EMF2 deficiency disrupts epigenetic and chromatin organization landscapes, blocking root regeneration competence in Arabidopsis.

Plant de novo organogenesis depends on callus formation, yet the epigenetic mechanisms governing organ regeneration remain poorly understood. EMBRYONIC FLOWER 2 (EMF2), a core component of Polycomb Repressive Complex 2, mediates transcriptional repression through H3K27me3 and is essential for root regeneration in Arabidopsis. Here, we investigate how EMF2-mediated H3K27me3 shapes chromatin architecture and gene expression during root regeneration. We combined time-course transcriptome profiling, H3K27me3 chromatin immunoprecipitation, in situ Hi-C, and a probe hybridization-based Capture Hi-C approach to analyze chromatin organization and gene expression dynamics in wild-type and emf2 calli during root induction. Although emf2 calli lost root regeneration capacity, they retained responsiveness to root induction signals. Despite large-scale reduction of H3K27me3 across the emf2 genome, many genes remained transcriptionally inactive, coinciding with the formation of new long-range chromatin interactions and weakened intra- and peri-domain contacts. Genes with low basal transcription were preferentially derepressed following extensive H3K27me3 loss. Our results demonstrate that large-scale reduction of H3K27me3 in emf2 drives dynamic reorganization of chromatin architecture in Arabidopsis callus, providing new insights into how histone modification and three-dimensional chromatin topology coordinately regulate gene expression during plant regeneration.