Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control

Abstract

The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX-labelled DNA damage foci in an ATM- and ATR-dependent manner. These γH2AX-labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co-localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta Genetic interaction between the RBR-silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity.

Keywords: Arabidopsis; RETINOBLASTOMA RELATED; BRCA1; DNA damage response; E2FA.

Figure 1. Silencing of RBR and CYCD3.1 overexpression both promote S‐phase entry but affect cell death response and DNA damage accumulation differently

1. Representative confocal laser scanning microscopy (CM) images of whole mount EdU‐labelled roots from 6‐day‐old (das) seedlings of Col‐0, amiRBR and Col‐0(CYCD3.1OE) lines with EdU (green) and DAPI (DNA, blue) staining. In the amiRBR and Col‐0(CYCD3.1OE) lines, the region of extra columella stem cell layers is labelled with a green bar in merged images. White vertical bar shows the region of cells where EdU counting was carried out. Images were taken in single median section, scale bar: 50 μm, arrow: QC position in each image.

2. CM images of propidium iodide (PI)‐stained root tips from 12 das seedling; genotypes indicated as in (A). Images were taken in single median section, scale bar: 50 μm, arrow: QC position in each image.

3. Number of EdU‐labelled cells as shown in (A) was counted in the epidermis, cortex and endodermis cell layers on both sides of the root. In each case, 10 roots (6 das) were quantified.

4. Cell death response in 6 and 12 das seedlings, total number of dead columella stem cells (CSC) and lateral root cap initials (LRC) and their descendants were counted in median sections as shown in (B), n > 2, N > 15. Note that in Col‐0(CYCD3.1OE) only 1–2 dead cells were detected in the analysed population. Quantification of the dead cell area in amiRBR is shown in Fig 2C.

5. Frequency of γH2AX‐labelled nuclei per total number of DAPI‐positive nuclei (%), n = 2, N > 6 root of 6 das seedlings, analysed nuclei > 1,000.

6. Representative CM images (single section) of γH2AX immune‐labelled cells of root tips from Col‐0, amiRBR and Col‐0(CYCD3.1OE). DAPI (blue), scale bar: 5 μm.

Data information: Values represent means with standard deviation (SD). In (C–E), a indicates significant difference around 1% confidence using Student's t‐test comparing amiRBR and Col‐0(CYCD3.1OE) to Col‐0. In (D), b indicates 99% significance (P < 0.01) between time points and in (E) ab indicates 99% significance (P < 0.01) to Col‐0 and amiRBR. n = biological repeat, N = sample per biological repeat.

Figure EV1. Both local silencing of RBR and overexpression of E2FA‐DPA and E2FB‐DPA result in extra S‐phase entry but only RBR silencing triggers cell death response

1. Differential interference contrast (DIC) microscopy images using Lugol staining to detect differentiated columella cells. Note the increased number of columella cell layers upon local reduction of RBR. Black arrow indicates the position of the dissection used to collect material for micro‐array analysis.

2. Confocal microscopy images (CM) of root tips after EdU staining (green, 2 h) at 4 das counterstained with DAPI (blue) and at 6 and 10 das using bright field. Yellow bar indicates the region with extra columella stem cell layers.

3. CM images of PI‐stained root samples from rRBr seedlings showing accumulation of cell death in time.

4. Representative CM images of whole mount EdU‐labelled (green) root tips of 6 das Col‐0, Col‐0(E2FA/DPAOE) and Col‐0(E2FB/DPAOE) seedlings; DNA was stained by DAPI.

5. Representative PI‐stained CM images of 12 das root tips from the genotypes indicated. Note that no cell death response was detected at any time point analysed.

Data information: Images were taken in median sections. Scale bars: 50 μm, genotype as indicated in the images. White arrows: QC position in each image.

Figure 2. Genotoxic stress upon RBR silencing leads to hypersensitive DNA damage response

1. A

   Representative (CM) images of Col‐0, rRBr and amiRBR root tips of 6‐ to 7‐day‐old seedlings after 16 h of mitomycin (MMC) and 20 h of zeocin treatment compared to non‐treated samples (Control).

2. B, C

   Cell death was quantified (B) by the number of the dead columella and lateral root cap stem cells (CSC, LRC) and their daughter cells, and (C) by measuring the area of dead vasculature above the QC in the presence of MMC for 16 h and zeocin for 20 h.

3. D

   Representative (CM) images of Col‐0 and amiRBR root tips of 6‐ to 7‐day‐old seedlings after 16 h of hydroxyurea (HU) treatment compared to non‐treated samples (control shown in A).

4. E

   Representative (CM) images of nuclei (single section) of Col‐0 and amiRBR 6 das root tips after 16 h of MMC treatment immune‐labelled for γH2AX (green). DAPI (blue), scale bar: 5 μm.

5. F

   Frequency (%) of γH2AX foci‐harbouring nuclei compared to total nuclei in 6 das Col‐0 and amiRBR root tip after 16 h of MMC treatment compared to non‐treated samples.

Data information: In (A and D), arrows indicate position of QC, scale bar: 50 μm. In (B, C and F), values represent mean with standard error, data are combined from n = 3 biological repeats, N > 15 roots for (B and C) and N > 5 in (F) of amiRBR and Col‐0, total nuclei > 1,000. a indicates significant difference within the 5 to 1% statistical confidence interval using Student's t‐test between amiRBR and rRBr versus Col‐0, and b indicates significant difference between treated versus non‐treated samples. n = biological repeats, N = samples per biological repeat.

Figure 3. RBR and E2FA nuclear focus formation depends on ATM/ATR kinases and coincides with γH2AX‐positive sites upon MMC and zeocin treatments

1. Representative CM images (single section) of nuclei with RBR foci at the γH2AX‐positive sites in Col‐0 upon 16 h of MMC and 3 h of zeocin treatment (white arrowheads); diffuse nuclear RBR signal is shown in the untreated control (RBR: green, γH2AX: red, DAPI: blue).

2. Partial co‐localisation of RBR and γH2AX foci shown on Imaris section of the nucleus (RBR: green, γH2AX: red). Main panel (z) shows a single z‐stack of the nucleus, right panel (y‐z) shows cross section by y plane perpendicular to z plane in the main panel, lower panel (x‐z) illustrates cross section by x plane perpendicular to z plane in the main panel. Scale bar: 1 μm; scale bar of magnified insets: 0.5 μm.

3. Representative CM images (single section) of nuclei showing accumulation of RBR (red) and E2FA‐GFP (green) in the same nuclear foci (white arrowheads) after 16 h of MMC and 3 h of zeocin treatment (RBR: red, E2FA‐GFP: green, DAPI: blue). RBR and E2FA‐GFP focus formation was not detected in untreated cells (control) or upon inhibition of ATM and ATR kinases (IATM+IATR+MMC). The activity of IATM and IATR inhibitors was followed on cell death response.

4. Imaris section of a nucleus showing co‐localisation of RBR (red) and E2FA‐GFP (green) in foci (DAPI: blue). Main panel (z) shows a single z‐stack of the nucleus, right panel (y‐z) shows cross section by y plane perpendicular to z plane in the main panel, and lower panel (x‐z) illustrates cross‐section by x plane perpendicular to z plane in the main panel. Scale bar: 1 μm; scale bar of magnified insets: 0.5 μm.

5. The range of Pearson correlation coefficients (PCCs) of RBR/E2FA‐ and RBR/γH2AX‐positive foci formed after 16 h of MMC treatment. PCCs are visualised in quartiles of ranked data (n = 30). While RBR/E2FA co‐localised in foci with high mean value of PCCs = 0.82, the RBR/γH2AX in foci showed PCCs ranging from 0.1 (side by side co‐localisation) to 0.75 (partial co‐localisation).

6. The effect of IATM and IATR inhibitors on cell death response upon 16 h of MMC treatment in Col‐0 was quantified by the number of columella stem cells (CSC) and lateral cap stem cells (LRC) and their descendants. Values represent mean with standard deviation, n = 2, N > 15 roots for each, a indicates significant difference within the 5 to 1% statistical confidence interval using Student's t‐test comparing samples treated with inhibitors (single or combined) and MMC to MMC only.

7. Representative CM image (single section) of a nucleus shows localisation of RBR and E2FA to a γH2AX‐positive site after 16 h of MMC treatment (white arrowheads, RBR: violet, γH2AX: red, E2FA‐GFP: green, DAPI; blue).

Data information: In the intensity profiles (A, C and G), the x‐axis shows length in μm measured from 1 and y‐axis illustrates relative intensity. Scale bars: 2 μm. n = biological repeats, N = samples per biological repeat.

Figure EV2. RBR nuclear foci can localise with condensed heterochromatin and CenH3

1. Representative CM images of nuclei (single section) of Col‐0 6 das root tips after 16 h of MMC treatment immunolabelled for RBR (green), γH2AX (red) and DAPI (blue). RBR foci localised at γH2AX‐positive sites and with DNA heterochromatin spots labelled by arrowheads, while RBR foci localised independently of condensed chromatin are marked by arrows and (a) and (b) illustrate intensity profiles for a section as given in merged images.

2. Representative CM image of nuclei (single section) showing localisation of RBR foci to CenH3‐labelled region (arrowheads) in 6 das Col‐0 root tips after 16 h of MMC treatment (RBR: green, CenH3: red, DAPI: blue).

Data information: In (A and B) intensity profiles: x‐axis shows length in μm measured from 1; y‐axis shows relative intensity. Scale bars: 2 μm. N > 3, n = 3. n = biological repeats, N = samples per biological repeat.

Figure 4. AtBRCA1 and RBR are recruited to γH2AX foci and partially co‐localise upon genotoxic stress, and locate to foci independent of each other

1. Representative CM images of PI‐stained root tips of Col‐0, Atbrca1‐1 (0, 16 h) and Atbrca1‐1(AtBRCA1 _pro :AtBRCA1 _gen :GFP) seedlings after 0, 4 and 16 h of MMC treatment. Arrows indicate position of QC, scale bar: 50 μm. Inset in the last image illustrates an enlarged nucleus with pronounced speckles.

2. CM images of PI‐stained root tips of rRBr;(AtBRCA1:GFP) showing AtBRCA‐GFP accumulation into foci in QC and the stem cell niche labelled with green arrowheads. Top and bottom images represent different root tips. Scale bar: 50 μm.

3. Representative CM images of nuclei (single section) with triple immunolabelling for RBR (violet), γH2AX (red) and AtBRCA1 (green) and stained for DAPI (blue) showing co‐localisation of AtBRCA1‐GFP with γH2AX (arrowheads), RBR with γH2AX (arrowheads) and RBR, γH2AX and BRCA‐GFP (arrowheads) after 16 h of MMC treatment. In the presence of ATM and ATR inhibitors (IATM+IATR+MMC), the γH2AX and AtBRCA1‐GFP nuclear signals and RBR foci formation were abolished. See also Table 1 for statistics.

4. Representative CM image (single section) of RBR foci localised with γH2AX‐positive sites (arrowheads) in nuclei of Atbrca1‐1 root meristematic cells after 16 h of MMC treatment (RBR: green, γH2AX: red, DAPI: blue).

Data information: In (C and D) intensity profiles: x‐axis shows length in μm measured from 1; y‐axis shows relative intensity. Scale bars: 2 μm. N > 3, n = 3. n = biological repeats, N = samples per biological repeat.

Figure EV3. The Atbrca1‐3 mutant, similarly to Atbrca1‐1, also shows hypersensitivity upon genotoxic stress

1. A

   Position of the T‐DNA insertion in Atbrca1‐1 (Reidt et al, 2006) and Atbrca1‐3 mutants. The sequence indicates the insertion in Atbrca1‐3, and difference in letter type shows the exon–intron border. Arrows depict position of forward (qF) and reverse (qR) primers used for qRT–PCR reactions.

2. B

   Expression level of the AtBRCA1 transcript in Col‐0, Atbrca1‐1 and Atbrca1‐3 alleles compared to the AtACTIN2 transcript level in Col‐0 in normal growth conditions. To control the inducibility of the transgenes, the alleles and Col‐0 were treated with MMC (+) and compared to non‐treated seedlings (−). The graph shows that genotoxic stress influenced the AtBRCA1 transcript level only in the control, but not in the alleles. However, neither of the Atbrca1 alleles are null alleles. n > 2, N > 100 seedlings (6 das) for each genotype and treatments.

3. C

   CM images of PI‐stained root tips of Col‐0, Atbrca1‐1 and Atbrca1‐3 6 das seedlings grown without (−MMC) and treated with MMC (+MMC) for 16 h. Scale bar: 20 μm, arrow: QC position in each image.

4. D, E

   Functional analysis of the (AtBRCA1 _pro :AtBRCA1 _gen :GFP) construct following cell death response in the introgressed line Atbrca1‐1(pgBRCA:GFP) compared to Col‐0, Atbrca1‐1 and Atbrca1‐3. (D) Cell death response was quantified in the distal stem cell region after 16 h of MMC treatment. Dead columella stem and daughter cells (CSC) and lateral root initials and their descendants (LRC) were counted in median section as shown in (C); n = 3, N > 15. (E) Ratio of PI‐stained area in the proximal meristem comparing mutants and the complementing line to Col‐0. PI‐stained area was measured in each experiment from N > 15 mutants and Col‐0, and then means and ratio were calculated. Finally, the mean of the different experiments/ratios (n = 3–4) was calculated and depicted.

Data Information: In (B, D and E), values represent means ± standard deviation. a indicates significant difference around 1% confidence using Student's t‐test comparing Atbrca1‐1 and Atbrca1‐3 to Col‐0, and b indicates 99% significance between Atbrca1‐1(AtBRCA1:GFP) and Atbrca1‐1. n = biological repeats, N = samples per biological repeat.

Figure 5. RBR and AtBRCA1 proteins can physically interact

1. Co‐immunoprecipitation of RBR with AtBRCA1 and E2FA proteins. Control: streptavidin beads, RBR: streptavidin beads bound with RBR‐biotin, AB‐GST: GST (anti‐glutathione‐S‐transferase) antibody, EA‐POD: Extravidin‐POD (peroxidase‐conjugated streptavidin) labelling RBR‐biotin‐containing complexes, GST‐BRCA1: GST‐labelled AtBRCA1, and GST‐E2FA: GST‐labelled E2FA proteins, in the input of the wheat germ extract.

2. BiFC assay in planta reveals physical interaction between AtBRCA1 and RBR (BRCA1‐N/RBR‐C). The RBR‐N/SCR‐C pair was used as a positive control, and BRCA1‐N/SCR‐C pair as a negative control. Young, growing tobacco leaves were infiltrated and analysed 36–48 h after infiltration. Scale bar: 50 μm, SCR: SCARECROW transcription factor.

Figure EV4. Lack of AtBRCA1 in conjunction with RBR silencing results in partially penetrant developmental arrest and suppresses cell death response, but does not influence extra stem cell division and S‐phase entry induced by RBR silencing in surviving individuals

1. Segregation (47 severe/49 survival) and growth habit of amiRBR;Atbrca1‐1 homozygous seedlings (F3).

2. Developmental defects in germinating seedlings, arrowheads point to missing primary leaves.

3. Relative transcript level of AtBRCA1 in amiRBR, Atbrca1‐1 and amiRBR;brca1‐1 compared to Col‐0, where the level of expression was set arbitrarily to 1 in non‐treated samples. Upon MMC treatment, the graph shows the ratio of expression between treated and non‐treated samples. Values represent mean ± SD, n > 2, N > 100. a: P < 0.05 shows significant increase in expression compared to Col‐0 untreated control using Student's t‐test.

4. Frequency (%) of EdU‐labelled nuclei (10‐min pulse) compared to total DAPI‐stained nuclei, a: P < 0.001, all compared to Col‐0 using Student's t‐test, n > 2, N > 10; error bars indicate ± SD.

5. WOX5_pro‐WOX5_gen‐3xGFP expression in the mutant lines showing QC division in the amiRBR;brca1‐1 compared to Col‐0 and Atbrca1‐1. Arrow indicates the position of the QC, scale bar: 20 μm.

6. Confocal images of amiRBR and amiRBR;brca1‐1 root tips of 12 das seedlings showing columella and stem cell layers (white dots). Arrow: QC position. Inset is showing the incorporation and presence of the amiRBR construct. Scale bar: 20 μm.

7. Quantification of the number of columella and stem cell layers of 4‐, 6‐ and 12‐day‐old roots from amiRBR, amiRBR;brca1‐1 and Col‐0. Values represent means ± SD, N > 15 for each mutant and Col‐0 (n = 3–4). a: P < 0.01 between the given genotype and Col‐0 at a given time point using Student's t‐test.

Data information: n = biological repeats, N = samples per biological repeat.

Figure 6. RBR and AtBRCA1 may act in a common process during DDR

1. Representative (CM) images of nuclei (single section) of amiRBR, Atbrca1‐1 and amiRBR;Atbrca1‐1 6 das root tips immunolabelled for γH2AX (green) and DAPI (blue). Scale bar: 5 μm.

2. Frequency (%) of γH2AX‐labelled nuclei to total DAPI‐stained nuclei in Col‐0, amiRBR, Atbrca1‐1, amiRBR;Atbrca1‐1 grown under normal conditions. Values represent means with SD, n = 3, and total nuclei > 1,000. a indicates significant difference within the 1% statistical confidence interval using Student's t‐test between amiRBR, Atbrca1‐1, amiRBR;Atbrca1‐1 versus Col‐0.

3. CM images of PI‐stained root tips from amiRBR, amiRBR;brca1‐1 and amiRBR;brca1‐3 of 12 das seedlings. Scale bar: 20 μm, arrow: QC position in each image.

4. Cell death response of amiRBR, amiRBR;brca1‐1, amiRBR;brca1‐3 seedlings at 4, 6 and 12 das. Values represent means with SD, N > 15 for each mutant and Col‐0 (n = 3–4) a: P < 0.01 between the given genotype and Atbrca1‐1, which did not develop cell death at any time point. b: P < 0.01 comparison between cross and amiRBR. The total number of dead columella stem and daughter cells (CSC), lateral root cap initials and their descendants (LRC) were counted in median sections as shown in (C).

Data information: n = biological repeats, N = samples per biological repeat.

Figure 7. Genes regulated by RBR are annotated to nucleosome assembly, replication and DDR; RBR protein is enriched on the AtBRCA1 promoter

1. A

   The pie chart represents the major processes regulated by RBR.

2. B, C

   Validation of transcriptome analysis for a selected set of up‐ and down‐regulated genes upon RBR silencing, respectively, using qRT–PCR on dissected root tips of 4‐day‐old rRBr and Col‐0 seedlings.

3. D

   Genes showing differential expression upon local RBR silencing are also de‐repressed in the constitutively silenced amiRBR line. Graph represents qRT–PCR on 4‐day‐old seedling material.

4. E

   Schematic representation of the AtBRCA1 promoter; black lines with numbers indicate the position and length of the amplified regions by qPCR analysis, the position of the start codon (ATG), the stop codon of the upstream neighbouring transcript and the position of putative E2F elements (red arrowheads) on the + and − strand, at positions −234 and −151, respectively, are indicated. Position of amplified regions: 1: −383 to −248; 2: −238 to −78; and 3: +313 to +455; positions are numbered from ATG (+1).

5. F

   Chromatin immunoprecipitation (ChIP) using RBR antibody; the graph shows fold enrichment calculated as a ratio of chromatin bound to the numbered section of the promoter with or without antibody. Values represent mean of three biological replicates with standard error, a: P < 0.01 compared to the negative control and b: P < 0.01 compared to the positive control using Student's t‐test. PCNA1 promoter was used as a positive control and IR (an intergenic region between At3g03360‐70) as a negative control. The enrichment on IR was arbitrarily set to 1. Numbers 1, 2 and 3 on the x‐axis refer to the regions labelled in (E).

Data information: In (B–D), values represent mean of fold change normalised to values of the relevant genes from Col‐0, and error bars indicate ± SD, n = 2, N > 100. All of the values were in the 1% statistical confidence interval using Student's t‐test. Abbreviations of genes are available in Appendix Table S1 and primers used in this study in Appendix Table S3. Data information: n = biological repeats, N = samples per biological repeat.

Figure 8. Spontaneous cell death upon RBR silencing is suppressed by E2FA and DNA damage response upon genotoxic stress is dependent on E2FA

1. Relative transcript level of AtBRCA1 in amiRBR, e2fa‐1, e2fa‐2, their double mutants and e2fb‐1, e2fb‐2 compared to Col‐0, where the level of expression was set arbitrarily to 1.

2. Relative transcript level of AtBRCA1 in Col‐0, e2fa‐1, e2fb‐1 and e2fb‐2 without and upon 16 h of MMC treatment. All the values are compared to the expression level measured in non‐induced Col‐0 which was set to 1.

3. ChIP using GFP antibody to chromatin isolated from Col‐0(AtE2FA‐GFP) seedlings; the graph shows fold enrichment on the AtBRCA1 promoter region 2 without and upon genotoxic treatment (MMC, 16 h). The graph illustrates a representative experiment. a: P < 0.01 without MMC, b: P < 0.01 in MMC compared to the non‐treated and IR control using Student's t‐test. The enrichment on IR was arbitrarily set to 1.

4. Quantitative analysis of cell death response in Col‐0, e2fa‐1, e2fa‐2, amiRBR, amiRBR;e2fa‐1 and amiRBR;e2fa‐2 mutants at 6 and 12 das. Values represent mean ± SD, at least two biological replicates testing more than 20 seedlings for each mutant. Note the absence and insignificant number of spontaneous cell death in the distal stem cell niche in Col‐0 and e2fa mutants, respectively, at these time points. a: P < 0.05 significance comparing single mutant to Col‐0 and b: P < 0.05 comparing double mutants to amiRBR using Student's t‐test. CSC: columella stem cells, LRC: lateral root cap initials and their descendents.

5. CM images of PI‐stained root tips in non‐treated e2fa‐1 mutant, and MMC‐treated Col‐0 and e2fa‐1 (6 das). Images were taken in median section, scale bar: 50 μm. Arrow: QC position in each image.

Data information: In (A and B), values represent mean ± SD, n > 2, N > 100 in each experiment. a: P < 0.05 comparing single mutant to Col‐0 and in (B) b: P < 0.05 comparing values upon MMC treatment using Student's t‐test. n = biological repeats, N = samples per biological repeat.

Figure EV5. Both transcription of AtBRCA1 and SMR4 upon RBR silencing and cell death response upon genotoxic stress are dependent of E2FA

1. Position of different T‐DNA insertions in AtE2FA, colours represent different domains: dark blue, N‐terminal; light blue, DNA‐binding domain; red, dimerisation domain; purple, marked box; lilac, transactivation domain; yellow, RBR binding domain. Drawing based on Magyar et al (2012).

2. Relative transcript level of RBR in amiRBR and amiRBR;e2fa‐1 and amiRBR;e2fa‐2 double mutants.

3. Cell death response in MMC‐treated 6 das seedlings of different e2fa alleles; total number of dead columella stem cells (CSC), lateral root cap initials (LRC) and their descendants were counted.

4. Quantification of cell death by measuring the area of dead vasculature (μm²) in the presence of MMC for 16 h. No cell death response was observed in non‐treated samples.

5. Relative transcript level of SMR4, SMR5, RAD51 and PARP2 in amiRBR, e2fa‐1, e2fa‐2 and double mutants compared to Col‐0, where the level of expression was set arbitrarily to 1. a: P < 0.05 significance between mutant versus Col‐0 using Student's t‐test; values represent mean of relative expression.

Data information: In (B–E), bars represent mean ± SD, n > 2, N > 10 seedlings in (C and D) and N > 100 in (B and E). a: P < 0.05 significance between different e2fa mutants versus Col‐0 using Student's t‐test. n = biological repeats, N = samples per biological repeat.

Figure EV6. Hypersensitive cell death response after genotoxic stress only partially depends on SOG1 upon RBR silencing

1. CM images of PI‐stained root tips from sog1‐1, amiRBR and amiRBR,sog1‐1 lines compared to Col‐0 as indicated above the columns. Images were taken in median section of 6 das seedlings treated with and without MMC (10 μg/ml). Scale bar: 50 μm, arrow: QC position in each image.

2. Representative images of 9 das seedlings showing cell death response after hydroxyurea (+HU, 1 mM) or zeocin (+zeo, 20 μg/ml) treatment. Scale bar: 50 μm, arrow: QC position in each image.

3. Relative RBR transcript level in amiRBR and amiRBR,sog1‐1 lines, taking mean of several independent lines. a: P < 0.05 significance genotypes versus Col‐0 using Student's t‐test.

4. Cell death response upon MMC treatment in sog1‐1, amiRBR and amiRBR,sog1‐1 lines compared to Col‐0. The total number of dead columella stem and daughter cells (CSC), lateral root cap initials (LRC) and their descendants were counted in median section as shown in (A). a: P < 0.05 significance genotypes versus Col‐0, b: P < 0.05 comparison of treated samples to non‐treated counterparts, c: P < 0.05 significance between amiRBR versus amiRBR,sog1‐1 using Student's t‐test. Note, that Col‐0 at 6 das and sog1‐1 at 6 and 12 das developed no dead cells.

Data information: Values represent mean ± SD, n = 3, N > 15 in (A, B and D) and N > 100 in (C) for each mutant and Col‐0. n = biological repeats, N = samples per biological repeat.

Figure 9. DNA damage response upon RBR silencing is independent of SOG1

1. CM images of PI‐stained root tips (12 das seedlings) from the genotypes indicated. Arrow indicates the position of the QC; scale bar: 50 μm.

2. Cell death response from 6 and 12 das seedlings, total number of dead columella stem cells (CSC), lateral root cap initials (LRC) and their descendants were counted as shown in (A). Note that neither Col‐0 at 6 das nor sog1‐1 at 6 and 12 das showed cell death. a: P < 0.05 at 6 das, b: P < 0.05 at 12 das mutants versus Col‐0 using Student's t‐test. Value represents mean ± SD, n > 3, N > 15 for each mutant and Col‐0. The genotype legend in the graph also holds for (C and D).

3. Relative expression level of DDR genes in sog1‐1, amiRBR and amiRBR,sog1‐1 lines (6 das) compared to Col‐0 (6 das), where the level of expression for each gene was set arbitrarily to 1. a: P < 0.05, mutants compared to Col‐0 using Student's t‐test.

4. Transcriptional induction of the indicated genes is depicted as fold change comparing MMC (16 h) to non‐treated samples of Col‐0, sog1‐1, amiRBR and amiRBR,sog1‐1. a: P < 0.05 amiRBR versus Col‐0 and b: P < 0.05 amiRBR,sog1‐1 to sog1‐1 using Student's t‐test.

Data Information: In (C) and (D), 6 das seedlings were analysed and data represent means with ± SD. At least three biological replicates were analysed, in each case around 100 seedlings for each mutant. For amiRBR,sog1‐1 the mean was calculated from the analysis of six independent transformants (T2 generation). n = biological repeats, N = samples per biological repeat.