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      Recondensation level of repetitive sequences in the plant protoplast nucleus is limited by oxidative stress

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          Abstract

          Protoplast cultures are remarkable examples of plant cell dedifferentiation. The state of dedifferentiation is evidenced by changes in cell morphology, genome organization, as well as by the capability of protoplasts to differentiate into multiple types of cells (depending on the type of the stimulus applied). The first change in the genome structure is connected with large-scale chromatin decondensation, affecting chromocentres involving various types of these repetitive sequences. This paper describes not only the de- and recondensation of satellite DNA type I and 5S rDNA repetitive sequences, but it also compares the recondensation level of chromatin with the levels of oxidative stress which were decreased by using an antioxidant, as well as the capabilities of the antioxidative systems within protoplasts, during the first 72 h of their culture. It is demonstrated that the treatment of protoplasts with ascorbic acid not only decreased the level of oxidative stress but also positively stimulated the expression of the ascorbate peroxidase and catalase. It also led to a greater recondensation of the chromatin (when compared to the untreated protoplasts); in addition, it supported cell proliferation. It is concluded that large-scale genome relaxation is more directly connected with oxidative stress than with large changes in the expression of genes; and further, that its recondensation is related to the start of (as well as the level of) protection by the antioxidative systems.

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          Most cited references23

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          Large-scale dissociation and sequential reassembly of pericentric heterochromatin in dedifferentiated Arabidopsis cells.

          Chromocenters in Arabidopsis thaliana are discrete nuclear domains of mainly pericentric heterochromatin. They are characterized by the presence of repetitive sequences, methylated DNA and dimethylated histone H3K9. Here we show that dedifferentiation of specialized mesophyll cells into undifferentiated protoplasts is accompanied by the disruption of chromocenter structures. The dramatic reduction of heterochromatin involves the decondensation of all major repeat regions, also including the centromeric 180 bp tandem repeats. Only the 45S rDNA repeat remained in a partly compact state in most cells. Remarkably, the epigenetic indicators for heterochromatin, DNA methylation and H3K9 dimethylation, did not change upon decondensation. Furthermore, the decondensation of pericentric heterochromatin did not result in transcriptional reactivation of silent genomic elements. The decondensation process was reversible upon prolonged culturing. Strikingly, recondensation of heterochromatin into chromocenters is a stepwise process. Compaction of the tandemly arranged 45S rDNA regions occurs first, followed by the centromeric 180 bp and the 5S rDNA repeats and finally the dispersed repeats, including transposons. The sequence of reassembly seems to be correlated to the size of the repeat domains. Our results indicate that different types of pericentromeric repeats form different types of heterochromatin, which subsequently merge to form a chromocenter.
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            Two phases of chromatin decondensation during dedifferentiation of plant cells: distinction between competence for cell fate switch and a commitment for S phase.

            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.
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              How cells dedifferentiate: a lesson from plants.

              The remarkable regenerative capacity displayed by plants and various vertebrates, such as amphibians, is largely based on the capability of somatic cells to undergo dedifferentiation. In this process, mature cells reverse their state of differentiation and acquire pluripotentiality--a process preceding not only reentry into the cell cycle but also a commitment for cell death or trans- or redifferentiation. Recent studies provide a new perspective on cellular dedifferentiation, establishing chromatin reorganization as its fundamental theme.
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                Author and article information

                Journal
                J Exp Bot
                jexbot
                exbotj
                Journal of Experimental Botany
                Oxford University Press
                0022-0957
                1460-2431
                May 2010
                2 April 2010
                2 April 2010
                : 61
                : 9
                : 2395-2401
                Affiliations
                [1 ]Department of Botany, Faculty of Science, Palacký University, Šlechtitelů 11, Olomouc 783 71, Czech Republic
                [2 ]Department of Biochemistry, Faculty of Science, Palacký University, Šlechtitelů 11, Olomouc 783 71, Czech Republic
                Author notes
                [* ]To whom correspondence should be addressed: E-mail: vladan.ondrej@ 123456upol.cz
                Article
                10.1093/jxb/erq067
                2877892
                20363868
                73973e4b-f811-4d4c-80f1-9c6d046374a4
                © 2010 The Author(s).

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

                History
                : 21 January 2010
                : 4 February 2010
                : 4 March 2010
                Categories
                Research Papers

                Plant science & Botany
                nuclear organization,protoplasts,oxidative stress,chromatin
                Plant science & Botany
                nuclear organization, protoplasts, oxidative stress, chromatin

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