5-Azacitidine Induces Cell Death in a Tissue Culture of Brachypodium distachyon.

Alexander Betekhtin,Lukasz Chajec,Ewa Kurczynska,Katarzyna Nowak,Robert Hasterok,Elzbieta Wolny,Magdalena Rojek,Jolanta Kwasniewska,Anna Milewska-Hendel

doi:10.3390/ijms19061806

Alexander Betekhtin, Lukasz Chajec + Show 7 more

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https://doi.org/10.3390/ijms19061806

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Abstract

Morphological and histological observations revealed that, at a concentration of 50 µM, 5-azacitidine (5-azaC) totally inhibited the induction of embryogenic masses (EM), while the cultivation of explants (zygotic embryos; ZEs) in the presence of 5 µM of 5-azaC led to the formation of a callus with EM in 10% of the cases. Transmission electron microscopy (TEM) analyzes revealed the presence of the morphological and ultrastructural features that are typical for the vacuolar type of cell death in the callus cells that were treated. A TUNEL assay confirmed the presence of DNA double-strand breaks for the callus cells that had been treated with both 5 and 50 µM 5-azaC concentrations. Analysis of the gene expression of selected cell death markers demonstrated a reduced expression of metacaspase, protein executer 1 (EX1), and thioredoxin (TRX) in the callus cells that had been treated compared to the control culture. The strongest increase in the gene activity was characteristic for glutathione S-transferase (GST). Our studies also included an analysis of the distribution of some arabinogalactan proteins (AGPs) and extensin epitopes, which can be used as markers of cells that are undergoing death in a Brachypodium distachyon tissue culture.

Highlights

Brachypodium distachyon (Brachypodium) has become a useful model system to study various aspects of a tissue culture in grasses
The results presented here show that after a week of cultivation on a callus induction medium (CIM), neither the explants in the control conditions with a developing callus nor those that had been grown in the presence of 5 and 50 μM of 5-azaC displayed any morphological differences
The Brachypodium embryogenic callus of the control explants (Figure 1a,d) and the explants that had been treated with 5 μM of 5-azaC (Figure 1b,e) were primarily composed of two different types of callus areas: (i) one that is vitreous and friable, which contained parenchymatous cells (PC) and (ii) a compact callus that was represented by embryogenic masses (EM) that were yellowish in color

Summary

Introduction

Brachypodium distachyon (Brachypodium) has become a useful model system to study various aspects of a tissue culture in grasses. The impact of DNA methylation on somatic embryogenesis (SE) in Arabidopsis thaliana (Arabidopsis) demonstrated a decrease in the global DNA methylation level during SE that accompanied with the upregulation of DNA methylases and the downregulation of DNA demethylases [11] These authors revealed significant repression of LEC2, LEC1, and BBM in vitro under the treatment of 5-azaC. Transcriptome analyzes of Arabidopsis seedlings treated with either 5-azaC or zebularine revealed a significant number of upregulated genes especially linked with transposable element genes. This showed that these two agents have a disproportionately large influence on the loci that are silenced by DNA methylation [14]

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