ISSN 00954527, Cytology and Genetics, 2013, Vol. 47, No. 1, pp. 29–33. © Allerton Press, Inc., 2013.

Original Russian Text © A.P. Kravets, D.A. Sokolova, G.S. Vengzhen, D.M. Grodzinsky, 2013, published in Tsitologiya i Genetika, 2013, Vol. 47, No. 1, pp. 37–43.

Corn Plant DNA Methylation Pattern Changes

at UV C Irradiation Fractionating
A. P. Kravets, D. A. Sokolova, G. S. Vengzhen, and D. M. Grodzinsky
Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine, Kyiv, Ukraine
email: kaplibra@gmail.com
Recieved March 7, 2012

Abstract—The relationship between changes in the methylation pattern of functionally different parts of
DNA and the chromosomal aberration’s yield was studied under the conditions of UVC irradiation fraction
ating. A combination of restriction analysis (HpaII, MspI, and MboI enzymes) with subsequent PCR (inter
nal transcribed spacer ITS1 and ITS4 and inter simple sequence repeat ISSR(14b) primers) was used. The
obtained results showed changes in the methylation pattern of the satellite and transcribed DNA part of DNA
at irradiation in the fractionating mode, depending on the fraction range. The role of a DNA methylation pat
tern changes in the development of radiation damage and induction of the organism’ protective reactions was
discussed.
DOI: 10.3103/S0095452713010052

INTRODUCTION and/or periodicity of a stress factor. The complexity of

DNA methylation is one of the most important and
multifunctional biological regulatory mechanisms,
which plays a key role in a series of epigenetic pro
cesses, such as genomic imprinting, differentiation,
apoptosis, morphogenesis, senescence, and regulation
of mobile elements’ activity [1–4]. Methylation of
cytosine residues is known to be simultaneously a nat
ural factor of mutagenesis [3] and a factor stabilizing
the DNA spatial structure that is necessary for enzy
matic reactions dealing with information processing
and reparation.
Plants occupy leading ranks with respect to the
content of methylated cytosine (up to 30%). DNA
methylation in plants is carried out by four classes of
methyltransferases of four classes [3], providing an
abundance and diversity of methylation sites. This
may play a key role in plant reactions to stress as the
alternative “run or fight” is commonly solved by plants
for the benefit of a “fight” rather than a “run” strategy
at all organization levels. A series of recent studies have
revealed changes in the level and pattern of DNA
methylation under biotic stress [4, 5], as well as under
different forms of abiotic stress conditions, such as
drought [4], salinization [4–7], and irradiation of dif
ferent intensities [8–13] and durations [8, 12, 13].
The reaction of organisms to stress is a system pro
cess, which involves damage development, full or par
tial recovery of the initial state, and changes in the
repeated stress sensitivity represented by either adap
tation or sensibilization of an organism.
Changes in stress sensitivity (either an increase or a
decrease) include a variety of reactions, the involve
ment of which depends on the intensity, duration,
interactions between stress reaction compounds is
caused by the hierarchical structural and functional
organization of an organism, in which different pro
cesses and structures demonstrate different sensitivity
levels and different periods required to change it. The
multifunctionality of DNA methylation provides dif
ferent forms of its participation in both damage devel
opment (for example, activation of genome mobile
elements and genome instability initiation) and initia
tion of protective mechanisms that deal with metabo
lism reorganization. Hence, reliably stated changes in
the level and/or pattern of DNA methylation observed
under stress conditions require further studies to clar
ify their biological role [4–13]. One of the essential
stages in the study of the role of this process in stress
reactions and formation of adaptive mechanisms is the
estimation of changes in the methylation patterns of
functionally different DNA sequences, i.e., satellite
and transcribed regions, under repeated stress.
The present research was aimed at studying
changes in methylation patterns in functionally differ
ent DNA sequences of corn at different intervals of
fractionated UVC irradiation. An estimation of the
methylation profile was performed in comparison with
the level of chromosome aberrations in meristematic
tissues which allowed us to assess the direction of plant
sensitivity changes.
MATERIALS AND METHODS
The objects of the study were seeds AND 3–7day
old corn seedlings (the Titan variety). Seeds were
couched on plates, the bottoms of which were covered

29
30 KRAVETS et al.
with wet filter paper and incubated in a thermostat at
23–24°C. To perform irradiation, an OBN150M
bactericidal irradiator (Ukraine) equipped with Phil
ips Special TUV30W lamps was used. Threedayold
seedlings were were exposed with 6.2–9 kJ/m2 frac
tionated UVC radiation (the dose rate was 6.2 W/m2)
with fraction range from 1 hour (h) to 1 day (d).
Apical root meristems were used for cytogenetic
assay. Samples for cytogenetic assay were collected on
the first, second, third, and fourth day after irradia
tion. After separation from the roots, the apexes were
placed in a Brodsky fixation solution (0.3 acetic acid :
1 ethanol : 3 formaldehyde mixture) for 2 h and then
washed with ethanol 3–4 times. Maceration was per
formed by alkaline hydrolysis with 20% NaOH for 2 h.
After that, the preparations were washed with distilled
water for 15 min.
Staining was performed with a mixture of acetoor
cein and hydrochloric acid (acetoorcein : 1M HCl, 1 : 1)
for 16–18 h. The stained material was washed with
45% CH3COOH, and squash preparations were pre
pared. To perform an analysis, ten parallel samples
were prepared and 5000–10000 cells were analyzed.
An analysis of chromosome aberrations was performed
by the anaphase–telophase method, taking into
account the specificity of plant tissues. The anaphase
cells were at least 300500 per each cellular prepara
tion.
Isolation of DNA was performed from the 6day
old corn seedlings by the STAB method [4], modified
for this culture. Deproteinization was performed with
a mixture of phenol and chloroform (1 : 1) [15].
The concentration of DNA in the obtained solu
tion was measured spectrophotometrically by a stan
dard methodology described in [16] using a BioPho
tometer Plus Eppendorf v.1.35 spectrophotometer.
A PCR analysis was performed in a fourchannel
Tertsik DNA amplifier (DNKtekhnologiya DNA
technology, Russia) with primers designed to minisat
ellite sequences ISSR (15soro, 5'ACACACAC
ACACACAC〈C〉3') and to the transcribed
sequences ITS1 (5'TCCGTAGGTGAACCT
GCGG3') and ITS4 (5'TCCTCCGCTTAT
TGATATGC3'). Both types of primers were syn
thesized by the Metabion company (Germany).
The reaction mixture for ISSR–PCR contained
1.25 units of Taq polymerase (recombinant), 5 μL 10 ×
Taq buffer, 5 mM MgCl2, 0.2 mM each dNTP, 0.5 μM
primer, 1.5 μg total genomic DNA, and 28.7 μL
deionized water (the total volume was 50 μL). The reac
tion mixture was covered with 20 μL of liquid petrola
tum [17]. Amplification with ISSR primers included
the following steps: 5 min initial denaturation at 94°C,
40 cycles; 45 s denaturation at 94°C; 45 s primer
annealing at 52°C; 90 s elongation at 72°C; and 7 min
final elongation at 72°C [18].
The reaction mixture for ITS–PCR contained
1.25 units of Taq polymerase (recombinant), 5 μL 10 ×
Taq Buffer, 4 mM MgCl2, 0.2 mM each dNTP, 0.5 μM
of each primer, 1.5 μg of total genomic DNA, and
26.2 μL of deionized water (the total volume was 50 μL).
The reaction mixture was covered with 20 μL of liquid
petrolatum [17]. The PCR analysis with ITS primers
included the following steps: 1.5 min initial denatur
ation at 94°C, 5 cycles; additional 40 cycles of dena
turation at 94°C, 15 s; primer annealing at 55°C, 15 s;
elongation at 72°C, 15 s; fixing, consisting of denatur
ation at 94°C for 10 s, primer annealing at 55°C for
10 s, elongation at 72°C for 10 s, and final elongation
at 72°C for 5 min [17].
Both restriction and amplification reactions were
carried out in a fourchannel Tertsik DNA amplifier
(DNKtekhnologiya DNAtechnology, Russia). Two
types of isoschisomer restrictases—HpaII (5'.C CGG.3')
and MspI (5'.C CGG.3')—and restrictase MboI (Fer
mentas, Germany) were used for restriction. Experi
ments were carried out in accordance with standard
protocols for restriction analysis provided by the man
ufacturer.
The reaction mixture for restriction analysis with
HpaII contained 0.2 units of the enzyme (0.3 μL),
2.0 μL of 10 × Buffer Tango, 1.5 μg of total genomic
DNA (5 μL), and 17.7 μL of deionized water (the total
volume was 25 μL). The mixture was covered with
20 μL of liquid petrolatum.
The reaction mixture for restriction analysis with
MspI contained 0.6 units of the enzyme (0.9 μL),
2.0 μL of 10 × Buffer Tango, 1.5 μg of total genomic
DNA (5 μL), and 17.1 μL of deionized water (the total
volume was 25 μL). The mixture was covered with
20 μL of liquid petrolatum.
The reaction mixture for restriction analysis with
MboI contained 0.2 units of the enzyme (0.3 μL),
2.0 μL of 10 × Buffer Tango, 1.5 μg of total genomic
DNA (5 μL), and 17.7 μL of deionized water (the total
volume was 25 μL). The mixture was covered with
20 μL of liquid petrolatum.
Restrictases and restriction sites
MspI 5'C…C*CG, C…5'
HpaII 5'…C*CGG…3'
3'G…G C*C…5'
MboI 5'…C*CGC…3'
3'…CT…AG *C…5'
The conditions of the restriction reaction were as
follows: incubation at 37°C for 16 h and reaction ter
mination by incubation at 65°C (for HpaII and MboI)
and 80°C (for MspI) for 20 min.
The obtained PCR and restriction products were
analyzed by electrophoresis in a 1% agarose gel sup
plemented with ethidium bromide in TBE buffer. The
obtained gels were visualized by UVtransilluminator.
CYTOLOGY AND GENETICS Vol. 47 No. 1 2013
 CORN PLANT DNA METHYLATION PATTERN CHANGES
To perform electrophoresis, the wells were filled with
equal volumes of PCR and restriction products per
10 μL. A FastRuler High Range DNA Ladder (Fer
mentas, Germany) containing fragments of 10000,
4000, 2000, 1000, and 500 bp and a FastRuler Low
Range DNA Ladder (Fermentas, Germany) contain
ing fragments of 850, 400, 200, and 50 bp were used as
molecular weight standards.
RESULTS AND DISCUSSION
The obtained cytogenetic data show display of frac
tionating effect. In other words, the sequential expo
sure resulted in a decrease in the object sensitivity with
respect to chromosomal aberration yield. However,
the effect was expressed differently in the time
depended manner between the fractions. An interpre
tation of this phenomenon should not only take into
account the possible changes in the activity of protec
tive mechanisms and reparation processes, which
occur after the first irradiation, but also special fea
tures of formative tissues of plants.
The meristem tissue is heterogenic by several
parameters. First, it is represented by an asynchronous
population of cells in different functional states, quite
evenly distributed throughout the cell cycle. There
fore, these cells demonstrate different sensitivity to
irradiation. Second, the meristem tissue contains qui
escent cells, such as stem cells of the quiescence center
and a small pool of diffusively distributed cells [19]. It
is noteworthy that the suppression of the irradiation
effect is more obvious at 1 and 4h fraction range
treatments fractions than at a 1day fraction range. If
the change in the meristem sensitivity had only been
caused by the stimulation of the protective and repar
ative mechanisms of a homogenous population of pro
liferating cells, one would have expected a monoto
nous increase in this parameter. The observed increase
in cell sensitivity may indicate the second fraction
treatment (1 day) of a group of cells that synchroni
cally turned from the quiescence to the division phase,
thus demonstrating high sensitivity to UVC irradia
tion. Therefore, the observed fractionation effect
reflects an interaction at least two groups of factors.
First, sequential irradiation fractions affect cell groups
of the asynchronous cell population. These cells ini
tially stay in different phases of the cell cycle, which
are characterized by different sensitivity. Therefore,
they undergo injury of different severity. At a 1day
fraction range, a synchronous switch of quiescent cells
to the division phase is possible that makes the cell
population extremely vulnerable. Second, it is possible
that different severity levels of chromosome damage in
cells, initially demonstrating different sensitivity, may
cause different efficacy of further changes in their sen
sitivity that do or do not require epigenetic modifica
tions.
The electrophoretic analysis results of the nativity
of the isolated DNA are shown in Fig. 2a. These
CYTOLOGY AND GENETICS Vol. 47 No. 1 2013
31
mitotic index
12
chromosomal aberrations
10
8
6
4
2
0
Control 1h 4h 1 day 0h
Fig. 1. Mitotic index and chromosome aberration’s yield
(vertical axis, %) at a fractionated UVC irradiation dose
of 7.2 kJ/m2 with different intervals between the fractions
(horizontal axis).
results were observed in all experimental variants. It
was demonstrated that direct UVC exposure did not
induce considerable DNA fragmentation at apoptosis
apoptosis.
An electrophoregram of amplification products of
native DNA with ISSR primers (Fig. 2b) shows no dif
ferences between the control and any of the experi
mental variants regardless of the treatment conditions.
The electrophoregrams of the amplification prod
ucts of the restriction products obtained with MboI
endonuclease (Fig. 2c) demonstrate insignificant dif
ferences in the methylation pattern of the control and
experimental samples. The most obvious differences,
which appear as highmolecularweight amplification
products, were observed between the control and
experimental variants after the fulldose irradiation
and a dayrange. These observations suggest a
decrease in the number of MboI endonuclease restric
tion sites. A comparison of this result with the data of
the cytogenetic analysis (Fig. 1) showed that these
variants corresponded to the highest yield of cytoge
netic abnormalities. An electrophoretic analysis of the
amplification products of the restriction reaction prod
ucts obtained with other endonucleases did not show
differences between experimental variants.
An electrophoretic analysis of the amplification
products of the transcribed part of the native DNA
(Fig. 2d) showed insignificant, but notable, differ
ences in their spectrum with respect to not only the
control and experimental samples, but also within the
variants, which underwent irradiation under different
regimes. The changes in the electrophoregram picture
are due to the appearance of amplification products
with a lower molecular weight. An explanation of this
phenomenon have to take into account the possible
damage of the primary DNA structure that corre
sponds to the increase in the yield of unstable chromo
somal aberrations (Fig. 1) in the meristem. This result
32
(a)
M1 1 2 3 4 5 M2 M1
(d)
M1 1 2 3 4 5 M2 M1
Fig. 2. Electrophoretic analysis of the nativity of isolated DNA (a), native DNA amplification products with ISSR primers (b),
amplification of MboI endonuclease restriction products with ISSR primers (c), native DNA amplification with ITS primers (d),
amplification of HpaII endonuclease restriction products with ITS primers (e): M1, highmolecularweight marker; 1, control
sample; 2, onehour fraction range; 3, fourhour; 4, oneday; 5, fulldose irradiation or no fraction range; M2, lowmolecular
weight marker.
does not contradict the data about the good nativity of
the isolated DNA (Fig. 2a). This only indicates the
preservation of the weight and charge of singular DNA
molecules, which may even belong to aberrant chro
mosomes. However, the participation of DNA mole
cules in PCR might have led to the occurrence of sin
gle breaks, the number and positions of which varied
with regard to the irradiation regime. One cannot deny
the possibility of a direct influence of hidden DNA
injuries on the DNA conformation that affects PCR
results. The connections between changes caused by
chromosomal aberrations, as well as the changes in the
methylation pattern, are confirmed by the data of
other authors, who observed the activation of homol
ogous recombination in response to stress [4].
Although these two events—homologous recombina
tion and chromosomal aberrations—have different or
even opposite biological significance, both of them
can be initialized by doublestrand breaks in DNA.
An electrophoregram of the amplification products
obtained by PCR of the HpaII restriction products
with ITS primers (Fig. 2e) demonstrated differences in
the methylation pattern of the control and exposured
variants. The most obvious differences were observed
between the control and experimental samples, which
corresponded to the fulldose irradiation. A simplifi
cation of the amplification product spectrum showed
a decrease in the number of HpaII restriction sites
after an irradiation dose of 7.2 kJ/m2.
Therefore, the fractionated UVC irradiation
caused rather complicated changes in methylation
patterns, each of which had its own biological signifi
KRAVETS et al.
(b) (c)
1 2 3 4 5 M2 M1 1 2 3 4 5 M2
(e)
1 2 3 4 5 M2
cance. It is known that satellite DNA is to a consider
able degree associated with heterochromatin and
undergoes more effective methylation than the tran
scribed part of DNA associated with euchromatin
[20, 21]. Changes in the methylation pattern of satel
lite DNA occur without changes in the primary struc
ture. This observation is confirmed by the similarity
between amplification products in electrophoresis
(Figs. 2b, 2c) and in turn confirms the lower sensitivity
of heterochromatin to UVC irradiation. Changes in
the satellite DNA methylation pattern may display or
reflect both the activation of mobile elements, which
are mostly associated with satellite DNA, and confor
mational changes in molecules that play protective
and adaptive roles [21].
Changes in the methylation pattern of the tran
scribed DNA part allow us to hypothesize that this
process has an epigenetic value affecting gene expres
sion. Presently, the role of changes in gene expression
in changes in organism sensitivity is poorly under
stood. According to the data of a fundamental study
carried out by Lekyavichus [22], a considerable role in
the adaptation process belongs to changes in the spec
trum of isoenzymes that are aimed at adjusting their
functional optimums to changes in the internal envi
ronment caused by stress. The observed changes in the
methylation pattern of the transcribed DNA part may
display or reflect modifications of epigenome, which
lead to active metabolic rearrangements related to
protective and reparation processes.
Therefore, the obtained data demonstrated
changes in the methylation patterns of both satellite
CYTOLOGY AND GENETICS Vol. 47 No. 1 2013
 CORN PLANT DNA METHYLATION PATTERN CHANGES
and transcribed DNA, caused by UVC irradiation. A
comparative analysis of these changes observed under
repeated stress conditions with changes in the number
of chromosome aberrations allows to think that they
may be connected with decreased sensitivity and
adaptation of plants to UVC irradiation. The changes
in the number of chromosome aberrations and the
DNA methylation pattern in the intervals between
sequential irradiation treatments made it possible to
assess interactions of different organization levels, i.e.,
tissue and cellular, in the adaptation of organisms to
stress.
ACKNOWLEDGMENTS
We thank the academician V.V. Morgun, director of
the Institute of Plant Physiology and Genetics,
National Academy of Sciences of Ukraine, for kindly
providing corn seeds. We also thank the head of the
laboratory of the Institute of Cell Biology and Genetic
Engineering B.V. Morgun and the researcher of the
Institute of Microbiology and Virology L.B. Zelenaya
for their kind help with methodology and discussion of
the obtained results.
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