Department of Plant Anatomy and Cytology, Faculty of Biology and Environmental Protection, University of Silesia in Katowice, 40-032 Katowice, Poland.
Department of Cell Biology, Faculty of Biology and Environmental Protection, University of Silesia in Katowice, 40-032 Katowice, Poland.
Int J Mol Sci. 2018 Jun 19;19(6):1806. doi: 10.3390/ijms19061806.
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 tissue culture.
形态学和组织学观察表明，在 50μM 的浓度下，5-氮杂胞苷（5-azaC）完全抑制了胚性体（EM）的诱导，而在 5μM 的 5-azaC 存在下培养外植体（合子胚；ZE），导致 10%的情况下形成具有 EM 的愈伤组织。透射电子显微镜（TEM）分析显示，在经过处理的愈伤组织细胞中存在典型的液泡型细胞死亡的形态和超微结构特征。TUNEL 检测证实，经 5 和 50μM 5-azaC 浓度处理的愈伤组织细胞存在 DNA 双链断裂。对选定的细胞死亡标记物的基因表达分析表明，与对照培养物相比，经处理的愈伤组织细胞中半胱天冬酶、蛋白执行器 1（EX1）和硫氧还蛋白（TRX）的表达减少。谷胱甘肽 S-转移酶（GST）的基因活性增加最为明显。我们的研究还包括对一些阿拉伯半乳聚糖蛋白（AGPs）和伸展素表位分布的分析，这些表位可作为组织培养中正在死亡的细胞的标志物。
