- The transition from leaf cells to protoplasts (plant cells devoid of cell walls) confers pluripotentiality coupled with chromatin reorganization. Here, we sought to identify remodeled chromosomal domains in Arabidopsis protoplasts by tracking DNA sequences undergoing changes in DNA methylation and by identifying up-regulated genes. We observed a reduction in DNA methylation at a pericentromeric region of chromosome 1, and up-regulation of several members of the NAC (NAM/ATAF1/CUC2) domain family, two of which are located near the telomeric region of chromosome 1. Fluorescence in situ hybridization (FISH) analysis demonstrated that both pericentromeric and telomeric subdomains underwent chromatin decondensation. This decondensation is subdomain-specific inasmuch as centromeric repeats remained largely unchanged, whereas the 18S rDNA underwent condensation. Within the pericentromeric subdomain, VIP1, a gene encoding a b-Zip nuclear protein required for Agrobacterium infectivity, was transcriptionally activated. Overexpression of this gene in tobacco resulted in growth retardation and inhibition of differentiation and shoot formation. Altogether, our data indicate that acquisition of pluripotentiality involves changes in DNA methylation pattern and reorganization of specific chromosomal subdomains. This change leads to activation of silent genes whose products are involved in acquisition or maintenance of pluripotentiality and/or the ensuing fate of the cell. Developmental Dynamics 230:12–22, 2004. © 2004 Wiley-Liss, Inc.