Unraveling the DNA Methylation in the rDNA Foci in Mutagen-Induced Brachypodium distachyon Micronuclei

Abstract

Many years have passed since micronuclei were first observed then accepted as an indicator of the effect of mutagens. However, the possible mechanisms of their formation and elimination from the cell are still not fully understood. Various stresses, including mutagens, can alter gene expression through changes in DNA methylation in plants. In this study we demonstrate for the first time DNA methylation in the foci of 5S and 35S rDNA sequences in individual Brachypodium distachyon micronuclei that are induced by mutagenic treatment with maleic acid hydrazide (MH). The impact of MH on global epigenetic modifications in nuclei and micronuclei has been studied in plants before; however, no in situ analyses of DNA methylation in specific DNA sequence sites are known. To address this problem, we used sequential immunodetection of 5-methylcytosine and fluorescence in situ hybridization (FISH) with 5S and 25S rDNA probes on the non-dividing cells of B. distachyon. Such investigations into the presence or absence of DNA methylation within specific DNA sequences are extremely important in plant mutagenesis in the light of altering gene expression.

Cytomolecular Organisation of the Nuclear Genome

“…Bara-Halama et al [ 19 ] is the first study in plants that provides some in situ insight into the epigenetic status of specific DNA sequences in micronuclei. They used B. distachyon , part of the model grass genus Brachypodium (see a recent comprehensive review [ 20 ] for more detail).…”

“…Its well-developed genomic, e.g., in [ 21 , 22 ] and cytogenetic resources, e.g., in [ 4 , 23 , 24 ] also make this genus an amenable model of advanced and extensive cytomolecular analyses of the chromosome and karyotype structure and evolution, e.g., in [ 25 , 26 , 27 , 28 , 29 ], interphase nucleus organisation in [ 30 ], selective rDNA inactivation (nucleolar dominance), e.g., in [ 31 , 32 , 33 ], and genome stability after an induced mutagenesis, e.g., in [ 34 , 35 ]. In their current research [ 19 ], the authors applied a sequential immunolocalisation of 5-methylcytosine and FISH with 5S and 25S rDNA probes to study the material subjected to a mutagenic treatment using maleic acid hydrazide. They observed several kinds of micronuclei at various frequencies, which differed in their 5S rDNA and 35S rDNA composition, and the methylation status of the foci occupied by these sequences.…”