Abstract
Main conclusion
Vernalization-mediated demethylation of BrCKA2 (casein kinase II α-subunit) and BrCKB4 (casein kinase II β-subunit) shorten the period of the clock gene BrCCA1 (circadian clock associated 1) in Brassica rapa.
Photoperiod and vernalization are two environmental cues involved in the regulation of floral transition, but the ways in which they interact remain unclear. DNA methylation is one of the main mechanisms involved in controlling the functional state of chromatin and gene expression in response to environmental signals. To study the interaction between photoperiod and vernalization in floral transition, we carried out a comparative genomic analysis of genome-wide DNA methylation profiles in normal (CK) and vernalized (CA) leaves from Brassica rapa using methylated-DNA immunoprecipitation sequencing (MeDIP-seq). Two subunits of casein kinase II (CK2), BrCKA2 (catalytic α-subunit of CK2) and BrCKB4 (regulatory β-subunit of CK2), exhibited gradual DNA demethylation and increased expression in vernalized B. rapa. DNA methylation-defective plants demonstrated the causal link between DNA demethylation changes and changes in gene expression. Virus-induced gene silencing (VIGS) of BrCKA2 and BrCKB4 in B. rapa resulted in no change to the period of BrCCA1 (circadian clock associated 1) and a 1-week late flowering time. Finally, we demonstrated that increased levels of BrCKA2 and BrCKB4 in vernalized B. rapa confer elevated CK2 activity, resulting in a shortened period of the clock gene BrCCA1, which plays an important role in perceiving photoperiod in plants. Thus, our results suggest that there is a direct interaction between photoperiod and vernalization through DNA methylation mechanisms.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (No. 31301782), Natural Science Foundation of Jiangsu Province of China (No. BK20130673), Opening Foundation of State Key Laboratory of Crop Genetics and Germplasm Enhancement (ZW2014006), China Postdoctoral Science Foundation (No. 2014M550294), and Special Foundation (No. 2015T80561). We thank Takato Imaizumi (Department of Biology, University of Washington, Seattle, WA, USA) for excellent comments on the manuscript. We are grateful to members of the System Biology of Chinese Cabbage Laboratory of Nanjing Agricultural University for discussions.
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W. Duan, H. Zhang and B. Zhang contributed equally to the paper.
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425_2016_2622_MOESM1_ESM.docx
Supplementary Table 1. Primers sequences used in the study. Supplementary Table 2. Summary of MeDIP-Seq Illumina data mapped to Brassica rapa genome. Supplementary Table 3. Methylated genes distribution in Brassica rapa duplicate genes. Supplementary Table 4. 1562 significantly differentially methylated genes. Supplementary Table 5. Intron.bed genes (down-methylation in the vernalized plant). Supplementary Table 6. CDS.bed genes (down-methylation in the vernalized plant). Supplementary Table 7. downstream2 k.bed genes (down-methylation in the vernalized plant). Supplementary Table 8. Upstream2 k.bed genes (down-methylation in the vernalized plant). Supplementary Table 9. Intron.bed gene (up-methylations in the vernalized plant). Supplementary Table 10. CDS.bed genes (up-methylation in the vernalized plant). Supplementary Table 11. downstream2 k.bed genes (up-methylation in the vernalized plant). Supplementary Table 12. Upstream2 k.bed genes (up-methylation in the vernalized plant). (DOCX 28 kb)
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Supplementary Fig. 1. Venn diagram of the distributions of methylated genes in vernalized and non-vernalized samples. (A) MeDIP-seq reads distribution in upstream2 k, CDS, Intron and downstream2 k elements of non-vernalized sample. (B) MeDIP-seq reads distribution in upstream2 k, CDS, Intron and downstream2 k elements of vernalized sample. (C) Different methylated reads distribution in upstream2 k, CDS, Intron and downstream2 k elements between vernalized and non-vernalized samples. (JPEG 578 kb)
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Supplementary Fig. 2. Degree of methylated duplication genes. (A) Upper: Venn diagram between duplicated genes and methylated genes in non-vernalized sample; bottom: Venn diagram between duplicated genes and methylated genes in vernalized sample. The duplicated genes in Brassica rapa genome were based on MCScanX program. (B) Percentage of the duplicated genes which were methylated in vernalized and non-vernalized samples. N: Non-vernalized; V: Vernalized. (JPEG 233 kb)
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Supplementary Fig. 3. GO classification of differentially methylated genes. The horizontal axis indicates GO items (biological process, cellular component and molecular function), the left vertical axis indicates the proportion of genes involved, and the right vertical axis indicates the exact number of genes. (TIFF 38671 kb)
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Supplementary Fig. 4. The shortened period of BrCCA1 is mainly caused by BrCKB4 in Brassica rapa post vernalization. (A) The DNA methylation level of BrCKA2 and BrCKB4 in CK and Aza-treated Brassica rapa seedlings. (B) qRT-PCR analysis of transcript abundance of BrCKA2 and BrCKB4 in CK and Aza-treated Brassica rapa seedlings. (C) Circadian oscillation of BrCCA1 RNA in BrCKA2 and BrCKB4 single (cka2, BrCKA2 single silenced line; ckb4, BrCKB4 single silenced line) and double-silenced plants (cka2ckb4) post vernalization. Closed diamonds: cka2ckb4 line; Closed squares: ckb4 line; Closed triangles: cka2 line. Results showed that the period and amplitude of BrCCA1 RNA was no difference between ckb4 and cka2ckb4. But cka2 shows shortened period of BrCCA1 post vernalization. (D) The flowering time of wild-type (CK), single (cka2, ckb4) and double (cka2ckb4) silenced Brassica rapa post vernalization. Asterisk (*) indicates p < 0.05. (JPEG 266 kb)
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Duan, W., Zhang, H., Zhang, B. et al. Role of vernalization-mediated demethylation in the floral transition of Brassica rapa . Planta 245, 227–233 (2017). https://doi.org/10.1007/s00425-016-2622-3
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DOI: https://doi.org/10.1007/s00425-016-2622-3