Molecular analysis and universality exploration of DNA damage-triggered reprogramming to stem cells

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Our study aimed to understand the molecular mechanism of DNA damage-triggered reprogramming from differentiated leaf cells into stem cells in Physcomitrium patens. To this end, we conducted single cell transcriptome analysis through single nucleus RNA-sequencing (snRNA-seq) to track transcriptional changes during the reprogramming process. We successfully obtained high quality snRNA-seq data. After the clustering of nuclear transcriptomes, we obtained a set of genes which were specifically expressed in each cluster. With fluorescence observation of promoter reporter lines of these specifically expressed genes, multiple clusters were annotated to specific tissues. Based on these cluster annotation results, we can extract the reprogramming trajectory of nuclei from leaf cells to newly formed protonema stem cells. From this analysis, we can identify stage-specific genes during DNA damage-triggered reprogramming and differentially expressed genes between reprogramming and non-reprogramming leaf cells at the late stage, which will provide a potential list of key genes driving the cellular reprogramming.To explore the universality of DNA damage-triggered reprogramming in land plants, we established culture conditions for multiple angiosperms and treated each plant with Zeocin. In most cases, the DNA damage reagent caused cell death in the tested plants, suggesting that efficient DNA damage repair is critical for successful cellular reprogramming.