Two Phases of Chromatin Decondensation during Dedifferentiation of Plant Cells

Abstract

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.A high proportion of mature plant cells retain characteristic features of totipotent stem cells, i.e. they have the capability to dedifferentiate, re-enter the cell cycle, and proliferate, eventually giving rise to the various organs that make up a new plant (1). In multicellular organisms, cellular dedifferentiation is the major process enabling the regeneration of complex tissues and organs as well as the establishment of new stem cell lineages; in animals it is often associated with carcinogenesis. The molecular mechanism(s) underlying re-entry of differentiated animal cells into the cell cycle and reactivation of DNA synthesis have been intensively studied. Yet, little is known about the early events that accompany cellular dedifferentiation, i.e. the establishment of competence for cell fate switch and the determination of cell fate.The tobacco protoplast system provides an outstanding experimental tool for the study of the biochemical and molecular basis for cellular dedifferentiation. The fully differentiated, non-dividing mesophyll cells of tobacco leaves can be separated from their original tissue by cell wall-degrading enzymes. This treatment results in the formation of a large population of protoplasts (plant cells devoid of cell wall) that, following treatment with phytohormones (auxin and cytokinin) can re-enter the cell cycle and proliferate (2-5). This system demonstrates a unique attribute of plant cells, totipotency, i.e. the capability for cloning in plants (1).Dedifferentiation of mature tobacco cells wa...

Structural and chemical study of callus formation from leaves of Rubia tinctorum

“…Interestingly, earlier findings showed that dedifferentiation of nuclei, chromatin decondensation and changes in density of nucleus staining took 72 -96 h, also in two distinct steps (Harikrishna et al 1989, Zhao et al 2001. In line with these steps the degradation of photosynthetic pigments and ultrastructural changes were observable, as well as the first accumulation steps of anthraquinone derivatives.…”

“…Middle lamella around the transfer cells turned into vesiculated and shiny coat. These structural changes might be related with the loss of original function and the isolation of cells entering cell cycle (Zhao et al 2001).…”

Phosphorylation of Histone H3 at Serine 10 Cannot Account Directly for the Detachment of Human Heterochromatin Protein 1γ from Mitotic Chromosomes in Plant Cells

“…Briefly, nuclei (75 l) were digested with increasing amounts (2, 5, and 10 units per 2 ϫ 10 6 nuclei) of micrococcal nuclease (MBI Fermentas) at room temperature for 12 min. A portion of the digested nuclei was used for DNA preparation as described (33). The remaining micrococcal nuclease (MNase)-digested nuclei were cooled on ice for 10 min before centrifugation at 13,000 ϫ g for 10 min at 4°C.…”

“…This also points to heterochromatin being a heterogeneous rather than a homogenous compartment, which can be specified not only by its level of compaction (33,50) but also by the molecular machinery involved in its formation.…”

In Vitro Regeneration of Foxtail Millet (Setaria italica (L.) Beauv.) cv. Buru Hotong

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