Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2011 Apr 6;108(17):7241–7246. doi: 10.1073/pnas.1103741108

CIRCADIAN CLOCK-ASSOCIATED 1 and LATE ELONGATED HYPOCOTYL regulate expression of the C-REPEAT BINDING FACTOR (CBF) pathway in Arabidopsis

Malia A Dong a,b, Eva M Farré b, Michael F Thomashow a,c,1
PMCID: PMC3084081  PMID: 21471455

Abstract

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 ELONGATED 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-REGULATED 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.

A general feature of plants from temperate environments is that they increase in freezing tolerance in response to low nonfreezing temperatures, a process called “cold acclimation” (1, 2). It now is well established that cold acclimation involves extensive changes in gene expression (3–6). The best understood cold-regulatory pathway is the CBF pathway. This pathway, which is widely conserved in plants (7), is best characterized in Arabidopsis (8, 9). When Arabidopsis plants are transferred from warm to cold temperature, C-REPEAT BINDING FACTOR 1 (CBF1), 2 (CBF2), and 3 (CBF3)—also known as DROUGHT RESPONSE ELEMENT BINDING FACTOR 1B (DREB1B), 1C (DREB1C), and 1A (DREB1A), respectively—are induced rapidly. These genes, which are linked physically in tandem array, encode transcription factors that are members of the AP2/ERF family of DNA-binding proteins (10). The CBF proteins bind to the CRT/DRE regulatory element present in the promoters of about 100 cold-regulated (COR) genes, known as the “CBF regulon,” and induce their expression (4, 6, 11). Constitutive overexpression of CBF1, CBF2, and CBF3 at warm temperature results in constitutive expression of the CBF regulon and an increase in freezing tolerance (12–14). The mechanisms whereby expression of the CBF regulon promotes freezing tolerance are not completely understood but involve the synthesis of low molecular weight cryoprotectants such as sucrose and raffinose and proteins that have cryoprotective properties (1, 2).

Given their importance in cold acclimation, efforts have been directed at understanding the mechanisms involved in cold-induction of CBF1, CBF2, and CBF3. To date, two positive regulators have been identified: INDUCER OF CBF EXPRESSION 1 (ICE1), a Myc family transcription factor that positively regulates CBF3 (15), and CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 3 (CAMTA3), a CAMTA family transcription factor that positively regulates CBF1 and CBF2 (16). The ICE1 and CAMTA3 genes are transcribed at warm temperature, indicating that their activities involve posttranscriptional regulatory mechanisms that are responsive to low temperature (16–18).

Another factor that affects the expression of CBF1, CBF2, and CBF3 is the circadian clock (19–22). At warm temperature, the transcript levels for CBF1, CBF2, and CBF3 oscillate with a peak at about 8 h after dawn (zeitgeber time 8; ZT8) and a trough at about ZT20. Moreover, cold-induction of CBF1, CBF2, and CBF3 is “gated” by the clock (22); if plants are exposed to low temperature at ZT4, the increase in CBF1, CBF2, and CBF3 transcript levels is much greater than if plants are exposed to low temperature at ZT16. These results indicate that cold induction of CBF1, CBF2 and CBF3 involves the integration of low-temperature and clock-regulatory pathways.

The circadian clock of Arabidopsis consists of multiple interlocking regulatory feedback loops (23, 24). Key components of the core feedback loop are CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), Myb transcription factors that have partially overlapping functions (25–28), and TIMING OF CAB 1 (TOC1), a PSEUDO RESPONSE REGULATOR (PRR) protein (29). Expression of CCA1 and LHY peaks just after dawn, whereas the expression of TOC1 peaks in the early evening. CCA1 and LHY bind to the Evening Element (EE) (19) present in the promoter of TOC1 and repress its transcription (30). TOC1 is necessary for the induction of both CCA1 and LHY (28). TOC1 is known to inhibit the repression of CCA1 by the TCP transcription factor CCA1 HIKING EXPEDITION 1 (CHE1) (31), but the means by which LHY expression is activated by TOC1 remains unknown. CCA1 and LHY also regulate expression of PSEUDO RESPONSE REGULATORS 7 (PRR7) and 9 (PRR9) (32, 33), two components of the morning regulatory loop. CCA1 and LHY bind to the promoters of these two genes to induce their expression, and the PRR7 and PRR9 proteins then negatively regulate CCA1 and LHY (32, 33).

It was reported recently that Arabidopsis plants carrying the prr5/prr7/prr9 triple mutation constitutively express CBF1, CBF2, and CBF3 at high levels and display constitutively high levels of freezing tolerance (34). Thus, it was proposed that PRR5, PRR7, and PRR9 might act as direct negative regulators of CBF1, CBF2, and CBF3 (34). Here we present results indicating that the clock also provides positive regulation of the CBF cold-response pathway and enhances freezing tolerance through action of the core clock components CCA1 and LHY.

Results

CCA1 and LHY Have a Direct Role in Circadian Regulation of CBF1, CBF2, and CBF3.

Consistent with previous reports (19–21), we found that CBF1, CBF2, and CBF3 are subject to circadian regulation (Fig. 1). Transcript levels for CBF1, CBF2, and CBF3 oscillated with a peak occurring at about ZT8 followed by a second peak about 24 h later. The oscillation patterns for the three CBF genes were similar, although we observed one consistent difference; whereas the ZT8 peak for CBF1 was lower than the second peak, the ZT8 peaks for CBF2 and CBF3 were higher than the second peak. Thus, the transition from dark to light may have a specific effect on the regulation of CBF1.

Fig. 1.

Fig. 1.

Open in a new tab

Effects of the cca1-11/lhy-21 double mutation on circadian regulation of CBF1, CBF2, CBF3, and CBF-targeted genes COR15A, COR47, and COR78. Wild-type Wassilewskija-2 (WS) and cca1-11/lhy-21 double-mutant plants were grown at 22 °C under a 12-h photoperiod to the four-leaf stage and then were transferred to constant light at zeitgeber time 0 (ZT0) (subjective day and night are indicated by white and gray bars, respectively). Plants were harvested every 2 h, and the transcript levels for the indicated genes were determined by qRT-PCR. Gene expression was normalized to ISOPENTENYL PYROPHOSPHATE (IPP2) for each sample. Gene expression is relative to one wild-type sample set to a value of 1 for each biological replicate. Values are averages from three independent biological experiments. Error bars indicate SEM.

Three lines of evidence led us to think that CCA1 and LHY might drive circadian regulation of CBF1, CBF2, and CBF3. First, the protein levels for CCA1 and LHY peak in the early morning (ZT1–3) (26, 35, 36), just before the time that the transcript levels of CBF1, CBF2, and CBF3 begin to increase. Second, the transcript levels for PRR7 (32, 36) and LIGHT-HARVESTING COMPLEX B (LHCB) (24, 35, 37), both of which are induced by CCA1 and LHY, peak similarly to the CBF genes. Last, the promoter regions of CBF1, CBF2, and CBF3 have several EE (AAAATATCT) (19) and CCA1-binding sites (CBS; AATCT) (35) (Fig. 2) that mediate binding of CCA1 and LHY (30, 35, 38) to target promoters. To determine whether CCA1 and LHY were involved in circadian regulation of CBF1, CBF2, and CBF3, we asked whether their expression was affected in plants carrying either the single cca1-11 or lhy-21 null mutations or the cca1-11/lhy-21 double mutation. We found that the single mutations had differing effects on the three CBF genes, whereas cycling of CBF1 was severely disrupted, CBF2 and CBF3 transcript levels cycled with approximately the same amplitudes as in the wild-type plants, although the peaks occurred about 2 h earlier than in the wild-type plants (Fig. S1). Period shortening of output genes such as LHCB has been observed previously in cca1 and lhy mutant plants (39, 40) as well as in our experiments (Fig. S2). The cca1-11/lhy-21 double mutations also had differing effects on the circadian regulation of the three CBF genes (Fig. 1); circadian regulation of CBF1 and CBF3 was essentially eliminated in the double-mutant plants, but CBF2 transcript levels clearly continued to cycle, although the amplitude was diminished, and the period was greatly shortened. Period shortening of output genes such as LHCB also has been observed previously in cca1-11/lhy-21 double-mutant plants (40, 41) and in our experiments (Fig. S3). From these results we concluded that circadian regulation of CBF1 and CBF3 is dependent on the action of either CCA1 or LHY and that circadian regulation of CBF2 involves action of CCA1 and LHY but can be driven to a considerable degree by other unknown factors.

Fig. 2.

Fig. 2.

Open in a new tab

Binding of CCA1 at the CBF1-3 locus. cca1-1 and cca1-1 CCA1p:CCA1-GFP plants were grown at 22 °C under a 12-h photoperiod to the four-leaf stage. Tissue was fixed at ZT4, and ChIP was performed using anti-GFP antibody. Immunoprecipitated DNA was quantified by qRT-PCR using primers specific to regions within the CBF1-3 locus (boxes A through O). The levels of immunoprecipitated DNA were normalized to the respective input DNA. Immunoprecipitation in cca1-1 CCA1p:CCA1-GFP plants (black bars) is relative to cca1-1 plants (gray bars) set to a value of 1. Primer pairs directed to the 3′ UTR of TOC1 (TOC1-3′UTR), ACTIN 7, and UBIQUITIN 10 (UBQ10) were used as negative controls (31). Primers near the EE element in the TOC1 promoter (TOC1-EE) were used as a positive control. Values represent the average of five independent biological experiments. Error bars indicate SEM. *P < 0.05 using a paired, one-tailed t test. In the CBF locus diagram, the transcribed regions are indicted by white boxes, and the approximate positions of CBS [(A2–4)TCT], and EE (AAAATATCT) motifs are indicated by gray circles and white squares, respectively.

CCA1 and LHY may impart circadian regulation of CBF1, CBF2, and CBF3 by binding to the EE and CBS motifs present in the CBF promoters and act as positive regulators stimulating transcription. To test this hypothesis, we conducted ChIP experiments to determine whether CCA1 binds directly to the promoter regions of CBF1, CBF2, and CBF3. To do so, we compared ChIP results obtained with plants carrying the cca1-1 mutation and cca1-1 plants that had been restored with a construct encoding the CCA1 protein tagged with GFP under the endogenous CCA1 promoter (31). Chromatin was isolated from plants harvested at ZT4, the point when CBF transcript levels begin to rise. In mock experiments where rabbit IG was used for precipitation, no specific binding was detected for any of the subregions (A to O in Fig. S4) of the CBF1-3 locus tested or for TOC1, ACTIN 7, or UBIQUITIN 10 (Fig. S4). In contrast, test experiments indicated that specific binding of the CCA1-GFP protein occurred throughout most of the CBF1-3 locus and also within the promoter region of TOC1, a positive control, but not in the promoters of ACTIN 7 or UBIQUITIN 10, two negative controls (31) (Fig. 2). Significant CCA1-GFP binding occurred in the promoter regions of CBF1, CBF2, and CBF3 (C, L, and G and I, respectively in Fig. 2). We also observed CCA1 associated with the coding region of CBF1 (E in Fig. 2), perhaps because of the tandem connection of the CBF genes and consequently close proximity to several CCA1 binding sites located in the adjacent CBF3 promoter (Fig. 2). Thus, although it is possible that there is CCA1 binding within the CBF1 transcript region, the actual binding site could be downstream in the CBF3 promoter. In sum, the results of our genetic and ChIP experiments support the model that circadian regulation of CBF1, CBF2, and CBF3 involves action of CCA1 and LHY binding to the promoters of these genes and up-regulating their transcription during the morning hours.

Rhythmic Expression of CBF Regulon COR Genes and Freezing Tolerance Are Impaired in Plants Carrying the cca1-11/lhy-21 Double Mutation.

Harmer et al. (19) suggested that the circadian regulation of CBF1, CBF2, and CBF3 could result in rhythmic expression of CBF regulon COR genes. To test this possibility, we examined the transcript levels for three CBF-inducible genes—COR15A, COR47, and COR78—in wild-type plants and in plants carrying the cca1-11/lhy-21 double mutation. The results indicated that the transcript levels for all three COR genes oscillated with a period of about 24 h in wild-type plants, although for COR15A and COR78 the amplitude of the second peak was much less than that of the first peak (Fig. 1). For all three genes, the first peak occurred at about ZT10, consistent with the transcript levels of CBF1, CBF2, and CBF3 peaking just before this time, at about ZT8 (19, 20) (Fig. 1). Moreover, the oscillation in COR transcript levels was largely reduced in the cca1-11/lhy-21 double-mutant plants (Fig. 1). These results were consistent with the model that circadian-regulated expression of CBF1, CBF2, and CBF3 imparts rhythmic expression of CBF-targeted COR genes at “basal” nonacclimating temperatures.

The decrease in expression of the CBF-targeted COR genes in the cca1-11/lhy-21 double-mutant plants could result in a decrease in basal freezing tolerance. We tested this possibility by using the electrolyte leakage assay to compare the freezing tolerance of wild-type and cca1-11/lhy-21 plants. The results indicated that the cca1-11/lhy-21 double mutation reduced freezing tolerance by about 50%; although the temperature at which cell damage results in release of 50% of total electrolytes (EL50) was about −4 °C in wild-type plants, it was about −2 °C in the cca1-11/lhy-21 mutant plants (Fig. 3). Thus, the circadian clock is required for maximum basal freezing tolerance.

Fig. 3.

Fig. 3.

Open in a new tab

Effect of the cca1-11/lhy-21 double mutation on plant freezing tolerance. cca1-11/lhy-21 double-mutant and wild-type Ws-2 (WS) plants were grown at 22 °C under a 12-h photoperiod for ∼18 d and were tested directly for freezing tolerance (nonacclimated plants; Upper) or were transferred at ZT4 to 4 °C for 7 d under a 12-h photoperiod and then tested for freezing tolerance (cold-acclimated plants; Lower). Freezing tolerance was tested using the electrolyte leakage test. The results presented are average values from three independent experiments. Error bars indicate SEM.

CCA1 and LHY Regulate Cold Induction of CBF1, CBF2, and CBF3.

Fowler et al. (22) reported that cold induction of CBF1, CBF2, and CBF3 is gated by the circadian clock. Given our results indicating a role for CCA1 and LHY in the circadian regulation of CBF1, CBF2, and CBF3, we asked whether these transcription factors also had a role in the gating phenomenon. As previously reported (22, 34), cold-induction of CBF1, CBF2, and CBF3 was much greater in the subjective day than in the subjective evening (Fig. 4). This cold induction was little affected by the single cca1-11 and lhy-21 mutations (Fig. S1) but was greatly reduced in the cca1-11/lhy-21 double mutants, and the period of cycling was shortened (Fig. 4), as was the period of cycling for LHCB (Fig. S3). Thus, CCA1 and LHY have a major role in the induction of CBF1, CBF2, and CBF3 expression in response to low temperature.

Fig. 4.

Fig. 4.

Open in a new tab

Effects of the cca1-11/lhy-21 double mutation on cold induction of CBF1, CBF2, CBF3, and CBF-targeted genes COR15A, COR47, and COR78. Wild-type Ws-2 (WS) and cca1-11/lhy-21 double-mutant plants were grown at 22 °(C under a 12- h photoperiod to the four- leaf stage and then were transferred to constant light at ZT0 (subjective day and night are indicated by white and gray bars, respectively). Plants were transferred to cold temperature (4 °C) for 2 h, every 2 h (CBF genes) or for 4 h every 4 h (COR genes) at the start of constant-light conditions. Transcript levels for the indicated genes were determined by qRT-PCR. The x axis represents the time (ZT) when plants were transferred to cold temperature. Gene expression was normalized to UBQ10 for each sample. Gene expression is relative to one wild-type sample set to a value of 1 for each biological replicate. Values are averages from three independent biological experiments. Error bars indicate SEM. Primer pair sequences are given in Table S1.

Cold induction of CBF-Targeted COR Genes and Freezing Tolerance Are Impaired in Plants Carrying the cca1-11/lhy-21 Double Mutation.

The finding that cold induction of CBF1, CBF2, and CBF3 was impaired in plants carrying the cca1-11/lhy-21 double mutation prompted us to determine whether the double mutation also impaired cold induction of CBF-targeted COR genes and the freezing tolerance of cold-acclimated plants. In wild-type plants, the degree to which COR15A, COR47, and COR78 were induced by low temperature cycled, with peaks in the late day and troughs in the late subjective evening (Fig. 4), times that were consistent with the cycling of CBF1, CBF2, and CBF3 cold induction (Fig. 4). In plants carrying the cca1-11/lhy-21 double mutation, the peak following the night-to-day transition was little affected, but the subsequent peaks were greatly diminished, and the period of cycling was shortened (Fig. 4). In addition, the freezing tolerance of cold-acclimated plants carrying the cca1-11/lhy-21 double mutation was about 1 °C less than that of cold-acclimated wild-type plants (Fig. 3). These results indicate that CCA1 and LHY are required for Arabidopsis plants to attain maximum levels of COR gene induction and freezing tolerance in response to low temperature.

Discussion

The CBF cold-response pathway is highly conserved among plants and has a major role in plant freezing tolerance (1–3, 9). Accordingly, there is considerable interest in understanding the mechanisms that control expression of this stress-response pathway. Here we establish that the CBF pathway is subject to positive regulation by the circadian clock components CCA1 and LHY. We show that these factors have roles in both circadian regulation and cold induction of the pathway and that they are required for plants to attain maximum freezing tolerance at both basal and cold-acclimating temperatures.

At basal growth temperature, the transcript levels for CBF1, CBF2, and CBF3 oscillate, with peaks and troughs occurring at about ZT8 and ZT20, respectively (19–21) (Fig. 1). Our genetic and ChIP analyses indicate that this circadian regulation is caused by the direct action of CCA1 and LHY binding at the CBF1-3 locus—presumably at the EE, CBS, and related motifs—and induces transcription of the CBF genes. In the morning hours, when CCA1 and LHY protein levels peak (25, 35, 36), the transcript levels of CBF1, CBF2, and CBF3 peak; in the evening hours, when CCA1 and LHY protein levels are low, the transcript levels for CBF1, CBF2, and CBF3 are low (Fig. 5). The finding that circadian regulation of CBF1 and CBF3 is nearly eliminated in plants carrying the cca1-11/lhy-21 double mutation indicates that no other regulatory proteins are sufficient to impart positive circadian regulation of these genes. In contrast, circadian regulation of CBF2 continues in plants carrying the cca1-11/lhy-21 double mutation, albeit with reduced amplitude and shortened periodicity. Thus, at least one additional regulatory protein appears to drive positive circadian regulation of CBF2. Prime candidates for this residual regulation are the four REVEILLE (RVE) proteins RVE1, RVE3, RVE4, and RVE8 (42). These Myb-like transcription factors fall into the CCA1 subfamily, bind to the EE motif, are circadian regulated, and, like CCA1 and LHY, have peak transcript levels at dawn (42).

Fig. 5.

Fig. 5.

Open in a new tab

Model for circadian regulation and gated cold induction of CBF1, CBF2, and CBF3. During the day, CCA1 and LHY bind throughout the CBF locus and promote CBF transcription. In the evening, CCA1 and LHY are at low levels and have little effect on CBF expression. Oscillations in CCA1 and LHY binding at the CBF locus largely account for the circadian regulation of the CBF genes. Circadian regulation also involves repression in the evening hours mediated by PIF7 (21) and PRR5, PRR7, and PRR9 (34). Transfer of plants to low temperature in the day or evening results in activation of ICE1 (15), CAMTA3 (CM3) (16), and possibly other transcription factors that stimulate transcription of the CBF genes. If the temperature drops during the day, the clock and cold-signaling pathways act synergistically to induce CBF expression to high levels. If plants are exposed to cold temperatures in the evening, there is no positive synergy between the two pathways, and there is repression by PRR5, PRR7, and PRR9 (34) leading to CBF induction at moderate levels. Relative transcript levels were calculated using peak and trough values obtained for the CBF genes in the experiments presented in Figs. 1 and 4 (the values obtained for plants grown at warm temperature and harvested in the evening were set to 1). See Discussion for details.

Kidokoro et al. (21) reported that circadian regulation of CBF1, CBF2, and CBF3 also involves negative regulation. These investigators found that PHYTOCHROME INTERACTING FACTOR 7 (PIF7) binds to a G-box element in the promoter of CBF2 and that this element is required for down-regulation of the CBF2 promoter during the subjective evening. In addition, they found that PIF7 physically interacts with TOC1 (21). Thus, circadian-controlled down-regulation of the CBF genes appears to involve action of a PIF7–TOC1 protein complex binding to G-box elements in their promoters (Fig. 5).

In addition to establishing a role for CCA1 and LHY in circadian regulation of CBF1, CBF2, and CBF3, our results indicate that CCA1 and LHY also act as positive regulators of CBF cold induction. This action is evidenced by the finding that cold induction of CBF1, CBF2, and CBF3 is greatly impaired in plants carrying the cca1-11/lhy-21 double mutation (Fig. 4). We propose that the gating of CBF1, CBF2, and CBF3 cold induction results, in part, from positive synergistic interaction between cold-signaling and clock-output pathways, the former mediated by ICE1 and CAMTA3 and the latter by CCA1 and LHY (Fig. 5). If the temperature drops in the morning, CCA1 and LHY are present at the CBF locus and can act with ICE1 and CAMTA3 to induce high-level expression of CBF1, CBF2, and CBF3. In contrast, if the temperature drops in the evening, CCA1 and LHY are at low levels; consequently there is little synergy between the low-temperature and clock pathways, and the induction of CBF1, CBF2, and CBF3 is low, approximating the peak levels obtained with circadian regulation (Fig. 5).

Cold induction of CBF1, CBF2, and CBF3 during the evening hours also may involve negative regulation. Such regulation would not appear to involve PIF7, because Kidokoro et al. (21) showed that the gating of CBF1, CBF2, and CBF3 expression is not impaired in plants carrying the pif7-2 mutation (21). However, Nakamichi et al. (34) found that circadian regulation of CBF1, CBF2, and CBF3 and the gating of their cold induction do not occur in plants carrying the prr9-11/prr7-10/prr5-10 triple mutation. When plants were grown at basal temperature, the transcript levels for CBF1, CBF2, and CBF3 remained high throughout the day in the triple-mutant plants (34). Similarly, the cold induction of CBF1, CBF2, and CBF3 in the triple-mutant plants remained at about the peak levels observed in wild-type plants regardless of the time of day at which the mutant plants were exposed to low temperature (34). Nakamichi et al. (34) concluded that PRR9, PRR7, and PRR5 are negative regulators of CBF1, CBF2, and CBF3 and proposed two possible mechanisms. One is that PRR9, PRR7, and PRR5 directly repress expression of the CBF genes. Alternatively, they suggested that aberrant expression of the CBF genes might result from the “circadian disorder” caused by the prr9-11/prr7-10/prr5-10 triple mutation. Our results provide no direct evidence in favor of or against the first model. However, the consistently high CBF expression may be explained in part by the constitutively elevated expression of CCA1 and LHY in the triple-mutant plants (34, 43).

A final point should be mentioned in regard to the role of the clock in freezing tolerance. Our results indicate that CCA1 and LHY are required for Arabidopsis to attain maximum levels of freezing tolerance at both non-acclimating and cold-acclimating temperatures (Fig. 3). Recently, Espinoza et al. (44) independently reached the same conclusion; they too found that the cca1-11/lhy-21 double mutation resulted in impaired freezing tolerance. Our results also indicate a mechanism whereby cold-signaling and clock-regulatory pathways are integrated to condition freezing tolerance: the positive regulation of the CBF cold-response pathway mediated through CCA1 and LHY binding at the CBF1–3 locus and inducing expression of CBF1, CBF2, and CBF3 (Fig. 5). Taken together, our results suggest that the integration of cold-signaling pathways with the circadian clock may have been an important evolutionary event that has contributed to plant adaptation to cold environments.

Materials and Methods

Plant Material and Growth Conditions.

Arabidopsis thaliana ecotype Ws-2 and mutants in this background were grown as described previously (16). Homozygous T-DNA mutant lines were obtained from the Arabidopsis Biological Resource Center (45). Null mutations were checked by quantitative RT-PCR (qRT-PCR). These lines were cca1-11(CS9378), lhy-21 (CS9379), and cca1-11/lhy-21(CS9380). Restored cca1-1 line, CCA1p:CCA1-GFP under the CCA1 endogenous promoter and cca1-1 (31), used in ChIP experiments, were generously donated by the Kay Laboratory (University of California, San Diego, La Jolla, CA).

All seeds were stratified for 3–5 d in the dark at 4 °C. Except for freezing-tolerance tests, plants were grown at 22 °C under sterile conditions on Gamborg's B5 medium (Caisson Laboratories) without sucrose at ∼100 μmol m−2 s−1 in a 12-h photoperiod. For circadian experiments, plants were sampled at 22 °C in 100 μmol m−2 s−1 constant light or at 4 °C in 35 μmol m−2 s−1 constant light. For electrolyte leakage experiments, plants were grown as described (16) at ∼100 μmol m−2 s−1 under a 12-h photoperiod. Cold- temperature treatment for plants grown on soil was at 4 °C in light at 35 μmol m−2 s−1 under a 12-h photoperiod.

RNA Analysis.

RNA extraction was performed as described in ref. 16. For qRT-PCR (Applied Biosystems 7500 FAST Real-Time PCR System in FAST mode), cDNA was made as described in ref. 16, except that total RNA of either 0.2 or 0.025 μg was used for a 40-μL reverse-transcription reaction. In the 10-μL PCR reactions, 2 μL of diluted cDNA was used. UBQ10 or IPP2 were used as reference genes. All primer sets are listed in Table S1.

ChIP.

ChIP experiments were carried out as described by Pruneda-Paz et al. (31) with a few modifications. CCA1p:CCA1-GFP and cca1-1 lines were sampled at ZT4 instead of ZT3. DNA was purified using the PCR Clean-Up Kit (Qiagen) instead of by phenol-chloroform extraction. Immunoprecipitated DNA was analyzed with Applied Biosystems FAST real-time PCR in FAST mode (using presets). For each biological replicate immunoprecipitated DNA was normalized to the input DNA as in ref. 31, and each of these values was expressed relative to the cca1-1 line set to a value of 1. A one-tailed paired t test was performed to assess the statistical significance of enrichment in the CCA1p:CCA1-GFP line compared with cca1-1 plants for each primer pair used across biological replicates. Primer pairs used in ChIP experiments are listed in Table S1.

Freezing-Tolerance Tests.

Electrolyte leakage assays were performed as described in ref. 16. For cold acclimation, plants were transferred to 4 °C at ZT4 for 7 d under a 12-h photoperiod. Assays for acclimated and nonacclimated plants started at ∼ZT2 in all biological replicates.

Supplementary Material

Supporting Information
supp_108_17_7241__index.html (803B, html)

Acknowledgments

We thank Steve Kay for providing the Arabidopsis lines carrying the cca1-1 mutation and the restored line expressing the CCA1-GFP fusion and are grateful to Sarah Gilmour for her help in preparing the manuscript. This research was funded by Grant DBI 0701709 from the National Science Foundation Plant Genome Project; by Grant DE-FG02-91ER20021from the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the US Department of Energy; and by the Michigan Agricultural Experiment Station.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1103741108/-/DCSupplemental.

References

  • 1.Thomashow MF. Plant cold acclimation: Freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol Plant Mol Biol. 1999;50:571–599. doi: 10.1146/annurev.arplant.50.1.571. [DOI] [PubMed] [Google Scholar]
  • 2.Ruelland EVM, Zachowski A, Vaughn H. Cold signaling and cold acclimation in plants. Adv Bot Res. 2009;49:36–54. [Google Scholar]
  • 3.Chinnusamy V, Zhu J, Zhu JK. Cold stress regulation of gene expression in plants. Trends Plant Sci. 2007;12:444–451. doi: 10.1016/j.tplants.2007.07.002. [DOI] [PubMed] [Google Scholar]
  • 4.Maruyama K, et al. Identification of cold-inducible downstream genes of the Arabidopsis DREB1A/CBF3 transcriptional factor using two microarray systems. Plant J. 2004;38:982–993. doi: 10.1111/j.1365-313X.2004.02100.x. [DOI] [PubMed] [Google Scholar]
  • 5.Hannah MA, Heyer AG, Hincha DK. A global survey of gene regulation during cold acclimation in Arabidopsis thaliana. PLoS Genet. 2005;1:e26. doi: 10.1371/journal.pgen.0010026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Fowler S, Thomashow MF. Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell. 2002;14:1675–1690. doi: 10.1105/tpc.003483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Chew YH, Halliday KJ. A stress-free walk from Arabidopsis to crops. Curr Opin Biotechnol. 2010;22:1–6. doi: 10.1016/j.copbio.2010.11.011. [DOI] [PubMed] [Google Scholar]
  • 8.Hua J. From freezing to scorching, transcriptional responses to temperature variations in plants. Curr Opin Plant Biol. 2009;12:568–573. doi: 10.1016/j.pbi.2009.07.012. [DOI] [PubMed] [Google Scholar]
  • 9.Thomashow MF. Molecular basis of plant cold acclimation: Insights gained from studying the CBF cold response pathway. Plant Physiol. 2010;154:571–577. doi: 10.1104/pp.110.161794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Riechmann JL, Meyerowitz EM. The AP2/EREBP family of plant transcription factors. Biol Chem. 1998;379:633–646. doi: 10.1515/bchm.1998.379.6.633. [DOI] [PubMed] [Google Scholar]
  • 11.Vogel JT, Zarka DG, Van Buskirk HA, Fowler SG, Thomashow MF. Roles of the CBF2 and ZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis. Plant J. 2005;41:195–211. doi: 10.1111/j.1365-313X.2004.02288.x. [DOI] [PubMed] [Google Scholar]
  • 12.Liu Q, et al. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell. 1998;10:1391–1406. doi: 10.1105/tpc.10.8.1391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, Schabenberger O, Thomashow MF. Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science. 1998;280:104–106. doi: 10.1126/science.280.5360.104. [DOI] [PubMed] [Google Scholar]
  • 14.Gilmour SJ, Fowler SG, Thomashow MF. Arabidopsis transcriptional activators CBF1, CBF2, and CBF3 have matching functional activities. Plant Mol Biol. 2004;54:767–781. doi: 10.1023/B:PLAN.0000040902.06881.d4. [DOI] [PubMed] [Google Scholar]
  • 15.Chinnusamy V, et al. ICE1: A regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes Dev. 2003;17:1043–1054. doi: 10.1101/gad.1077503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Doherty CJ, Van Buskirk HA, Myers SJ, Thomashow MF. Roles for Arabidopsis CAMTA transcription factors in cold-regulated gene expression and freezing tolerance. Plant Cell. 2009;21:972–984. doi: 10.1105/tpc.108.063958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Miura K, et al. SIZ1-mediated sumoylation of ICE1 controls CBF3/DREB1A expression and freezing tolerance in Arabidopsis. Plant Cell. 2007;19:1403–1414. doi: 10.1105/tpc.106.048397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Knight H, Trewavas AJ, Knight MR. Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation. Plant Cell. 1996;8:489–503. doi: 10.1105/tpc.8.3.489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Harmer SL, et al. Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. Science. 2000;290:2110–2113. doi: 10.1126/science.290.5499.2110. [DOI] [PubMed] [Google Scholar]
  • 20.Bieniawska Z, et al. Disruption of the Arabidopsis circadian clock is responsible for extensive variation in the cold-responsive transcriptome. Plant Physiol. 2008;147:263–279. doi: 10.1104/pp.108.118059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Kidokoro S, et al. The phytochrome-interacting factor PIF7 negatively regulates DREB1 expression under circadian control in Arabidopsis. Plant Physiol. 2009;151:2046–2057. doi: 10.1104/pp.109.147033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Fowler SG, Cook D, Thomashow MF. Low temperature induction of Arabidopsis CBF1, 2, and 3 is gated by the circadian clock. Plant Physiol. 2005;137:961–968. doi: 10.1104/pp.104.058354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Pruneda-Paz JL, Kay SA. An expanding universe of circadian networks in higher plants. Trends Plant Sci. 2010;15:259–265. doi: 10.1016/j.tplants.2010.03.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Harmer SL. The circadian system in higher plants. Annu Rev Plant Biol. 2009;60:357–377. doi: 10.1146/annurev.arplant.043008.092054. [DOI] [PubMed] [Google Scholar]
  • 25.Wang ZY, Tobin EM. Constitutive expression of the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene disrupts circadian rhythms and suppresses its own expression. Cell. 1998;93:1207–1217. doi: 10.1016/s0092-8674(00)81464-6. [DOI] [PubMed] [Google Scholar]
  • 26.Schaffer R, et al. The late elongated hypocotyl mutation of Arabidopsis disrupts circadian rhythms and the photoperiodic control of flowering. Cell. 1998;93:1219–1229. doi: 10.1016/s0092-8674(00)81465-8. [DOI] [PubMed] [Google Scholar]
  • 27.Mizoguchi T, et al. LHY and CCA1 are partially redundant genes required to maintain circadian rhythms in Arabidopsis. Dev Cell. 2002;2:629–641. doi: 10.1016/s1534-5807(02)00170-3. [DOI] [PubMed] [Google Scholar]
  • 28.Alabadí D, Yanovsky MJ, Más P, Harmer SL, Kay SA. Critical role for CCA1 and LHY in maintaining circadian rhythmicity in Arabidopsis. Curr Biol. 2002;12:757–761. doi: 10.1016/s0960-9822(02)00815-1. [DOI] [PubMed] [Google Scholar]
  • 29.Matsushika A, Makino S, Kojima M, Mizuno T. Circadian waves of expression of the APRR1/TOC1 family of pseudo-response regulators in Arabidopsis thaliana: Insight into the plant circadian clock. Plant Cell Physiol. 2000;41:1002–1012. doi: 10.1093/pcp/pcd043. [DOI] [PubMed] [Google Scholar]
  • 30.Alabadí D, et al. Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock. Science. 2001;293:880–883. doi: 10.1126/science.1061320. [DOI] [PubMed] [Google Scholar]
  • 31.Pruneda-Paz JL, Breton G, Para A, Kay SA. A functional genomics approach reveals CHE as a component of the Arabidopsis circadian clock. Science. 2009;323:1481–1485. doi: 10.1126/science.1167206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Farré EM, Harmer SL, Harmon FG, Yanovsky MJ, Kay SA. Overlapping and distinct roles of PRR7 and PRR9 in the Arabidopsis circadian clock. Curr Biol. 2005;15:47–54. doi: 10.1016/j.cub.2004.12.067. [DOI] [PubMed] [Google Scholar]
  • 33.Nakamichi N, et al. PSEUDO-RESPONSE REGULATORS 9, 7, and 5 are transcriptional repressors in the Arabidopsis circadian clock. Plant Cell. 2010;22:594–605. doi: 10.1105/tpc.109.072892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Nakamichi N, et al. Transcript profiling of an Arabidopsis PSEUDO RESPONSE REGULATOR arrhythmic triple mutant reveals a role for the circadian clock in cold stress response. Plant Cell Physiol. 2009;50:447–462. doi: 10.1093/pcp/pcp004. [DOI] [PubMed] [Google Scholar]
  • 35.Wang ZY, et al. A Myb-related transcription factor is involved in the phytochrome regulation of an Arabidopsis Lhcb gene. Plant Cell. 1997;9:491–507. doi: 10.1105/tpc.9.4.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Hamilton EE, Kay SA. SnapShot: Circadian clock proteins. Cell. 2008;135:368–368e1. doi: 10.1016/j.cell.2008.09.042. [DOI] [PubMed] [Google Scholar]
  • 37.Mockler TC, et al. The DIURNAL project: DIURNAL and circadian expression profiling, model-based pattern matching, and promoter analysis. Cold Spring Harb Symp Quant Biol. 2007;72:353–363. doi: 10.1101/sqb.2007.72.006. [DOI] [PubMed] [Google Scholar]
  • 38.Green RM, Tobin EM. Loss of the circadian clock-associated protein 1 in Arabidopsis results in altered clock-regulated gene expression. Proc Natl Acad Sci USA. 1999;96:4176–4179. doi: 10.1073/pnas.96.7.4176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Hall A, et al. The TIME FOR COFFEE gene maintains the amplitude and timing of Arabidopsis circadian clocks. Plant Cell. 2003;15:2719–2729. doi: 10.1105/tpc.013730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Gould PD, et al. The molecular basis of temperature compensation in the Arabidopsis circadian clock. Plant Cell. 2006;18:1177–1187. doi: 10.1105/tpc.105.039990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Locke JCW, et al. Extension of a genetic network model by iterative experimentation and mathematical analysis. Mol Syst Biol. 2005 doi: 10.1038/msb4100018. 10.1038/msb4100018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Gong W, et al. The development of protein microarrays and their applications in DNA-protein and protein-protein interaction analyses of Arabidopsis transcription factors. Mol Plant. 2008;1:27–41. doi: 10.1093/mp/ssm009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Fukushima A, et al. Impact of clock-associated Arabidopsis pseudo-response regulators in metabolic coordination. Proc Natl Acad Sci USA. 2009;106:7251–7256. doi: 10.1073/pnas.0900952106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Espinoza C, et al. Interaction with diurnal and circadian regulation results in dynamic metabolic and transcriptional changes during cold acclimation in Arabidopsis. PLoS ONE. 2010;5:e14101. doi: 10.1371/journal.pone.0014101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Alonso JM, et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science. 2003;301:653–657. doi: 10.1126/science.1086391. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information
supp_108_17_7241__index.html (803B, html)
1103741108_pnas.201103741SI.pdf (628KB, pdf)

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES