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Tyutereva, E.V.; Strizhenok, A.D.; Kiseleva, E.I.; Voitsekhovskaja, O.V. Applications of the Comet Assay in Plant Studies. Encyclopedia. Available online: https://encyclopedia.pub/entry/55660 (accessed on 18 April 2024).
Tyutereva EV, Strizhenok AD, Kiseleva EI, Voitsekhovskaja OV. Applications of the Comet Assay in Plant Studies. Encyclopedia. Available at: https://encyclopedia.pub/entry/55660. Accessed April 18, 2024.
Tyutereva, Elena V., Aleksei D. Strizhenok, Elizaveta I. Kiseleva, Olga V. Voitsekhovskaja. "Applications of the Comet Assay in Plant Studies" Encyclopedia, https://encyclopedia.pub/entry/55660 (accessed April 18, 2024).
Tyutereva, E.V., Strizhenok, A.D., Kiseleva, E.I., & Voitsekhovskaja, O.V. (2024, February 28). Applications of the Comet Assay in Plant Studies. In Encyclopedia. https://encyclopedia.pub/entry/55660
Tyutereva, Elena V., et al. "Applications of the Comet Assay in Plant Studies." Encyclopedia. Web. 28 February, 2024.
Applications of the Comet Assay in Plant Studies
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Contrarily to chronic stresses, acute (i.e., fast and dramatic) changes in environmental factors like temperature, radiation, concentration of toxic substances, or pathogen attack often lead to DNA damage. Some of the stress factors are genotoxic, i.e., they damage the DNA via physical interactions or via interference with DNA replication/repair machinery. However, cytotoxic factors, i.e., those that do not directly damage the DNA, can lead to secondary genotoxic effects either via the induction of the production of reactive oxygen, carbon, or nitrogen species, or via the activation of programmed cell death and related endonucleases. The extent of this damage, as well as the ability of the cell to repair it, represent a significant part of plant stress responses. Information about DNA damage is important for physiological studies as it helps to understand the complex adaptive responses of plants and even to predict the outcome of the plant’s exposure to acute stress. Single cell gel electrophoresis (Comet assay) provides a convenient and relatively inexpensive tool to evaluate DNA strand breaks in the different organs of higher plants, as well as in unicellular algae. Comet assays are widely used in ecotoxicology and biomonitoring applications.

DNA damage plant stress response detection of DNA breakage neutral and alkaline Comet assay

1. Introduction

The maintenance of genome integrity is crucial for unicellular and multicellular plants to reach their full lifespan. The stability of DNA is required for proper growth and development, as well as for the faithful transmission of genetic material from one generation to the next. Because of their sessile nature, plants are constantly exposed to unfavorable conditions and cope with numerous DNA damaging factors, both endogenous (spontaneous, pre-programmed, or metabolically derived) and exogenous (e.g., atmospheric radiation, heat, desiccation, allelochemicals, and pollutants). Hundreds to thousands of DNA lesions are generated daily in each cell via different threats [1]. Breaks in a single strand or in both strands of the DNA represent a danger for the existence of the plant cell. However, almost all DNA lesions are rapidly and efficiently fixed through cellular DNA repair mechanisms.
Both the regulated destruction of DNA and the undesired events disturbing DNA integrity are associated with different aspects of plant life. They can occur during developmental events such as cell differentiation or the establishment and release of seed dormancy, and during stress responses induced, e.g., by pathogens, allelochemicals, or various abiotic factors. In all cases, genotoxic effects [2] can lead to short-term reversible genome damage or to the irreversible dismantling of the nuclear DNA in the course of the execution of programmed cell death (PCD).
For almost 40 years, single cell gel electrophoresis (SCGE), also known as the Comet assay, the single cell gel assay (SCG), or microgel electrophoresis (MGE), has remained one of the main cytogenetic tools for investigations of DNA lesions (strand breaks) and repair pathways in eukaryotic cells. SCGE was proposed in 1984 as a method for the detection of radiation-induced DNA breaks and was initially restricted to animal/mammalian systems [3]. Comet assays combine agarose electrophoresis methodology with fluorescence microscopy in order to observe and quantify DNA strand breakage at the level of single cells. The Comet technique was based on the following principles: Cells with damaged DNA exhibit increased migration of the chromosomal DNA from the nucleus in an electric field. The DNA migration pattern has a typical ‘comet’ shape, consisting of a head and a tail. The bulk DNA, also called the nucleoid (“the head”), moves from the cathode to the anode during electrophoresis more slowly than the short, broken DNA fragments (“the tail”), and the DNA then resembles a comet moving with the tail forward. Nowadays, this method is applicable for the detection of many DNA defects and is not limited to DNA strand break analysis.
Koppen and Verschaeve (1996) were the first researchers who adapted the Comet assay for the analysis of genotoxic effects in plants. Vicia faba roots were treated with seven mutagenic agents, and the isolated nuclei were evaluated for the extent of DNA migration [4]. From this experiment, it became clear that the Comet assay is well suited for applications in plants, and that the sensitivity of the Comet test is comparable to, or even higher than, the traditional chromosome aberration test or micronucleus test [5][6], as reviewed in [7]. By now, Comet assays have been applied to plants exposed to different adverse conditions, and described in a number of reviews and protocols [8][9][10][11][12][13][14]. The adaptation of the method to plants has led to a burst in research in the field of ecotoxicology, while highly valuable results were obtained also in phytopathology, embryology, and plant cell biology. The protocol of the Comet assay has been revised many times and modified for different objects and tasks, with ever-increasing reliability and reproducibility [14][15][16][17].

2. Comet Assays in Plant Ecotoxicology and Biological Monitoring

The predominant application of Comet assays to studies of plants over the world is in tests of the mutagenic and genotoxic effects of pollutants. Comet assays have revolutionized the field of genetic ecotoxicology, or eco-genotoxicology (reviewed in [11][18][19]), as they provide a non-specific, sensitive, rapid, and low-cost tool for the detection of genetic damage in natural biota, and for the biomonitoring of toxicants both in aquatic and terrestrial ecosystems. Compared to assays based on animal studies, plant-based bioassays are easy to perform, while providing information on the induction of DNA strand breaks in somatic and germ cells that is relevant for the whole biota. Traditional plant bioassays include the detection of chromosomal aberrations or exchanges between sister chromatids; tests for point mutations with visible phenotype are also useful (e.g., chlorophyll mutations, waxy mutations, or embryo mutations of Arabidopsis; reviewed in [20]). In recent years, even more analyses are being performed on plants using Comet assays [11][18][19]. The advantages and limitations of Comet assays applied to animal models for eco-genotoxicology and biomonitoring have been discussed [21]; most of them are also valid for plants.
An example of the successful use of the Comet assay in a commercial application is in the estimation of drinking water quality, a vitally important parameter for human health and longevity. The genotoxicity of perfluoroalkylated and polyfluoroalkylated substances (PFASs), a group of man-made chemicals contaminating drinking water along the entire supply chain, to a Crustacean, an alga, a plant, a bacterium, and a human leukocyte culture was evaluated in a study by Alias [18]. The authors concluded that the Comet assay is a highly promising tool for use in water quality control services, in order to provide the necessary quality characteristics of raw water in critical situations [18].
The leaves of deciduous plants can be used as bioindicators of the DNA damage caused by various genotoxic factors such as radiation, chemicals, allelochemicals, heavy metals, nanoparticles, or complex contaminants originating from the atmosphere and soil. Ligustrum vulgare (common privet) has been used as a model to monitor the air pollution using Comet assays: analyses of leaves harvested at three locations with different levels of air pollution in Bosnia and Herzegovina, including two urban and one rural location, showed that DNA damage measured as tail intensity depended on the sampling period, leaf position, and growth stage, and showed clear differences between urban and rural locations [22]. Furthermore, this model proved sensitive to seasonal variations in air pollution levels since DNA damage in L. vulgare leaves was proportional to the average concentration of particulate matter of a size ranging from 0.001 to 2.5 µm in diameter (PM2.5) and depended on indoor vs. outdoor conditions [22].
Another expanding area of Comet assay applications is the evaluation of the genotoxic effects of newly developed herbicides. For example, cytotoxic and genotoxic effects of imazethapyr, an imidazolinone herbicide, were investigated using root meristem cells of Allium cepa [23]. The results indicated that imazethapyr exhibits cytotoxic activity and induces DNA damage in a dose-dependent manner. Comet assays were used to examine the cytotoxic and genotoxic effects of the herbicides penoxsulam, pinoxaden, and clopyralid on A. cepa roots. All three compounds showed a cytotoxic effect by reducing the root growth and mitotic index, and a genotoxic effect by increasing chromosome aberrations and DNA damage, as compared to control roots [24][25][26]. Furthermore, the genotoxic effects of two herbicides representing synthetic auxins, picloram and dicamba, on root meristems of A. cepa were evaluated utilizing Comet assays in roots grown either under conditions simulating tissue culture (i.e., aseptic conditions using Murashige and Skoog (MS) medium) or in bidistilled water [27]. Both herbicides induced a more severe stress and more pronounced DNA damage in the cells of roots grown under tissue culture conditions. This study confirmed the genotoxic effects of two growth regulators on plant cells [27].

3. Comet Assays in Plant Physiological Studies

The Comet assay has proved a valuable research tool and, as such, has been used in plant radiation biology, embryology, and studies on plant development and responses to abiotic and biotic stresses, as well as in experimental data modeling. In plant molecular studies, Comet assays are utilized to characterize the mutants impaired in nuclear DNA repair functions, in DNA damage sensing/signaling, and chromatin remodeling [14][28][29][30][31][32]. Furthermore, Comet assays can be applied to studies of DNA replication in chloroplasts. In chloroplasts, transcription–replication conflicts can lead to the formation of R-loops, temporary hybrids between template DNA and nascent mRNA, which can block replication fork progression and provide a major source of genomic instability. In a recent study, the Comet assay (neutral version) was used to characterize the mechanism involved in R-loop formation via assessing the chloroplast genome integrity in mutants [33].
Comet assays are also very useful for the complex characterization of instability induced in plant genomes by various stress factors [34]. Moreover, the alkaline Comet assay provides an efficient tool for differentiating between agents that represent sources of genotoxic and purely cytotoxic damage. For instance, Comet assays performed with human leukocytes showed that camptothecin, an inhibitor of topoisomerase I, or actinomycin D, an inhibitor of RNA synthesis, both provoked DNA strand breaks (i.e., comets were observed in the assays), while chemicals that do not bind DNA such as cordycepin, an mRNA synthesis inhibitor that blocks the elongation of the growing RNA chain, fluorodeoxyuridine, blocking the action of thymidilate synthetase, or puromycin, an inhibitor of protein synthesis, did not induce the formation of comets [35]. Below, the researchers discuss how the Comet assay can be used for studies on plants exposed to stress factors causing, directly or indirectly, damage to DNA.

3.1. Use of the Comet Assay in Studies on Plants Exposed to Genotoxic Stress Factors

Common genotoxic stress factors include ionizing radiation such as gamma irradiation, X-rays, and short-waved UV-C, as well as exposure to radiomimetics, a large group of compounds acting directly on DNA-like alkylating agents and antibiotics of the bleomycin family, or drugs inhibiting DNA synthesis and repair like, e.g., antimetabolites [36][37].
Gamma rays are the most effective ionizing radiation used in agricultural programs, for instance, for the surface sterilization of agricultural products in order to increase their conservation time via reducing pathogen propagation. The effects of γ-radiation on the nuclear DNA of Vicia faba seeds either without treatment or treated with ZnO nanoparticles were studied using flow cytometry, an alkaline Comet assay, and the random amplification of polymorphic DNA (RAPD)-PCR; all three assays confirmed that the treatment of the seeds with ZnO nanoparticles preserved their DNA integrity [38]. Furthermore, γ-radiation is an attractive alternative to the chemicals used against an obligate endoparasite, the root-knot nematode Meloidogyne incognita that can infect most vegetables, fruits, and ornamental plants all over the world [39]. Analysis of the effects of low doses of γ-radiation on the infectivity of M. incognita, growth of tomato and pepper host plants, and DNA breakage in leaf cells using neutral Comet assays showed that the treatment of seedlings with γ-irradiation is a promising technique for nematode control without any suppressive effects on the host plants [39].
The effects of seed pre-treatment with cold atmospheric-pressure air plasma on DNA and on the induction of a positive adaptive response in seedlings were studied in pea seedlings [40]. The DNA damage was analyzed using alkaline comet assays, and the induced adaptive response was tested using the toxic concentrations of a DNA double-strand break inducer, the glycopeptide antibiotic zeocin. At all exposure times studied, seed pre-treatment with plasma exerted a protective effect on seeds and subsequently led to a reduction in DNA damage in zeocin-treated pea seedlings, in comparison to seedlings germinated from control seeds without plasma treatment. These results not only confirmed that plasma can be safely used in agriculture for seed treatment, but also showed the existence of a plasma-induced adaptive response [40].
The exposure of plants to heavy metals can decrease the growth and yield of crops, and produce toxic, mutagenic, and carcinogenic effects in natural biological systems. Prolonged exposure to physiological concentrations of heavy metals, e.g., chromium, can inhibit the enzymes of DNA repair mechanisms and cause the formation of DNA adducts, leading to direct DNA damage like SSBs, DSBs, base modifications, and DNA–DNA/protein cross-linking, resulting in genotoxic effects [41]. Furthermore, the exposure of plants to heavy metals initiates ROS formation and other cytotoxic effects in the cytosol that may also lead to DNA damage [42][43][44]. Alkaline Comet assays were used to evaluate the extent of DNA damage in leaf and root cells of Indian mustard (Brassica juncea) and yellow lupin (Lupins luteus) caused by lead [45][46]. The accumulation of lead inside cells was proportional to the amount of DNA fragments migrating away from the tail of DNA comets, as well as to the degree of cell damage. Another study using neutral Comet assays showed that exogenously applied jasmonic acid decreased the DNA damage incurred by the lead treatment [47].
The DNA damage and chromatin degradation were evaluated using alkaline Comet assays and fluorescent immunolabeling following the exposure of the roots of Allium cepa and Vicia faba to the organophosphate insecticides fenthion and malathion and to two heavy metal salts, nickel nitrate and potassium dichromate [48]. Severe DNA damage in the cells of the roots treated with the highest doses of both stressors was associated with the induction of apoptosis-like programmed cell death [48].
Arsenic toxicity was evaluated using alkaline Comet assays [49][50]. In both the leaves and roots of V. faba and Cucumis melo grown hydroponically in liquid medium supplemented with disodium hydrogen arsenate, a loss of DNA integrity was shown. In another study, neutral Comet assays showed that vanadium treatment leads to DNA fragmentation in Allium cepa roots with the level of DSBs increasing depending of the VCI3 dose, possibly indicating the induction of PCD [51].
A study comparing the effects of four heavy metals (cadmium, zinc, copper, and lead), the preservative sodium benzoate, and wastewater on DNA integrity was performed with the roots of Allium cepa using the neutral Comet assay [52]. The results showed that the DNA damage, depending on the comet tail length, was highest in the presence of cadmium and decreased in the presence of the other pollutants in the order cadmium > zinc > sodium benzoate > copper > lead > wastewater. Interestingly, the pretreatment of barley seedlings with a non-toxic dose of cadmium chloride prior to exposure to MNU reduced the frequency of chromatid aberrations and the formation of micronuclei and aneuploid cells, as well as the amount of DNA in comet tails [53].

3.2. Use of the Comet Assay in Studies on Plants Exposed to Cytotoxic Stress

Salinity is one of the major abiotic stress factors and affects the productivity of agricultural crops worldwide. Both Na+ and Cl ions cause a number of cytotoxic effects on cells and organelles [54][55]. Furthermore, an increase in ROS production and a decrease in cytosolic K+ can lead to the induction of PCD; one of the important steps in this pathway is the activation of the endonucleases that introduce dsDNA breaks [56][57][58]. Below, the researchers discuss several studies showing, on the basis of the Comet assay, that exposure to NaCl can be accompanied by damage to DNA; more examples are listed in Table 1.
Table 1. Some examples of the successful application of Comet assays to studies of plant stress responses.
Neutral Comet assays performed with the leaves of barley genotypes with different salt tolerance showed that after two weeks of plant growth in a hydroponic medium supplemented with 300 mM NaCl, DNA damage in leaf cells of a salt-sensitive cultivar significantly increased; this was accompanied by a rise in ROS production and a drop in the activities of antioxidant systems [73].
The alkaline version of the Comet assay was successfully used to show that pretreatment of Vigna radiata (mungbean) seeds with sublethal doses of NaCl can alleviate the injurious effects of the later application of NaCl stress on roots and leaves of 7 d old seedlings [72]. As confirmed by Comet assays, an increase in DNA damage was caused by a NaCl-induced rise in ROS in a dose-dependent manner [72]. Alkaline Comet assays also revealed the cytoprotective role of the brassinosteroid 24-Epibrassinolide (EBL) in Brassica juncea (Indian mustard) under salinity stress. Exogenously applied EBL diminished the deleterious effect of 100 mM NaCl on DNA, as evidenced via a decrease in tail length and tail moment of the comets. The EBL-induced protective mechanisms were based on the prevention of the accumulation of ROS [74].
The role of DNA polymerase λ (OsPolλ) in DNA repair and in plant tolerance to salinity and drought stress was studied on three Oryza sativa cultivars [75]. The damage to genomic DNA was assessed using neutral Comet assays. Both OsPolλ gene expression and enzymatic activity were enhanced in response to these stresses, and higher DNA damage was associated with higher OsPolλ expression and enzyme activity.
Interestingly, pre-treatment with L-carnitine reduced the genotoxic effects of salinity, as shown by a study of seed germination and cell division in the root meristem cells of barley seedlings [76]. L-carnitine facilitates the transport of long chain fatty acids from the cytosol into the mitochondria and acts in lipid metabolism, presumably through the management of specific acyl-CoA pools [88]; the exogenous application of L-carnitine can enhance plant growth [89]. Alkaline Comet assays’ results showed that exogenously applied L-carnitine alleviated the harmful effects of salt stress as it led to the increased mitotic activity of root meristem cells and reduced the levels of DNA damage.
Allelopathic compounds, also called allelochemicals, are biologically active secondary metabolites produced by ‘donor’ plants in order to suppress the growth of their competitors—‘recipient’ plants of the same or different species. In many studies of plant–plant interactions, a reduction in DNA integrity was observed in recipient plants. This corresponds well to the fact that in many cases, allelochemicals induce ROS-mediated PCD in the recipient plants (e.g., [90]).
Narciclasine, an Amaryllidaceae alkaloid isolated from Narcissus tazetta bulbs, inhibits root growth due to cell cycle arrest, and causes the accumulation of ROS and an increase in DNA damage in the cells of lettuce roots [86]. A forest tree, Eucalyptus globulus, is able to produce allelochemicals, which accumulate in rhizosphere soil at high concentrations. When the fine powder of dried Eucalyptus leaves was added to a soil mixture for soybean, an increase in DNA damage was revealed through alkaline Comet assays, accompanied by an enhancement of the transcript levels of a legumain-like cysteine protease VPE3, indicating that the Eucalyptus leaf powder induced an apoptotic response to allelopathic stress [85].
The genus Solanum is the largest in the family Solanaceae, and Solanum species produce various allelochemicals, e.g., steroidal glycoalkaloids. The allelopathic effects of the hydroalcoholic extracts from two Solanum species, S. muricatum and S. betaceum, on lettuce were investigated; alkaline Comet assays indicated that both extracts induced DNA damage in leaf cells [87].
Strikingly, Comet assays have revealed a link between the plant immune response and DNA damage repair. Accumulation of DSBs in Arabidopsis was observed following infection by plant pathogens: a bacterium, a hemibiotrophic oomycete, and a necrotrophic fungus. Non-pathogenic E. coli did not produce this effect and thus served as negative control [68]. The genotoxicity of microbial infections could be attributed mainly to the effectors produced by pathogens and not to host-generated ROS [68]. Potato virus X (PVX) induced DNA damage in the nuclei of its host, tobacco (Nicotiana tabacum var. xanthi) [69]. Neutral Comet assays detected chronic genotoxic stress in an insertional mutant of Arabidopsis impaired in the nuclear DNA mismatch repair mechanism; the phenotype included the activation of DDR-associated genes and an increased resistance against Pseudomonas syringae [71]. Thus, the constitutive expression of DDR and DSB repair genes seems to contribute to an increased plant resistance to pathogens [71].
Heat stress is one of the major abiotic stress factors that induces heavy DNA damage in plants including nucleotide modifications, formation of SSBs and DSBs, and changes in chromatin architecture (reviewed in [61]); it also inhibits DNA repair mechanisms [91]. In higher eukaryotes, hyperthermia leads to the activation of highly conserved defensive mechanisms known as the heat stress response, in which the heat shock transcription factors (HSF) enhance the transcription of heat shock proteins’ genes (HSP) via directly binding to the heat response elements in their promoters (reviewed in [61]). Recently, the high expression of osmotically responsive genes 1 (HOS1), an E3 ubiquitin ligase, was shown to play a role in the establishment of basal thermotolerance. As was shown by neutral Comet assays, HOS1 induced basal thermotolerance via the activation of DNA repair genes, acting as a transcriptional co-regulator [61]. Contrarily to basal thermotolerance, acquired thermotolerance depends on the heat stress memory that is often “encoded” by chromatin modifications [61]. Thus, under heat stress, DNA repair is mediated by the thermoresponsive HOS1-dependent pathway, in addition to the ATM-dependent pathway for repairing DSBs and, possibly, the ATR-dependent pathway for repairing SSBs.
Comet assays were used to monitor DNA damage in plant cells under heat stress in several more studies (see also Table 1). Experiments on Vicia faba leaves showed that DNA damage increased with increasing levels of heat stress, correlated with the in situ production of ROS, the number of dead cells, and the activity of proline synthesis [59]. Comet assays revealed the important role of the temperature enhanced lesion spots 1 (hes1) mutation in rice in the activation of the heat stress response; the mutation led to ROS production, development of necrotic spots, and a high thermosensitivity of hes1 plants [60].
Last but not least, the dual role of ROS physiology should be kept in mind. Some developmental changes involve a sharp increase in ROS production, which represents an intrinsic source of DNA damage in plants, and therefore has to be kept within a limit called the “oxidative window”. For instance, during the period of seed imbibition, cells need to maintain a fine balance between the production of ROS acting as oxidative signals promoting germination, and germination-delaying oxidative damage [92]. As shown in a study on Medicago truncatula seeds using alkaline Comet assays on radicles and 4-day-old seedlings, DNA repair is critical for the successful emergence of the radicle during the initial rehydration of the seeds, since DNA damage accumulates during maturational seed drying and storage due to poor repair capacities [93]. In another study, neutral Comet assays showed that the priming of the seeds of Solanum melongena with low NaCl concentrations enhanced the germination rate, leading to an increase in ROS levels but a decrease in DNA damage levels [79].
Note that, although conditions and protocols of the Comet assay were identical, the procedures of the isolation of the nuclei were not, and subtle differences can be seen between the two species, illustrating the necessity of the optimization of the whole method for every plant species and organ/tissue. While in non-treated control samples from both species, comets and debris of degraded nuclei were absent, heat shock-treated nuclei were losing their round shape, and “haloed” nuclei were observed where strand breaks had led to the release of supercoils and DNA relaxation, as well as comets with the accumulation of fragmented DNA in the comet tails. Zeocin-mediated DNA cleavage resulted in the formation of comets with relatively short tails of less than half of the total comet length. The high level of background debris in zeocin-treated barley samples is thought to be related to the strong disintegration of the nuclei. Note also that in barley control samples, some of the nuclei show protrusions indicative of the beginning of DNA relaxation; the reason for this might be the release of proteolytic and/or nucleolytic enzymes during the isolation of the nuclei, indicating that the procedure should be further optimized.

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