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. 2023 Feb 1;12:e84594. doi: 10.7554/eLife.84594

Figure 3. Protein levels of CBFs increase during the vernalization process.

(A) Transcript levels of CBF1, 2, and 3 under short-term cold exposure. Wild-type plants were subjected to 0, 1, 3, 6, 12, and 24 hr of 4 °C cold treatment. Relative levels of CBF1, 2, and 3 were normalized to that of UBC. Values have been represented as mean ± SEM of three biological replicates. The blue shadings indicate cold periods. Significant differences have been marked using different letters (a–c; p < 0.05; one-way ANOVA followed by Tukey’s post-hoc test). (B) Transcript levels of CBF1, 2, and 3 after 20V and 40V. Wild-type plants were treated with 4 °C vernalization under an SD cycle and collected at ZT4. Relative levels of CBF1, 2, and 3 were normalized to that of UBC. Values have been represented as mean ± SEM of three biological replicates. The blue shadings denote periods under cold. Significant differences have been marked using different letters (a–c; p < 0.05; one-way ANOVA followed by Tukey’s post-hoc test). (C) Daily rhythms of CBF1, 2, and 3 transcript levels in NV or 40V plants. Col-0 plants grown under an SD cycle were collected every 4 hr between ZT0 and ZT24. Relative transcript levels of CBF1, 2, and 3 were normalized to that of PP2A. Values have been represented as mean ± SEM of three biological replicates. The white and black bars represent light and dark periods, respectively. Asterisks indicate a significant difference between NV and 40V (***, p < 0.001; two-way ANOVA). (D) Dynamics of CBF3 protein level under short-term cold exposure. The pCBF3:CBF3-myc transgenic plants were subjected to 0, 1, 3, 6, 12, and 24 hr of 4 °C cold treatment. CBF3 proteins were detected using anti-myc antibodies. Rubisco was considered the loading control. Numbers below each band indicate relative signal intensity compared to 6 hr. The mean values of two biological replicates are presented. (E) Increase of CBF3 protein level during the vernalization process. The pCBF3:CBF3-myc transgenic plants, subjected to 4 °C vernalization, were collected at ZT4 of the indicated time point. CBF3 proteins were detected using anti-myc antibodies. Rubisco was considered the loading control. Numbers below each band indicate relative signal intensity compared to 1V. The mean values of three biological replicates are presented.

Figure 3—source data 1. Uncropped labeled blot images and the original image files for the immunoblots.

Figure 3.

Figure 3—figure supplement 1. Vernalization increases CBF3 protein levels with a minor effect on the daily rhythm.

Figure 3—figure supplement 1.

Figure 3—figure supplement 1—source data 1. Uncropped labeled blot images and the original image files for the immunoblots.