Epigenetic changes involve DNA methylation, and modification of histones and small regulatory RNAs [1] . In plants, epigenetic regulation is involved in the development, differentiation, plant responses to the environment, such as biotic and abiotic stress [2] -[4] , and DNA methylation, which is an epigenetic marker involved in silencing of transposable elements and genomic imprinting [5] -[10] .

Epigenetic modifications affect chromatin dynamics, and determine whether they are in an active euchromatin or an inactive or silenced heterochromatin state. Changes in chromosome density influence the access of proteins to binding sites on the DNA, including RNA transcriptases and various transcription factors, subsequently modulating gene expression [2] [11] , thus allowing or preventing gene transcription through the modification of epigenetic markers.

These epigenetic modifications consist of changes at key sites in the molecules that form the chromatin, particularly in the loose amino acid chains found in histones, and in the DNA bases themselves. This commonly involves methylation of position five on the pyrimidine ring in cytosine. Chromatin remodeling and nuclear reorganization have also been shown to be involved in plant responses to stress [12] , and stress can affect chromatin spatial disposition [13] , thus interfering directly with the individual plasticity of an organisms’ phenotype.

Programs aiming to improve fig trees using conventional procedures to obtain new cultivars are rare in many countries, such as Brazil. This is especially due to the low genetic variability and difficulties in obtaining plants from gamete fusion, as the wasp Blastophaga psenes that is responsible for natural pollination is not found in Brazil [14] . Thus, mutagenic genetic improvement is an important field of research in crop science; and obtaining information about this species, especially regarding its genetic variability is necessary so that appropriate propagation and management projects can be developed.

Low dose radiation can stimulate various biological processes and has been applied in different fields to produce mutant plants that can be used to improve crops [15] . From fig cuttings of the cultivar “Roxo-de-Valinhos” irradiated with gamma rays at a dose of 30 Gy [16] , it was possible to select plants based on their unique and divergent morphological characteristics, and agronomic interest [17] . However, no polymorphisms were observed in irradiated figs subjected to molecular analysis, including random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) techniques, indicating that no genetic modifications had occurred. This suggested that epigenetic changes were responsible for the different characteristic observed between treatments following gamma radiation [18] . Following irradiation, changes in global DNA methylation were detected using methylation-sensitive amplification polymorphism (MSAP) molecular markers [19] . This study evaluated global DNA methylation and chromatin in irradiated fig selections and compared it with that observed in the commercial cultivar “Roxo-de-Valinhos”.

The experiment was conducted at the Epigenetics and Reproduction Laboratory of Medicine School of Ribeirão Preto (São Paulo, Brazil), using leaves of five selected irradiated plants based on their unique morphological characteristics [17] . The specimens consisted of cuttings irradiated with gamma rays, at the dose of 30 Gy, with 238 Gy∙hour⁻¹ dose rate, at the Energy Nuclear Center in Agriculture (Piracicaba, Brazil). The treatments are listed below:

1) Roxo-de-Valinhos cultivar―control.

2) Irradiated Plant Selection―440 (IP-440).

3) Irradiated Plant Selection―433 (IP-433).

4) Irradiated Plant Selection―189 (IP-189).

5) Irradiated Plant Selection―214 (IP-214).

6) Irradiated Plant Selection―301 (IP-301).

To determine whether the plasticity of DNA methylation is related to stress induced by irradiation, we investigated global DNA methylation in response to radiation treatment, using Imprint™ Methylated DNA Quantification technology (Sigma-Aldrich), which assesses the spatial distribution of 5-methylcytosine sites and thus detects methylated cytosine residues.

According to the analysis of variance, an F-test for a 95% level of confidence, there was a statistically significant difference in the percentage of methylated DNA between the treatment groups. A post-hoc analysis of the average percentages showed that treatment three (IP 433-long stalk) leads to lower levels of global methylation, than any other medium, is not, however, statistically different, of the treatment non-irradiated control “Roxo-de- Valinhos” (Table 1).

The irradiated control had a lower level of global methylation compared with that observed following other treatments, suggesting that radiation is an external stressor that is capable of altering methylation patterns in plants.

The use of an ELISA assay to quantify global DNA methylation is a relatively new method in plants, which so far, has only been used to quantify DNA methylation in the cork oak genome [22] . In the present study, changes in global DNA methylation were detected after irradiation imposition. Hypo- and hypermethylated DNA modulates gene transcription in plants and occurs under conditions of environmental stress induced by cold, heat and/or salinity [23] [24] .

Differences in the level of global DNA methylation were found between rice varieties in response to salt stress using an ELISA assay. Mutations in epigenetic regulators affected specific phenotypic parameters related

Means followed by the same letter do not differ by Tukey’s test at a 95% level of confidence.

to salinity tolerance, such as the root length and biomass, reinforcing the need for a better understanding of the association between epigenetic networks and plant responses to environmental stresses [25] .

Physical (different types of radiation) and chemical mutagens can be used in vivo or in vitro during plant breeding to increase genetic variability and allow genotypes of interest to be obtained [26] . In figs, dormant buds were irradiated with gamma-rays and the LD50 (dose causing 50% lethality) was determined as 25 Gy; however, a preliminary test should be performed for each genotype before the start of any study [27] . Cuttings of axillary buds were treated with gamma rays (50 - 100 Gy) and small, earliness mutants were obtained. These were tested for use in production, and one of them (Bol) was subsequently released to producers [28] .

Using different doses of gamma radiation in Arabidopsis thaliana, a decrease in DNA methylation was observed with an increasing radiation dose. These results showed that radiation at a dose of 200 Gy was associated with a substantial reduction in the transcriptional silencing of genes, probably due to induction of genomic hypomethylation [29] .

Analysis of global DNA methylation in irradiated figs showed that the effects of radiation do not affect the genomes of different selections in a homogeneous manner. However, demethylation of parts of the genome is likely, since the most polymorphic bands were observed in this condition [19] .

DNA methylation in Arabidopsis thaliana as a repressive mark is often associated with the expression of transcriptionally inactive repetitions, such as repetitive sequences, transposons, and heterochromatic [30] [31] . Methylated DNA loci are often followed by small interfering RNAs (RNAi) [32] [33] , which regulates heterochromatin function and plays an important role in DNA methylation, such as the control of silencing [34] [35] [1] .

In the present study, treatment three showed that radiation affects the silencing system. This breaks the equilibrium certainly caused an increase in expression of these satellite sequences, forcing the RNAi system to an expression control. Recruitment of the silencing system to a great extent the genome may have caused an imbalance in the silencing coding and/or promoters regions of genes, namely a strong demethylation of these sequences.

DNA methylation can be established and maintained in plants and animals. When methylation pathways are inactivated, the effects of DNA methylation are diluted following cellular replication leading to passive DNA demethylation. In other cases, however, DNA methylation is removed by active DNA demethylation. Active or passive DNA demethylation simultaneously reduces the level of DNA methylation at specific developmental stages [30] .

The genome of superior plants contains many transposable elements that potentially disturb the stability of the genome. In plants, DNA methylation usually occurs in these transposable elements. Loss of DNA methylation in mutant genes responsible for it leads to the disruption of transcriptional silencing of transposable elements and other repetitive DNA sequences, thus generating different phenotypes [35] .

Variation in the level of DNA methylation in different selections of fig irradiated with gamma rays is observed, indicating that this abiotic factor can affect the epigenome of plants, leading to the generation of different phenotypes, and being a pioneer of new data in fig crops.

This research was supported by Research Support Foundation of São Paulo State (FAPESP) with a PhD scholarship to M. G. F. Rodrigues, and the Post Graduate Program in Genetics of University of Sao Paulo Medical School. We also acknowledge Dra. Fabiani Gai Frantz and Luana Silva Soares for their support completing the ELISA analysis.

Maria GabrielaFontanetti Rodrigues,Murilo RacySoares,Ester SilveiraRamos,SilvanaGiuliatti, (2015) Analysis of Global DNA Methylation in Irradiated Fig Selections. American Journal of Plant Sciences,06,2257-2262. doi: 10.4236/ajps.2015.614228