Throughout their lifecycle, plants are subjected to DNA damage from various sources, both environmental and endogenous. Investigating the mechanisms of the DNA damage response (DDR) is essential to unravel how plants adapt to the changing environment, which can induce varying amounts of DNA damage. Using a combination of whole-mount single-molecule RNA fluorescence in situ hybridization (WM-smFISH) and plant cell cycle reporter lines, we investigated the transcriptional activation of a key homologous recombination (HR) gene, RAD51, in response to increasing amounts of DNA damage in Arabidopsis thaliana roots. The results uncover consistent variations in RAD51 transcriptional response and cell cycle arrest among distinct cell types and developmental zones. Furthermore, we demonstrate that DNA damage induced by genotoxic stress results in RAD51 transcription throughout the whole cell cycle, dissociating its traditional link with S/G2 phases. This work advances the current comprehension of DNA damage response in plants by demonstrating quantitative differences in DDR activation. In addition, it reveals new associations with the cell cycle and cell types, providing crucial insights for further studies of the broader response mechanisms in plants.

Here we provide all the raw data images for the manuscript: Kutashev et al. 2024 New Phytologist (DOI: 10.1111/NPH.19875).

Description of the data and file structure

Files are organized according to figure numbers as described in the manuscript (DOI: 10.1111/NPH.19875). Please refer to the methods section of the manuscript for details on the imaging settings.

Figure 1 - Raw microscopy images for Immunofluorescence (anti-gammaH2AX) and single-molecule RNA FISH (detection of RAD51 transcripts).

Figure 2 - Raw microscopy images for wholemount single molecule RNA FISH (detection of RAD51 transcripts).

Figure 3 - Raw microscopy images for wholemount single molecule RNA FISH using RAD51-GFP line (detection of RAD51 protein and RNAs).

Fugure 4- RAW microscopy images for cell cycle investigation (CDT1a-CFP; Cytrap; EdU; WM-smFISH/EdU)

Fig. S1. Raw microscopy images used for evaluation of RAD51 and PP2A transcription using single molecule RNA FISH

Fig. S2. Raw microscopy images used for quantification of RAD51 transcription sites and mRNAs per cell from Arabidopsis roots using WM-smFISH.

Fig. S3. Raw microscopy images used for quantification of RAD51 mRNA molecules in Arabidopsis roots exposed to increasing amounts of DNA damage using WM-smFISH.

Fig. S4. Magnified representative images from Arabidopsis root cells with different number of RAD51 mRNAs obtained by WM-smFISH.

Fig. S5. Raw microscopy images used for quantification of RAD51 mRNA molecules per cell in stem cell cells of Arabidopsis roots.

Fig. S7. Raw microscopy images of RAD51 WM-smFISH in meristematic and elongation zones of Arabidopsis roots .

Fig. S8. Raw microscopy images used for quantification of RAD51-GFP mRNA and RAD51-GFP protein signals in roots of Arabidopsis RAD51-GFP line treated with 0 μM and 10 μM zeocin.

Fig. S9. Raw microscopy images used for quantification of RAD51-GFP mRNA and RAD51-GFP protein signals in roots of Arabidopsis RAD51-GFP line treated with 50 μM of zeocin.

Fig. S10. Raw microscopy images used for quantification of RAD51-GFP mRNA and RAD51-GFP protein signals in roots of Arabidopsis RAD51-GFP line treated with 170 μM of zeocin.

Fig. S11. Raw microscopy images used for evaluation of cell cycle arrest in Arabidopsis roots using EdU staining.

Fig. S12. Raw microscopy images used for evaluation of cell cycle changes using Cytrap line in roots after exposure different concentrations of zeocin.

Fig. S13. Raw microscopy images used for quantification of DAPI signal intensity in Arabidopsis roots exposed to different concentrations of zeocin.

Fig. S14. Raw microscopy images used for evaluation of cell cycle changes using PlaCCI line in roots after exposure to different concentrations of zeocin.

Fig. S15. Raw microscopy images used for assessment of RAD51 mRNA molecule half-life.

Fig. S16. Raw microscopy images used for quantification of RAD51 mRNAs cell in cells with (+) and without (-) EdU staining.

Table S2. Raw microscopy images used for quantification of RAD51 transcription sites.

Data from: Differences in RAD51 transcriptional response and cell cycle dynamics reveal varying sensitivity to DNA damage among Arabidopsis thaliana root cell types

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Abstract

Description of the data and file structure

Data from: Differences in RAD51 transcriptional response and cell cycle dynamics reveal varying sensitivity to DNA damage among Arabidopsis thaliana root cell types

Data files

Abstract

README: Data from: Differences in RAD51 transcriptional response and cell cycle dynamics reveal varying sensitivity to DNA damage among Arabidopsis thaliana root cell types

Description of the data and file structure

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