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Petr Cápal & Vladan Ondřej
Cellular dedifferentiation is a process in which cells change from a given differentiated state into a transient, stem cell-like state and reenter into the cell cycle (Grafi 2004). In plants, cellular dedifferentiation is exemplified by protoplastization. Here, the cell wall of differentiated leaf cells is degraded by given enzymes that finish by protoplasts’ formation (Zhao et al. 2001). The protoplasts assume a unique transient state with features resembling animal stem cells (Grafi et al. 2011). The global chromatin decondensation is characterized for plant protoplasts (Zhao et al. 2001; Tessadori et al. 2007; Ondřej et al. 2009b, 2010) that is also a distinguishing feature of animal stem cells. Various stimuli, that include phytohormones like auxins and cytokinins, induce protoplasts to reenter the cell cycle and proliferate. The reprogrammed cells could form calli, which under appropriate conditions can form shoots and roots, somatic embryos, and eventually fertile plant (Grafi et al. 2011). However, application of auxins induces endoreduplication that could thwart future cell development; absence of these growth factors in culture of dedifferentiating protoplast cells causes their death with hallmark events of programmed cell death (Zhao et al. 2001). The most dynamic and large-scale chromatin rearrangement occurs during protoplastization, where the majority of heterochromatin decondenses, which in nuclei of Cucumis spp. is clearly visible as disappearance of chromocenters (heterochromatic domains intensively stained by DAPI). Chromocenters contain non-coding DNA sequences, transposable elements, and also inactive ribosomal genes (Ondřej et al. 2009a, b). This decondensation of heterochromatin affects nearly all nuclei and involves the most repetitive sequences of the genome including 180-bp and 5S ribosomal DNA (rDNA) repeats, transposons (Tessadori et al. 2007), and also subtelomeric satellite II repeats (Ondřej et al. 2009b; Ondřej et al. 2010). It is evident that dedifferentiated cells need the balance between heterochromatin and euchromatin to be established, together with epigenetic changes and changes in expression profile in order to survive and proliferate. Changes in expression could alter production of transcription factors, their target genes, stress-induced genes, and miRNAs that are expressed from non-coding regions of the genome that are decondensed in protoplast nuclei. Nuclear DNA can be modified by post-replicative methylation of its cytosine residues. In plants, up to 80% of cytosinesin CpG sites are methylated. The chromocenters of Arabidopsis nuclei, for example, contain the majority of these methylated sequences (Probst et al. 2003). This epigenetic modification of DNA plays an important role in normal embryo and germ cell development in mammals and plants. DNA methylation is involved in gene silencing, as well as transposon inactivation (Heard et al. 1997; Feil and Khosla 1999; Martienssen and Colot 2001; Saze et al. 2003). Several experiments have studied global changes in the protoplast transcriptome (Grafi et al. 2011; Xiao et al. 2012). Using macroarray technology in cotton protoplasts, Yang et al. (2008) showed expression of ESTs corresponded to genes mainly involved in cell wall regeneration, stress-defense response, metabolism and energy, plus protein metabolism and storage. But, the data from gene expression studies were not given in context of DNA methylation patterns. In view of this fact, we present here expression of selected genes together with their DNA methylation level. This approach could contribute not only to understanding cell reprogramming but also to protoplast recalcitrance in regeneration in some plant species. Cucumbers, that were used in presented experiments, and related species from the genus Cucumis show recalcitrance in protoplast regeneration. Despite many attempts to overcome this issue, cucumber protoplasts are only able to divide and form calli (Gajdová et al. 2007) and fail to complete plant regeneration. Thus, we studied changes in gene expression and DNA methylation to try to understand processes leading to inability of cucumber protoplasts to fully regenerate into plants. The genes studied were involved in molecular analysis of cell proliferation, cell wall synthesis, and oxidative stress. We also compared these genes with methylation patterns of repetitive sequences and whole genome methylation during the first 3 d of cucumber protoplast cultivation.
