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. 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 protoplasts isolation ? following treatment with cell-wall degrading enzymes - while the second occurs only after protoplasts are induced with phytohormones to reenter 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 protoplasts 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, followed, under appropriate conditions, by a second, proteasome-dependent phase, representing a commitment for the mitotic cycle. These findings might have implications to a wide range of dedifferentiation-driven cellular processes in higher eukaryotes.