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Dedifferentiation signifies the capacity of somatic cells to acquire stem cell-like properties. This process can be induced during normal development and as a response to various stimuli, such as pathogen infection and wounding. Dedifferentiation also characterizes the transition of differentiated leaf cells into protoplasts (plant cells devoid of cell walls), a transition accompanied by widespread chromatin decondensation. Transcriptome profiling of dedifferentiating protoplast cells revealed striking similarities with senescing cells; both display a large increase in the expression of genes of specific transcription factor (TF) families, including ANAC, WRKY, bZIP, and C2H2. Further analysis showed that leaves induced to senesce by exposure to dark display characteristic features of dedifferentiating cells, including chromatin decondensation, disruption of the nucleolus, and condensation of rRNA genes. Considering that premature senescence can be induced by various stress conditions both in plant and animal cells, our results suggest that the response of plant and also animal cells to certain stresses converges on cellular dedifferentiation whereby cells first acquire stem cell-like state prior to acquisition of a new cell fate (e.g., reentry into the cell cycle or death).

Cellular dedifferentiation underlies topical issues in biology such as regeneration and nuclear cloning and has common features in plants and animals. In plants, this process characterizes the transition of differentiated leaf cells to protoplasts (plant cells devoid of cell walls) and is accompanied by global chromatin reorganization associated with reprogramming of gene expression. A screen for mutants defective in proliferation and callus formation identified kyp-2—a mutant in the KRYPTONITE (KYP)/SUVH4 gene encoding a histone H3 lysine 9 (H3K9) methyltransferase. Analysis of telomere length revealed stochastic telomerase-independent lengthening of telomeres in wild type but not in kyp-2 protoplasts. In kyp-2 mutant, telomeric repeats were no longer associated with dimethylated H3K9. The Arabidopsis telomerase reverse transcriptase (tert) mutant displayed accelerated proliferation despite its short telomeres, though it also showed accelerated cell death. Microarray analysis uncovered several components of the ubiquitin proteolytic system, which are downregulated in kyp-2 compared to wild-type protoplasts. Thus, our results suggest that histone methylation activity is required for the establishment/maintenance of the dedifferentiated state and/or reentry into the cell cycle, at least partly, through activation of genes whose products are involved in the ubiquitin proteolytic pathway. In addition, our results illuminate the complexity of cellular dedifferentiation, particularly the occurrence of DNA recombination that can lead to genome instability.

Plants possess a single gene for the structurally related HETEROCHROMATIN PROTEIN1 (HP1), termed LIKE-HP1 (LHP1). We investigated the subnuclear localization, binding properties, and dynamics of LHP1 proteins in Arabidopsis thaliana cells. Transient expression assays showed that tomato (Solanum lycopersicum) LHP1 fused to green fluorescent protein (GFP; Sl LHP1-GFP) and Arabidopsis LHP1 (At LHP1-GFP) localized to heterochromatic chromocenters and showed punctuated distribution within the nucleus; tomato but not Arabidopsis LHP1 was also localized within the nucleolus. Mutations of aromatic cage residues that recognize methyl K9 of histone H3 abolished their punctuated distribution and localization to chromocenters. Sl LHP1-GFP plants displayed cell type–dependent subnuclear localization. The diverse localization pattern of tomato LHP1 did not require the chromo shadow domain (CSD), whereas the chromodomain alone was insufficient for localization to chromocenters; a nucleolar localization signal was identified within the hinge region. Fluorescence recovery after photobleaching showed that Sl LHP1 is a highly mobile protein whose localization and retention are controlled by distinct domains; retention at the nucleolus and chromocenters is conferred by the CSD. Our results imply that LHP1 recruitment to chromatin is mediated, at least in part, through interaction with methyl K9 and that LHP1 controls different nuclear processes via transient binding to its nuclear sites.

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.

The remarkable regeneration capacity of plant cells is based on their capability to dedifferentiate. We recently reported that cellular dedifferentiation proceeds through two distinct phases, each accompanied by chromatin decondensation: acquisition of competence for fate switch followed by a signal-dependent reentry into S phase. The purpose of this study was to (1) characterize changes in chromatin factors associated with chromatin decondensation, and (2) study the relationship between chromatin decondensation and transcriptional activation of pRb/E2F-regulated genes. We show that plant cells competent for fate switch display a disruption of nucleolar domain appearance associated with condensation of 18S ribosomal DNA, as well as modifications of histone H3 and redistribution of heterochromatin protein 1 (HP1). We further show that the pRb/E2F-target genes RNR2 and PCNA are condensed and silent in differentiated leaf cells but become decondensed, although not yet activated, as cells acquire competence for fate switch; transcriptional activation becomes evident during progression into S phase, concomitantly with pRb phosphorylation. We propose that chromatin reorganization is central for reversion of the differentiation process leading to resetting of the gene expression program and activation of silent genes.

Cellular dedifferentiation is the major process underlying totipotency, regeneration, and formation of new stem cell lineages in multicellular organisms. In animals it is often associated with carcinogenesis. Here, we used tobacco protoplasts (plant cells devoid of cell wall) to study changes in chromatin structure in the course of dedifferentiation of mesophyll cells. Using flow cytometry and micrococcal nuclease analyses, we identified two phases of chromatin decondensation prior to entry of cells into S phase. The first phase takes place in the course of protoplast isolation, following treatment with cell wall degrading enzymes, whereas the second occurs only after protoplasts are induced with phytohormones to re-enter the cell cycle. In the absence of hormonal application, protoplasts undergo cycles of chromatin condensation/decondensation and die. The ubiquitin proteolytic system was found indispensable for protoplast progression into S phase, being required for the second but not the first phase of chromatin decondensation. The emerging model suggests that cellular dedifferentiation proceeds by two functionally distinct phases of chromatin decondensation: the first is a transitory phase that confers competence for cell fate switch, which is followed, under appropriate conditions, by a second proteasome-dependent phase representing a commitment for the mitotic cycle. These findings might have implications for a wide range of dedifferentiation-driven cellular processes in higher eukaryotes.

The Rbc S multigene family in hexaploid wheat, Triticum aestivum , is composed of at least 22 genes that were classified into four subfamilies (SFs). In this study, the relative expression of three SFs was analysed, (1) in various plant organs at different developmental stages of one hexaploid line and two tetraploid lines—a wild and a cultivated one—and (2) in fully expanded flag leaves of 16 diploid, 54 tetraploid, and 40 hexaploid lines. The expression of Rbc S genes was found to be tissue-specific (highest expression in leaves and stems) and negatively correlated with organs’ age. In all cases—at all ploidy levels, in all plant organs and developmental stages, and in most light treatments—SF-1 genes accounted for at least 80% of the Rbc S mRNA. Similar to other plant species, wheat Rbc S genes were found to be light regulated, with the expression of SF-1 genes being less affected by light than the other two SFs. A negative correlation between the expression level and RFLP was found. Accordingly, the SF with the highest level of expression (SF-1) was the most conserved, suggesting a higher selection pressure for the more active Rbc S genes.

Three different 3' noncoding sequences of wheat rubisco small subunit (SSU) genes (RbcS) were used as probes to identify the gene members of different RbcS subfamilies in the common wheat cultivar Chinese Spring (CS). All genes of the wheat RbcS multigene family were previously assigned to the long arm of homoeologous group 5 and to the short arm of homoeologous group 2 chromosomes of cv CS. Extracted DNA from various aneuploids of these homoeologous groups was digested with four restriction enzymes and hybridized with three different 3' noncoding sequences of wheat SSU clones. All RbcS genes located on the long arm of homoeologous group 5 chromosomes were found to comprise a single subfamily, while those located on the short arm of group 2 comprised three subfamilies. Each of the ancestral diploid genomes A, B, and D has at least one representative gene in each subfamily, suggesting that the divergence into subfamilies preceded the differentiation into species. This divergence of the RbcS genes, which is presumably accompanied by a similar divergence in the 5' region, may lead to differential expression of various subfamilies in different tissues and in different developmental stages, in response to different environmental conditions. Moreover, members of one subfamily that belong to different genomes may have diverged also in the coding sequence and, consequently, code for distinguishable SSU. It is assumed that such utilization of the RbcS multigene family increases the adaptability and phenotypic plasticity of common wheat over its diploid progenitors.

Methyl-CpG binding domain (MBD) proteins in Arabidopsis thaliana bind in vitro methylated CpG sites. Here, we aimed to characterize the binding properties of AtMBDs to chromatin in Arabidopsis nuclei. By expressing in wild-type cells AtMBDs fused to green fluorescent protein (GFP), we showed that AtMBD7 was evenly distributed at all chromocenters, whereas AtMBD5 and 6 showed preference for two perinucleolar chromocenters adjacent to nucleolar organizing regions. AtMBD2, previously shown to be incapable of binding in vitro–methylated CpG, was dispersed within the nucleus, excluding chromocenters and the nucleolus. Recruitment of AtMBD5, 6, and 7 to chromocenters was disrupted in ddm1 and met1 mutant cells, where a significant reduction in cytosine methylation occurs. In these mutant cells, however, AtMBD2 accumulated at chromocenters. No effect on localization was observed in the chromomethylase3 mutant showing reduced CpNpG methylation or in kyp-2 displaying a reduction in Lys 9 histone H3 methylation. Transient expression of DDM1 fused to GFP showed that DDM1 shares common sites with AtMBD proteins. Glutathione S-transferase pull-down assays demonstrated that AtMBDs bind DDM1; the MBD motif was sufficient for this interaction. Our results suggest that the subnuclear localization of AtMBD is not solely dependent on CpG methylation; DDM1 may facilitate localization of AtMBDs at specific nuclear domains.

Heterochromatin protein 1 (HP1) controls heterochromatin formation in animal cells, at least partly through interaction with lysine 9 (Lys-9)-methylated histone H3. We aimed to determine whether a structurally conserved human HP1 protein exhibits conserved heterochromatin localization in plant cells and studied its relation to modified histone H3. We generated transgenic tobacco plants and cycling cells expressing the human HP1gamma fused to green fluorescent protein (GFP) and followed its association with chromatin. Plants expressing GFP-HP1gamma showed no phenotypic perturbations. We found that GFP-HP1gamma is preferentially associated with the transcriptionally "inactive" heterochromatin fraction, a fraction enriched in Lys-9-methylated histone H3. During mitosis GFP-HP1gamma is detached from chromosomes concomitantly with phosphorylation of histone H3 at serine 10 and reassembles as cells exit mitosis. However, this phosphorylation cannot directly account for the dissociation of GFP-HP1gamma from mitotic chromosomes inasmuch as phosphorylation does not interfere with binding to HP1gamma. It is, therefore, possible that phosphorylation at serine 10 creates a "code" that is read by as yet an unknown factor(s), eventually leading to detachment of GFP-HP1gamma from mitotic chromosomes. Together, our results suggest that chromatin organization in plants and animals is conserved, being controlled at least partly by the association of HP1 proteins with methylated histone H3.