Low temperature induction of Arabidopsis CBF1, 2, and 3 is gated by the circadian clock

Abstract

Exposing Arabidopsis (Arabidopsis thaliana) plants to low temperature results in rapid induction of CBF1, 2, and 3 (CBF1-3; also known as DREB1B, C, and A, respectively), which encode transcriptional activators that induce expression of a battery of genes that increase plant freezing and chilling tolerance. Recently, it has been shown that basal levels of CBF3 transcripts and those of certain CBF-regulated genes exhibit circadian cycling. Here, we further explored the regulation of CBF1-3 by the circadian clock. The results indicated that the extent to which CBF1-3 transcripts accumulated in response to low temperature was dependent on the time of day that the plants were exposed to low temperature and that this was regulated by the circadian clock. The highest and lowest levels of cold-induced CBF1-3 transcript accumulation occurred at 4 and 16 h after subjective dawn, respectively. An analysis of CBF2 promoter-reporter gene fusions indicated that this control included transcriptional regulation. In addition, the cold responsiveness of RAV1 and ZAT12, genes that are cold induced in parallel with CBF1-3, was also subject to circadian regulation. However, whereas the maximum level of cold-induced RAV1 transcript accumulation occurred at the same time of day as did CBF1-3 transcripts, that of ZAT12 was in reverse phase, i.e. the highest level of cold-induced ZAT12 transcript accumulation occurred 16 h after subjective dawn. These results indicate that cold-induced expression of CBF1-3, RAV1, and ZAT12 is gated by the circadian clock and suggest that this regulation likely occurs through at least two nonidentical (though potentially overlapping) signaling pathways.

Figure 1.

The extent to which CBF1-3 transcripts accumulate in response to low temperature depends on the time of day at which plants are exposed to the cold. Arabidopsis plants were grown in a 12:12 photoperiod at 24°C. Plates were transferred to low temperature (4°C) at either ZT4 or ZT16 and samples harvested after the indicated times along with samples from plates that had been maintained at 24°C. RNA gel blots were prepared from total RNA and hybridized with gene-specific probes for CBF1-3. rRNA stained with ethidium bromide was used to compare loading.

Figure 2.

The circadian clock gates CBF1-3 expression levels in response to low temperature. Arabidopsis plants were grown in a 12:12 photoperiod at 24°C and then released into LL at ZT0. Plants were then transferred to low temperature (4°C) at 6-h intervals beginning at ZT4. Samples were harvested from the cold-treated plants (4°C) and also from plants held at 24°C after 1, 4, 8, and 24 h (shown left to right above each temperature label). A, RNA gel blots were prepared from total RNA and hybridized with a full-length CBF2 probe (CBF2-FL) that cross-hybridizes with CBF1 and CBF3 transcripts. Results are presented as a proportion of the highest value after normalization with respect to eIF4a expression levels. White and hatched boxes indicate subjective day and night, respectively. The first lane for each ZT time sample represents samples harvested before temperature treatment. B, RNA gel blots prepared from selected RNA samples derived from a repeat of experiment (A) above were hybridized with gene-specific probes for CBF1-3.

Figure 3.

Constitutive expression of CCA1 abolishes gating by the circadian clock of CBF1-3 induction in response to low temperature. CCA1-OX plants were grown under a 12-h photoperiod at 24°C, transferred to LL at ZT0, and then exposed to low temperature (4°C, LL) for the indicated periods at 12-h intervals beginning at ZT4. These samples were grown and harvested concurrently with the wild-type samples shown in Figure 1. Total RNA was isolated and RNA blots prepared, which were hybridized with the CBF2-FL probe. In the bottom portion of the figure, the hybridization results are presented as a proportion of the highest value after normalization with respect to 25S rRNA expression levels.

Figure 4.

Circadian gating of CBF1-3 cold responsiveness involves transcriptional regulation. Transgenic Arabidopsis plants, carrying the GUS reporter gene under the control of 1-kb and 155-bp fragments of the CBF2 promoter, CBF2::GUS and 155::GUS, respectively (see text), were grown and treated as described in the legend for Figure 3. RNA gel blots, prepared from total RNA, were hybridized with probes for GUS and CBF2-FL. A, RNA gel blot showing cold responsiveness of the CBF2::GUS reporter gene. B, Graph of data from A showing mean expression of GUS at time points where cold induction of the CBF genes is maximal (1 and 4 h at 4°C). Mean expression levels are presented as a proportion of the highest value after normalization with respect to 25S rRNA expression levels. C, RNA gel blot showing cold responsiveness of the 155::GUS reporter gene. D, Graph of data from C showing mean expression of GUS at time points where cold induction of the CBF genes is maximal (1 and 4 h at 4°C). Mean expression levels are presented as a proportion of the highest value after normalization with respect to 25S rRNA expression levels. The subtle differences in the kinetics observed for cold-induced accumulation of the endogenous CBF and reporter GUS transcripts at ZT4, 28, and 52 are likely to involve differences in transcript stability and, potentially, differences in promoter activity.

Figure 5.

Cold induction of the RAV1 and ZAT12 genes is gated by the circadian clock. Wild-type Arabidopsis plants were grown and treated as described in the legend for Figure 3. A, RNA gel blots prepared from total RNA were hybridized with probes for RAV1 and ZAT12. B, Graph of data from A showing mean expression of RAV1 and ZAT12 at time points where cold induction of these genes is maximal (i.e. 1 and 4 h at 4°C). Mean expression levels are presented as a proportion of the highest value after normalization with respect to 25S rRNA expression levels.

Figure 6.

CBF target genes show attenuated circadian gating in response to low temperature. Wild-type Arabidopsis plants were grown and treated as described in the legend for Figure 3, except that plants were transferred to 4°C at 12-h intervals beginning at ZT12. A, RNA gel blots prepared from total RNA were hybridized with probes for COR78, COR6.6, and CBF2-FL. B, Graph of data from A showing mean expression of COR78 and COR6.6 at time points where these genes show induction by cold (4 h, 8, and 24 h at 4°C). Mean expression levels are presented as a proportion of the highest value for each gene after normalization with respect to 25S rRNA expression levels.