Present address: Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, PO Box 7080, SE-75007 Uppsala, Sweden.
CK2-defective Arabidopsis plants exhibit enhanced double-strand break repair rates and reduced survival after exposure to ionizing radiation
Article first published online: 11 JUN 2012
© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd
The Plant Journal
Volume 71, Issue 4, pages 627–638, August 2012
How to Cite
Moreno-Romero, J., Armengot, L., Mar Marquès-Bueno, M., Britt, A. and Carmen Martínez, M. (2012), CK2-defective Arabidopsis plants exhibit enhanced double-strand break repair rates and reduced survival after exposure to ionizing radiation. The Plant Journal, 71: 627–638. doi: 10.1111/j.1365-313X.2012.05019.x
- Issue published online: 6 AUG 2012
- Article first published online: 11 JUN 2012
- Accepted manuscript online: 8 APR 2012 11:45PM EST
- Received 21 April 2011; revised 28 March 2012; accepted 2 April 2012; published online 11 June 2012.
Figure S1. Influence of auxin transport on IR hypersensitivity.(a) Five-day-old seedlings were transferred to a medium containing 1 μM Dex and either 1 μM IAA or 10 μM NPA, for 48 h. Dex was then removed from the medium and seedlings were γ-irradiated with 100 Gy. Pictures show 20-days-old plantlets submitted to those treatments, and their corresponding controls (-Dex). (b) Quantification of seedlings fresh weights from the experiments in (a). Pairwise comparison were assessed using the non-parametric Kruskal–Wallis test and significant differences (P < 0.05) are marked (*).
Figure S2. Expression and activity of CK2 subunits after genotoxic treatments. (a) IR-induced transcriptional changes of CK2-encoding genes. Seven-day-old plants were irradiated with 100 Gy and transcript levels measured by qRT-PCR. TSO2 gene (inset), which is radiomodulated, was used as a positive control. The data represent fold-changes in irradiated versus non-irradiated plants using the nomenclature for CK2-encoding genes as in (Salinas et al., 2006). Data were normalized to those of EIF-1-α gene. (b) Transcriptional changes of CK2-encoding genes after bleomycin plus mitomycin treatments. The data were obtained from public arrays (ArrayExpress), and shoots and roots were analysed separately. (c) Time-course of CK2 activity after IR treatments. Seven-day-old wt plantlets were irradiated with 100 Gy. Data shown are the mean of biological triplicates (±SD).
Figure S3. Validation of array data. Upper panel: qRT-PCR quantification of transcripts for some of the well characterized radiomodulated genes. CK2mut plants were grown under the same experimental conditions as those in the arrays. a.u.: arbitrary units. Bottom panel: data from the array.
Figure S4. DNA damage response and cell cycle. (a) Cell-cycle transcriptional regulation of genes involved in HR and NHEJ pathways. Data were obtained from public arrays published in (Menges et al., 2005), using Genevestigator (https://www.genevestigator.com). (b) Expression of core cell-cycle genes in CK2mut plants (data from array). Fold-changes in expression of +dex versus –dex CK2mut plants are represented with the same colour code as in Figure 2. Only genes with significant changes (ANOVA P-value <0.001) are shown.
Figure S5. DSB repair proficiency. (a) Comet assays in neutral conditions (N/N protocol) from seedlings incubated with 10 μM bleomycin for the indicated times. Bar plots show the % of tail DNA in the population of nuclei. (b) Comet assays in alkali solution (A/A protocol) in seedlings irradiated with 100 Gy. Data are represented as in (a). Arabidopsis atm mutant was used as a DNA-repair deficient control in both experiments. Asterisks mean significant differences at P < 0.005 (Student’s t-test). Data shown are the mean values of at least two independent experiments.
Figure S6. MNase sensitivity. (a) Gel electrophoresis of nuclei digested with 2.5 U/ml of MNase at 37°C for the indicated times. (b) Plots of pixel intensities from the scanned gel in (a) versus relative gel front. Arrows point to the accumulation of low molecular weight fragments.
Figure S7. Transcript profiling of chromatin-related GO categories and of heterochromatic silenced regions. (a) Fold-changes of gene expression in chromatin-related GO categories. Fold changes were calculated and represented as in Figure 2e. (ANOVA P-value <0.001). (b) Transcriptional gene silencing at telomeric regions. Distance to telomeres was measured as number of genes, and median signal ratios for genes with identical distances are shown. Horizontal black and gray lines denote the median and median absolute deviation, respectively, based on the signal ratio of all genes in the mutant. (c) Transcriptional gene silencing at the pericentromeric regions of chromosomes 2 and 4. Box plots show the distribution of signal ratio for all genes (open boxes) or for genes from the pericentromeric regions (pale gray filled boxes correspond to chromosome 2, and dark gray boxes to chromosome 4). Closed circles represent the 5th and 95th percentiles. Arabidopsis met1-3 mutant (Mathieu et al., 2007), which shows release of silencing in those regions, was used as control. (d) Transcriptional gene silencing of transposable-related elements. A plot of signal ratios (log10) of +Dex versus -Dex CK2mut plants is shown. Only 30 of 1155 transposable elements present in the array (transposable-related elements from a list published in (Slotkin et al., 2009)) show significant expression changes (ANOVA P < 0.001).
Mathieu, O., Reinders, J., Caikovski, M., Smathajitt, C. and Paszkowski, J. (2007) Transgenerational stability of the Arabidopsis epigenome is coordinated by CG methylation. Cell, 130(5), 851-862.
Menges, M., de Jager, S. M., Gruissem, W. and Murray, J. A. (2005) Global analysis of the core cell cycle regulators of Arabidopsis identifies novel genes, reveals multiple and highly specific profiles of expression and provides a coherent model for plant cell cycle control. Plant J., 41(4), 546-566.
Salinas, P., Fuentes, D., Vidal, E., Jordana, X., Echeverria, M. and Holuigue, L. (2006) An extensive survey of CK2 alpha and beta subunits in arabidopsis: Multiple isoforms exhibit differential subcellular localization. Plant Cell Physiol., 47(9), 1295-1308.
Slotkin, R.K., Vaughn, M., Borges, F., Tanurdzic, M., Becker, J. D., Feijo, J. A. and Martienssen, R. A. (2009) Epigenetic reprogramming and small RNA silencing of transposable elements in pollen. Cell, 136(3), 461-472.
Table S1. (A) Global changes of gene expression in response to IR. GO categories enrichment analysis from the significant upregulated and downregulated genes. Higher order groups (pale blue grouped) are composed by different GO categories. Data correspond to those shown in the schematic representation of Figure 2B. (B) Comparison of transcript profiles of “Gamma effect” and “CK2mut + Gamma effect”. List of genes corresponding to Venn diagrams shown in Figure 2C. (C) Fold-changes of transcript levels of DNA-repair and chromatin-related genes. List of genes corresponding to the pairwise comparisons shown on the scheme (numbered 1 to 4). These data are represented in Figure 2E (repair genes) and Figure S4A (chromatin-related genes).
Table S2. Primers used to amplify gene transcripts by real-time RT-PCR. The annealing temperature was 60°C in all cases.
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