DEMETER DNA demethylase reshapes the global DNA methylation landscape to control cell identity transition during plant regeneration

Plants possess high potential for somatic cell reprogramming. Despite extensive studies on the molecular network and key genetic factors during regeneration, the underlying epigenetic landscape remains incompletely understood. Here, we explored methylome and transcriptome dynamics during two-step plant regeneration. During leaf-to-callus transition, genic CG methylation shifts, while pericentromeric regions undergo substantial CG and extensive CHH hypomethylation. Upon shoot regeneration, methylation increases across all cytosine contexts, particularly in pericentromeric regions, coinciding with RNA-directed DNA methylation (RdDM) pathway activation. However, the DEMETER (DME)-deficient dme-2 mutant exhibited significant genic CG redistribution and global non-CG hypomethylation, partly due to RdDM downregulation. The dme-2 mutant affects gene expression in pluripotency and shoot meristem development, promoting regeneration through a reprogrammed state by pericentromeric hypomethylation. Our study demonstrates the intimate linkage between DME demethylase and RdDM underlying pericentromeric non-CG methylation maintenance, providing insight into the epigenetic mechanisms balancing pluripotency states and genome stability during plant regeneration.