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TUNEL Assay to Assess Extent of DNA Fragmentation and Programmed Cell Death in Root Cells under Various Stress Conditions

Abstract

DNA damage is one of the common consequences of exposure to various stress conditions. Different methods have been developed to accurately assess DNA damage and fragmentation in cells and tissues exposed to different stress agents. However, owing to the presence of firm cellulosic cell wall and phenolics, plant cells and tissues are not easily amenable to be subjected to these assays. Here, we describe an optimized TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay-based protocol to determine the extent of DNA fragmentation and programmed cell death in plant root cells subjected to various stress conditions. The method described here has the advantages of simplicity, reliability and reproducibility.

Keywords: DNA fragmentation, Free DNA termini, Genotoxic, Programmed cell death (PCD), Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling

Background

Exposure to various stresses generally leads to at least some degree of DNA damage resulting in various lesions such as thymine dimerization, alkylation of bases, single stranded nicks, and double-stranded breaks (Bray and West, 2005; Manova and Gruszka, 2015). Of all types of DNA damage, DNA fragmentation is of particular concern during stress conditions, which may either be a direct effect of the stress (as observed, for example, upon treatment with genotoxic agents) or an indirect effect (predominantly, via excessive generation of reactive oxygen species) or may even be a cumulative consequence of both (Bray and West, 2005; Kapoor et al., 2015). This DNA damage must be accurately repaired by the cell’s repair machinery, failing which there may be deleterious consequences including cell death. For maintaining the normal state, cells utilize the DNA damage response which relies on three non-exclusive events viz. detection/recognition of the damage, its access by the repair machinery and finally its repair (Smerdon, 1991).

One of the major molecular mechanisms of stress adaptation at the cellular level involves the resistance to DNA damage and/or efficient repair of the damaged DNA caused due to stress. Therefore, to assess the stress adaptability of a genotype, accurate assessment of DNA damage is often needed. Two widely-used assays to detect DNA fragmentation in plants are Single Cell Gel Electrophoresis–also known as Comet assay (Santos et al., 2015), and TUNEL [Terminal deoxynucleotidyl Transferase (TdT)-mediated dUTP Nick-End Labeling] assay. In comet assay, the tissue of interest is sliced and the resulting cell suspension containing nuclei is embedded in an agarose matrix followed by its alkaline electrophoresis and staining with DAPI/ethidium bromide. After electrophoresis, micrographs show the appearance of broken DNA like a tail similar to that of a comet while the undamaged and condensed DNA appears like a spherical mass forming the head of the comet (Wang et al., 2013). Comet assay, though quite useful, has a few limitations. For instance, it requires isolated nuclei, and hence gives no information on the distribution of DNA damage in a given tissue as well as regarding programmed cell death (PCD). The other widely-used assay–TUNEL assay, can be used to detect in situ DNA strand breaks. TUNEL assay is based on incorporation of labeled dUTP in the DNA (mediated by the enzyme terminal deoxynucleotidyl transferase) which occurs only at the regions with free 3’ termini (i.e., breaks or extreme ends of the chromosome) (Gavrieli et al., 1992). Besides, as breaks in inter-nucleosomal DNA often lead to programmed cell death, TUNEL assay provides significant information about PCD. TUNEL assay, in its basic form, also offers the advantages of simplicity and can give an idea about the distribution of DNA fragmentation (TUNEL-positive cells) in the tissue being studied.

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