction of the C-REPEAT BINDING FACTOR 1 (CBF1), CBF2, and CBF3 genes, which encode transcriptional activators, followed by induction of the CBF-targeted genes known as the "CBF regulon." Expression of the CBF regulon results in an increase in freezing tolerance. Previous studies established that CBF1, CBF2, and CBF3 are subject to circadian regulation and that their cold induction is gated by the circadian clock. Here we present the results of genetic analysis and ChIP experiments indicating that both these forms of regulation involve direct positive action of two transcription factors that are core components of the clock, i.e., CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELON-GATED HYPOCOTYL (LHY). In plants carrying the cca1-11/lhy-21 double mutation, cold induction of CBF1, CBF2, and CBF3 was greatly impaired, and circadian regulation of CBF1 and CBF3 was essentially eliminated; circadian regulation of CBF2 continued, although with significantly reduced amplitude. Circadian regulation and cold induction of three CBF regulon genes, i.e., COLD-REGU-LATED GENE15A (COR15A), COR47, and COR78, also were greatly diminished in plants carrying the cca1-11/lhy-21 double mutation. Furthermore, the cca1-11/lhy-21 double mutation resulted in impaired freezing tolerance in both nonacclimated and cold-acclimated plants. These results indicate that CCA1/LHY-mediated output from the circadian clock contributes to plant cold tolerance through regulation of the CBF cold-response pathway.

Cultured guard cell protoplasts (GCP) of tree tobacco (Nicotiana glauca) comprise a novel system for investigating the cell signaling mechanisms that lead to acquired thermotolerance and thermoinhibition. At 32°C in a medium containing an auxin (1-naphthaleneacetic acid [NAA]) and a cytokinin (6-benzylaminopurine), GCP expand, regenerate cell walls, dedifferentiate, and divide. At 38°C, GCP acquire thermotolerance within 24 h, but their expansion is limited and they neither regenerate walls nor reenter the cell cycle. These putative indicators of auxin insensitivity led us to hypothesize that heat suppresses induction of auxin-regulated genes in GCP. Protoplasts were transformed with BA-mgfp5-ER, in which the BA auxin-responsive promoter regulates transcription of mgfp5-ER encoding thermostable green fluorescent protein (GFP) or with a similar 35S-cauliflower mosaic virus constitutive promoter construct. Heat suppressed NAA-mediated activation of BA. After 21 h at 32°C in media with NAA, 49.0% 6 3.9% of BA-mgfp5-ER transformants strongly expressed GFP; expression percentages were similar to those of 35S-mgfp5-ER transformants at 32°C or 38°C. After 21 h at 38°C in media with NAA, 7.9% 6 1.6% of BA-mgfp5-ER transformants weakly expressed GFP, similar to GCP cultured at 32°C in media lacking NAA. Expression at 38°C was not increased by incubating for 48 h or increasing NAA concentrations 20-fold. After 9 to 12 h at 38°C, BA was no longer activated when cells were transferred to 32°C. Heat-stressed cells accumulate reactive oxygen species, and hydrogen peroxide (H 2 O 2 ) suppresses auxin-responsive promoter activation in Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts. H 2 O 2 did not suppress BA activation at 32°C, nor did superoxide and H 2 O 2 scavengers prevent BA suppression at 38°C.