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Background: Plants possess a high potential for somatic cell reprogramming, enabling the transition from differentiated tissue to pluripotent callus, followed by the formation of de novo shoots during plant regeneration. Despite extensive studies on the molecular network and key genetic factors involved in this process, the underlying epigenetic landscape remains incompletely understood.
Results: Here, we explored the dynamics of the methylome and transcriptome during the two-step plant regeneration process. During the leaf-to-callus transition in Arabidopsis Ler, CG methylation shifted across genic regions, exhibiting a similar number of differentially methylated regions (DMRs) for both hypo- and hypermethylation. Pericentromeric regions underwent substantial CG and extensive CHH hypomethylation, alongside some CHG hypermethylation. Upon shoot regeneration from callus, genic regions displayed extensive reconfiguration of CG methylation, while pericentromeric methylation levels highly increased across all cytosine contexts, coinciding with the activation of the RNA-directed DNA methylation (RdDM) pathway. However, mutation in DEMETER (DME) DNA demethylase gene resulted in significant genic CG redistribution and global non-CG hypomethylation in pericentromeric regions, particularly during shoot regeneration. This non-CG hypomethylation observed in dme-2 mutants was, at least partly, due to RdDM downregulation. The dme-2 mutants affected gene expression involved in pluripotency and shoot meristem development, resulting in enhanced shoot regeneration through a reprogrammed state established by pericentromeric hypomethylation compared to wild type.
Conclusion: Our study demonstrates epigenetic changes, accompanied by transcriptome alterations, during pluripotency acquisition (leaf-to-callus) and regeneration (callus-to-de novo shoot). Additionally, it highlights the functions of the DME demethylase, particularly its close association with the RdDM pathway, which underlies pericentromeric non-CG methylation maintenance. These results provide important insights into the epigenetic reconfiguration associated with cell identity transition during somatic cell reprogramming.
