Cápal, P., Ondřej, V.
IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY
50:
789-794,
2014
Keywords:
Abstract:
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 cytosines
in 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.
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IEB authors: Petr Cápal
