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.

Plant tissues are not easily amenable to some of the steps of TUNEL assay. The major reasons for this are: difficulty in permeabilization due to the presence of cellulosic cell wall and potential inhibition of TdT-catalyzed reaction by phenolics present in the plant cells. Due to these reasons, TUNEL assay is not a frequently utilized procedure for assessment of DNA fragmentation and PCD in plants. A few recent studies, nonetheless, have shown the application of TUNEL assay in rice (Kwon et al., 2013) and Arabidopsis (Phan et al., 2011; Yang et al., 2014). However, most of these studies have used microtomy/ultramicrotomy and ‘paraffin section’ preparation–a procedure which is not very easy, and requires somewhat expensive instrumentation and technical expertise. Given the range of information which TUNEL assay can provide, especially when determining the stress adaptability of plant genotypes, and its advantages in comparison to other methods, there is a need to develop a standardized, easy-to-follow and relatively inexpensive protocol for TUNEL assay using plant tissues.

Here, we describe an optimized TUNEL assay-based protocol to assess the extent of DNA fragmentation and programmed cell death in plant root cells under various stress conditions. The protocol presented here describes, in detail, a more generalized version of the methodology used for TUNEL assay in our recent study (Tripathi et al., 2016). While we often use this method to study DNA damage and PCD in root tissue from rice and Arabidopsis, it can also be utilized to study these phenomena in root tissue from other herbaceous plants with some minor modifications as detailed in the ‘Procedure’ section. The method presented here is quite easy-to-follow, reliable and reproducible.