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TUNEL Assay to Assess Extent of DNA Fragmentation and Programmed Cell Death in Root Cells under Various Stress Conditions
各种胁迫条件下根细胞DNA片段化和程序性细胞死亡的TUNEL法评估   

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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(DNA片段化), Free DNA termini(游离DNA末端), Genotoxic(遗传毒性), Programmed cell death (PCD)(程序性细胞死亡(PCD)), Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(末端脱氧核苷酸转移酶介导的dUTP缺口末端标记)

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.

Materials and Reagents

  1. 1-200 μl pipet tips (DNase-free) (Corning, USA)
  2. 0.2-2 μl pipet tips (DNase-free) (Corning, USA)
  3. 100-1,000 μl pipet tips (DNase-free) (Corning, USA)
  4. Razor blade (any standard make)
  5. 1.5 ml and 2 ml microcentrifuge tubes (DNase-free) (Corning, USA)
  6. 15 ml and 50 ml centrifuge tubes (DNase-free) (Corning, USA)
  7. Aluminium foil (any standard make)
  8. Glass slides (any standard make)
  9. Cover slips (any standard make)
  10. 1-4 weeks old rice (Oryza sativa cv. IR64) seedlings (see Note at the beginning of the ‘Procedure’ section)
  11. Ethanol (Sigma-Aldrich, catalog number: 24102 )
    Note: This product has been discontinued.
  12. ProLong® Gold Antifade mountant with DAPI (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36931 )
  13. DeadEndTM Fluorometric TUNEL System (Promega, catalog number: G3250 )
    Note: *In case the DeadEndTM Fluorometric TUNEL System (Promega, catalog number: G3250 ) is being used, then the chemicals/reagents marked with an asterisk (*) need not be procured. See Note 2 below.
  14. Sodium chloride (NaCl) (AMRESCO, catalog number: 0241 )
  15. Potassium chloride (KCl) (AMRESCO, catalog number: 0395 )
  16. Sodium phosphate dibasic (Na2HPO4) (AMRESCO, catalog number: 0404 )
  17. Potassium phosphate monobasic (KH2PO4) (AMRESCO, catalog number: 0781 )
  18. Paraformaldehyde powder (Sigma-Aldrich, catalog number: 158127 )
  19. Citric acid monohydrate (Sigma-Aldrich, catalog number: C1909 )
  20. Trisodium citrate dihydrate (Sigma-Aldrich, catalog number: S1804 )
  21. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  22. Tris(hydroxymethyl)aminomethane (Sigma-Aldrich, catalog number: 252859 )
  23. Sodium cacodylate* (Sigma-Aldrich, catalog number: C4945 )
  24. Cobalt(II) chloride hexahydrate* (Sigma-Aldrich, catalog number: C8661 )
  25. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 10708984001 )
    Manufacturer: Roche Diagnostics, catalog number: 10708984001 .
  26. Bovine serum albumin (BSA) (AMRESCO, catalog number: 0332 )
  27. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: 03620 )
  28. Fluorescein-12-dUTP* (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0101 )
  29. dATP* (Promega, catalog number: U1202 )
  30. Terminal Deoxynucleotidyl Transferase, Recombinant (rTdT)* (Promega, catalog number: M1871 )*
  31. Sodium citrate
  32. Hydrochloric acid (HCl)
  33. Propidium iodide (PI) (Sigma-Aldrich, catalog number: 81845 )
  34. DNase-free proteinase K (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2544 )
  35. 1x phosphate buffered saline (PBS), pH 7.4 (see Recipes)
  36. Fixative buffer (see Recipes)
  37. 100 mM citric acid solution (see Recipes)
  38. 100 mM trisodium citrate dihydrate solution (see Recipes)
  39. 100 mM sodium citrate buffer, pH 6.0 (see Recipes)
  40. Permeabilization solution (see Recipes)
  41. Proteinase K (see Recipes)
  42. Equilibration buffer (see Recipes)
  43. Nucleotide mix (see Recipes)
  44. TUNEL reaction mix (see Recipes)
  45. 20x saline-sodium citrate (SSC) buffer (see Recipes)
  46. 2x saline-sodium citrate (SSC) buffer (see Recipes)
  47. Propidium iodide stock solution (1 μg/μl) (see Recipes)

Equipment

  1. 0.2-2 μl pipette (Gilson, PIPETMAN Classic®)
  2. 2-200 μl pipette (Gilson, PIPETMAN Classic®)
  3. 100-1,000 μl pipette (Gilson, PIPETMAN Classic®)
  4. Glass beakers of volume 20 ml, 50 ml, and 100 ml (Schott Duran, Germany)
  5. Reagent bottles, glass of volume 50 ml, 100 ml, 500 ml, and 1,000 ml (any standard make)
  6. Laboratory fume hood (any standard make)
  7. Magnetic stirrer (Genetix Brand, India)
  8. Water bath (any standard make)
  9. Confocal microscope (Nikon, model: Nikon A1R )

Software

  1. ImageJ Standard version 1.46r (http://imagej.net/mbf/)
  2. NIS Elements AR (Nikon, Japan) or a comparable software for confocal microscope image acquisition and analysis

Procedure

Note: The steps mentioned below have been described for carrying out in situ TUNEL assay in samples obtained from root tissue of 1-4 weeks old rice (Oryza sativa cv. IR64) seedlings. For root tissue from rice plants older than 4 weeks, or for those from other herbaceous plant species, the volume of the vessels and reagents can be adjusted accordingly.

  1. Germination of seeds, growth conditions and processing of samples
    Seeds may be germinated in hydroponic system, or in vermiculite or on solid medium. For rice, the suggested growth conditions are 28 ± 2 °C and 16 h/8 h photoperiod (Tripathi et al., 2015). After growth for desired number of days (for example, 15 days), root tissue should be separated from the seedling by cutting using razor blades.

  2. Fixation
    1. Fix root samples from the plants in which DNA fragmentation and PCD have to be assessed by incubating roots in fixative buffer for 16 h at 4 °C. Ensure that the roots are completely immersed in the fixing solution. Fixation may be performed in 50 ml tubes with their caps closed properly.
      Note: Fixative buffer contains paraformaldehyde–a bronchial, eye and skin irritant. Please see Note regarding preparation and disposal of the fixative buffer in the Recipes section.
    2. Wash the fixed roots with absolute ethanol (by adding absolute ethanol and gentle shaking for 10-15 sec) twice at room temperature. After washing, incubate the root samples in 70% ethanol for 24 h at 4 °C. Decant the solution (see Note 1).
    3. Incubate the samples in 1x PBS (see Recipes) for 20 min at room temperature. Remove the solution and repeat this step thrice with fresh PBS to completely remove traces of ethanol.

  3. Permeabilization
    1. Take out the root sample from the PBS solution and incubate the sample in 100 mM sodium citrate buffer (pH 6.0) (see Recipes) in a fresh tube for 15 min at room temperature.
    2. Subject root samples immersed in the sodium citrate buffer to microwave (350 W) for a duration of 30-60 sec depending on the thickness of the root tissue. For subjecting samples to microwave radiation, the root samples may be placed in 200 ml microwave-safe glass beakers. The volume of citrate buffer in these beakers should be sufficient enough to ensure complete immersion of the samples. For root samples obtained from 1-2 week-old rice seedlings, buffer volume of 100 ml is usually sufficient. Cool the samples rapidly by adding equal volume of distilled water.
    3. Remove the solution and incubate the samples in permeabilization solution (see Recipes) at 37 °C for 30 min in water bath with intermittent shaking.
    4. To digest the proteins, add DNase-free Proteinase K (see Recipes) to a final concentration of 20 µg/ml followed by incubation at 37 °C for 30 min in a water bath with intermittent shaking.
    5. Remove the solution and wash the root samples thrice with 1x PBS at room temperature.

  4. TUNEL reaction
    1. For in situ TUNEL assay, cut root samples approximately 1 cm length from the root tip using sharp razor blades and glass plates. Collect the cut root tips in microcentrifuge tubes (2 ml), one tube for each of the samples. TUNEL reaction can now be performed in these tubes.
      Note: While cutting the root samples, avoid mechanical damage, for that reason the razor blades should be sharp.
    2. Prepare TUNEL reaction mix (see Recipes) and add 110 µl of the mix (see Note 2 below) to each of the microcentrifuge tubes. The reaction should be carried out at 37 °C for 1 h. The microcentrifuge tubes should be covered with aluminum foil so as to avoid exposure to light.
      Note: The step C2 should be carried out immediately after C1.
    3. Stop the reaction by adding 1 ml of 2x saline-sodium citrate (2x SSC) buffer (pH 7.0, see Recipes).
    4. Remove 2x SSC solution and stain root tips with propidium iodide by addition of 100 μl of 1 μg/ml of propidium iodide solution (final concentration, see Recipes) and incubation at 37 °C for 10 min. Remove the PI solution and wash thrice with 1x PBS.
      Note: The microcentrifuge tubes should be covered with aluminum foil so as to avoid exposure to light.
    5. Mount the root tips onto slides with an Antifade mounting reagent. For this piece of study, we have used ProLong® Gold Antifade reagent with DAPI (Thermo Fisher Scientific, USA).
    6. Visualize the slides under a confocal microscope using 405 nm- (for DAPI) and 488 nm- (for PI and fluorescein) -lasers, as applicable, and channel settings based on emission range of the respective dyes. Representative images have been shown in Figure 1.


      Figure 1. Micrographs showing salt stress-treated rice root tissue subjected to TUNEL assay to assess in situ DNA fragmentation. Root tissue from 15-day old rice seedlings was subjected to salinity stress (200 mM NaCl) for 60 h followed by TUNEL assay as per the protocol described above. F: Fluorescein (green), PI: Propidium Iodide (red), DAPI: 4’,6-diamidino-2-phenylindole (blue). Samples were mounted on slides in a mountant (ProLong® Gold Antifade with DAPI, Thermo Fisher Scientific) and the slides were examined under Nikon A1R (Nikon, Japan) confocal microscope using a 20x objective. NIS Elements AR software (Nikon, Japan) was used to acquire and process the images. DAPI and PI were used to stain DNA (both damaged and undamaged). Fluorescein fluorescence (green) is due to incorporation of fluorescein-12-dUTP during the TUNEL reaction and would correspond to the number of free DNA ends. Scale bars = 100 µm.

Data analysis

The incorporation of labelled dUTP during the TUNEL reaction would theoretically be directly proportional to the frequency of free DNA termini (which are generated majorly due to breaks in DNA) and therefore, would indicate the extent of DNA fragmentation which in turn can provide a rough estimate of PCD. The extent of DNA fragmentation can be quantified through measurement of fluorescence intensity in the micrographs (as shown in Figure 1) in a well-defined region of interest (ROI) using various micrograph analysis software like ImageJ (http://imagej.net/mbf/) or NIS Elements (Nikon, Japan). Analysis of fluorescence intensity through ImageJ has been described in the documentation section of ImageJ and can be found at the URL: https://imagej.nih.gov/ij/docs/. One of the paths for choosing ROI and analyzing the image intensity is Analyze>Tools>ROI Manager, through which ROI Manager can be accessed. A selection tool may be chosen and the ROI can be selected by drawing. Next, to analyze the intensity, in the ROI manager window click ‘Add [t]’ then ‘Measure’ which will give the values of maximum, minimum and mean intensity along with area of the ROI. Detailed instructions regarding image processing and analysis can be found in ImageJ documentation section and in the Tutorials and Examples section (https://imagej.nih.gov/ij/docs/examples/index.html). Alternatively, when longitudinal sections of roots are used for TUNEL assay, the frequency of TUNEL-positive cells can be determined via counting the number of cells showing fluorescein-fluorescence and the total number of cells in the defined ROI. Irrespective of the method chosen, the capture settings of microscope, and the area of the ROI should be kept identical for the samples which are to be compared. When examining the stress adaptability of different plants, a relative assessment of the extent of DNA fragmentation by analyzing the fluorescence intensity often serves the purpose.

Notes

  1. Incubation of whole root samples in buffers or ethanol is to be carried out in beakers of appropriate volume. During the incubation period, the beakers/tubes should be properly covered with aluminum foil. Decanting/removal of solution after washing or incubation should be carefully carried out so as not to disturb the root samples and the next step should be carried out immediately to avoid drying of the samples.
  2. To carry out the TUNEL reaction, for our recent study (Tripathi et al., 2016), we used equilibration buffer, nucleotide mix (containing fluorescein-12-dUTP) and recombinant terminal deoxynucleotidyl transferase (rTdT) supplied with the DeadEndTM Fluorometric TUNEL System (from Promega, USA). However, buffers and reaction mixes prepared using reagents (molecular biology-grade), along with separately procured terminal deoxynucleotidyl transferase (TdT) enzyme and fluorescently labelled dUTP, can also be used to carry out the TUNEL reaction. Buffer preparation and composition of the reaction mixes have been provided below.

Recipes

  1. 1x phosphate buffered saline (PBS), pH 7.4 (1,000 ml)
    8 g NaCl
    0.2 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    Dissolve in 800 ml deionized water and bring the volume to 1,000 ml with deionized water. Store at room temperature
  2. Fixative buffer (4% paraformaldehyde [prepared in 1x PBS, pH 7.4])
    20 g paraformaldehyde is to be weighed and 1x PBS is added so as to bring the volume to 500 ml. For proper dissolution, heat the closed bottle at 65 °C in a water bath for 1.5-2 h. The solution may be stored at 4 °C
    Note: It may be noted that paraformaldehyde is quite hazardous and is a bronchial, eye and skin irritant and therefore, it should be weighed in a fume hood. The fixative buffer after use should be discarded by emptying in a hazardous waste container.
  3. 100 mM citric acid solution
    2.10 g citric acid monohydrate in 100 ml deionized water. The solution may be stored at 4 °C
  4. 100 mM trisodium citrate dihydrate solution
    2.94 g trisodium citrate dihydrate in 100 ml deionized water. The solution may be stored at 4 °C
  5. 100 mM sodium citrate buffer (100 ml), pH 6.0
    Mix 11.5 ml of 100 mM citric acid solution and 88.5 ml of 100 mM trisodium citrate dihydrate solution. Ensure the pH of the resulting solution is 6.0. The solution may be stored at 4 °C
  6. Permeabilization solution
    0.1% Triton X-100 in 100 mM sodium citrate buffer, pH 6.0. The solution should be prepared fresh
  7. Proteinase K
    20 mg/ml prepared in 10 mM Tris-Cl, pH 7.5. The enzyme solution should be stored at -20 °C
  8. Equilibration buffer*
    200 mM sodium cacodylate (pH 6.5)
    25 mM Tris-Cl (pH 6.5)
    2.5 mM cobalt chloride
    200 µM dithiothreitol
    250 µg/ml bovine serum albumin
    The solution should be prepared fresh
  9. Nucleotide mix*
    10 mM Tris-HCl (pH 7.5)
    1 mM EDTA
    60 μM fluorescein-12-dUTP
    100 μM dATP
    The mix should be stored at -20 °C
    *Note: In case the DeadEndTM Fluorometric TUNEL System (Promega, USA) is being used, then the reagents marked with an asterisk (*) need not be prepared as similar reagents are supplied with the kit. See Note 2 above.
  10. TUNEL reaction mix (per reaction)
    100 µl equilibration buffer
    10 µl nucleotide mix
    60 U (2 µl) of rTdT (Promega, USA)
    The mix should be freshly prepared
  11. 20x saline-sodium citrate (SSC) buffer (100 ml), pH 7.0
    3 M (17.54 g) sodium chloride (NaCl)
    0.3 M (8.82 g) sodium citrate
    Dissolved in 80 ml deionized water; pH is adjusted to 7.0 with hydrochloric acid (HCl) and final volume is to be brought to 100 ml with deionized water. The solution may be stored at room temperature
  12. 2x saline-sodium citrate (SSC) buffer (100 ml)
    10 ml 20x SSC, pH 7.0
    90 ml deionized water
  13. Propidium iodide stock solution (1 mg/ml)
    10 mg of propidium iodide dissolved in 10 ml 1x PBS
    Note: This solution is to be stored at 4 °C in dark. Besides, propidium iodide is potentially carcinogenic. Avoid contact with skin and wear proper protective clothing while handling the solution. Propidium iodide solution after use should be discarded by emptying in a hazardous waste container.

Acknowledgments

Research in our laboratory is supported by funds from the Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India, and ICGEB. AKT has been a recipient of senior research fellowship from DBT, Government of India, which is gratefully acknowledged. The protocol presented here describes, in detail, a more generalized version of the methodology used for TUNEL assay to assess DNA fragmentation and programmed cell death in root tissue under various stress conditions in our recent study (Tripathi et al., 2016) published in the journal Plant Physiology.

References

  1. Bray, C. M. and West, C. E. (2005). DNA repair mechanisms in plants: crucial sensors and effectors for the maintenance of genome integrity. New Phytol 168(3): 511-528.
  2. Gavrieli, Y., Sherman, Y. and Ben-Sasson, S. A. (1992). Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119(3): 493-501.
  3. Kapoor, D., Sharma, R., Handa, N., Kaur, H., Rattan, A., Yadav, P., Gautam, V., Kaur, R., and Bhardwaj, R. (2015). Redox homeostasis in plants under abiotic stress: role of electron carriers, energy metabolism mediators and proteinaceous thiols. Front Environ Sci 3: 13.
  4. Kwon, Y. I., Abe, K., Endo, M., Osakabe, K., Ohtsuki, N., Nishizawa-Yokoi, A., Tagiri, A., Saika, H. and Toki, S. (2013). DNA replication arrest leads to enhanced homologous recombination and cell death in meristems of rice OsRecQl4 mutants. BMC Plant Biol 13: 62.
  5. Manova, V. and Gruszka, D. (2015). DNA damage and repair in plants - from models to crops. Front Plant Sci 6: 885.
  6. Phan, H. A., Iacuone, S., Li, S. F. and Parish, R. W. (2011). The MYB80 transcription factor is required for pollen development and the regulation of tapetal programmed cell death in Arabidopsis thaliana. Plant Cell 23(6): 2209-2224.
  7. Santos, C., Pourrut, B., and Ferreira de Oliveira, J. (2015). The use of comet assay in plant toxicology: recent advances. Front Genet 6: 216.
  8. Smerdon, M. J. (1991). DNA repair and the role of chromatin structure. Curr Opin Cell Biol 3(3): 422-428.
  9. Tripathi, A. K., Pareek, A. and Singla-Pareek, S. L. (2016). A NAP-family histone chaperone functions in abiotic stress response and adaptation. Plant Physiol 171(4): 2854-2868.
  10. Tripathi, A. K., Singh K., Pareek, A. and Singla-Pareek, S. L. (2015). Histone chaperones in Arabidopsis and rice: genome-wide identification, phylogeny, architecture and transcriptional regulation. BMC Plant Biol 15: 42.
  11. Wang, Y., Xu, C., Du, L. Q., Cao, J., Liu, J. X., Su, X., Zhao, H., Fan, F. Y., Wang, B., Katsube, T., Fan, S. J. and Liu, Q. (2013). Evaluation of the comet assay for assessing the dose-response relationship of DNA damage induced by ionizing radiation. Int J Mol Sci 14(11): 22449-22461.
  12. Yang, Z. T., Wang, M. J., Sun, L., Lu, S. J., Bi, D. L., Sun, L., Song, Z. T., Zhang, S. S., Zhou, S. F. and Liu, J. X. (2014). The membrane-associated transcription factor NAC089 controls ER-stress-induced programmed cell death in plants. PLoS Genet 10(3): e1004243.

简介

DNA损伤是暴露于各种压力条件的常见后果之一。 已经开发了不同的方法来准确评估暴露于不同应激剂的细胞和组织中的DNA损伤和碎裂。 然而,由于纤维素细胞壁和酚类物质的存在,植物细胞和组织不容易进行这些测定。 在这里,我们描述了优化的TUNEL(末端脱氧核苷酸转移酶介导的dUTP切口标记)测定方法,以确定经受各种应激条件的植物根细胞中DNA片段化和程序性细胞死亡的程度。 这里描述的方法具有简单,可靠和重复性好的优点。
【背景】暴露于各种压力通常导致至少一定程度的DNA损伤,导致各种损伤,例如胸腺嘧啶二聚化,碱基烷基化,单链缺口和双链断裂(Bray和West,2005; Manova和Gruszka,2015)。在所有类型的DNA损伤中,DNA片段化在应激条件下特别令人关注,这可能是应激的直接影响(如用基因毒素治疗方法所观察到的)或间接作用(主要是通过过度产生的活性氧),甚至可能是两者的累积结果(Bray和West,2005; Kapoor等,2015)。这种DNA损伤必须由细胞的修复机械精确修复,否则可能会导致细胞死亡。为了维持正常状态,细胞利用依赖于三个非排他事件的DNA损伤反应。检测/识别损坏,其通过维修机械的访问,最后修复(Smerdon,1991)。
   在细胞水平上应力适应的主要分子机制之一涉及对由于应激引起的受损DNA的DNA损伤和/或有效修复的抗性。因此,为了评估基因型的应激适应性,通常需要对DNA损伤进行准确评估。两种广泛用于检测植物DNA断裂的测定法是单细胞凝胶电泳 - 也称为彗星测定(Santos等人,2015)和TUNEL [末端脱氧核苷酸转移酶(TdT)介导的dUTP尼克端标记]测定。在彗星测定中,将感兴趣的组织切片,将含有细胞核的所得细胞悬浮液包埋在琼脂糖基质中,随后进行碱性电泳并用DAPI /溴化乙锭染色。电泳后,显微照片显示类似于彗星的尾部破裂的DNA的外观,而未损伤和冷凝的DNA看起来像形成彗星头部的球形质粒(Wang等,2013)。彗星测定虽然非常有用,但有一些限制。例如,它需要分离的细胞核,因此不提供关于给定组织中DNA损伤分布的信息以及关于程序性细胞死亡(PCD)的信息。其他广泛使用的测定-TUNEL测定法可用于检测原位DNA链断裂。 TUNEL测定基于在仅具有游离3'末端的区域(即,染色体的断裂或末端)上发生的DNA(由酶末端脱氧核苷酸转移酶介导)中引入标记的dUTP(Gavrieli等人,1992 )。此外,由于核内DNA脱落常常导致程序性细胞死亡,TUNEL测定提供了有关PCD的重要信息。 TUNEL测定在其基本形式中也提供简单的优点,并且可以提供关于正在研究的组织中DNA片段化(TUNEL阳性细胞)的分布的想法。
   植物组织不容易适应TUNEL测定的一些步骤。其主要原因是:由于纤维素细胞壁的存在而难以渗透,并且由植物细胞中存在的酚类物质引起的TdT催化反应的潜在抑制。由于这些原因,TUNEL测定不是用于评估植物中DNA片段化和PCD的常用方法。尽管如此,最近的一些研究已经显示了TUNEL测定在水稻中的应用(Kwon等,2013)和拟南芥(Phan et al。,2011; Yang et al。,2014)。然而,大多数这些研究已经使用切片术/超薄切片术和“石蜡切片”制备 - 这种方法不是很容易,并且需要一些昂贵的仪器和技术专长。鉴于TUNEL测定可以提供的信息范围,特别是当确定植物基因型的胁迫适应性时,其优点与其他方法相比,需要开发一种标准化,易于遵循和相对便宜的TUNEL协议测定使用植物组织。
   在这里,我们描述了一种优化的基于TUNEL测定的方案,以评估在各种胁迫条件下植物根细胞中DNA片段化和程序性细胞死亡的程度。本文提出的方案详细描述了我们最近的研究(Tripathi等,2016)中用于TUNEL测定的更广泛的方法。虽然我们经常使用这种方法研究来自水稻和拟南芥根组织中的DNA损伤和PCD,但也可以用于在其他草本植物的根组织中研究这些现象,并在“程序”部分中详细描述了一些微小的修改。这里提出的方法是非常容易遵循,可靠和可重现的。

关键字:DNA片段化, 游离DNA末端, 遗传毒性, 程序性细胞死亡(PCD), 末端脱氧核苷酸转移酶介导的dUTP缺口末端标记

材料和试剂

  1. 1-200μl移液管吸头(无DNA酶)(康宁,美国)
  2. 0.2-2μl移液管吸头(不含DNase)(康宁,美国)
  3. 100-1,000μl移液管吸头(无DNA酶)(康宁,美国)
  4. 剃刀刀片(任何标准品)
  5. 1.5ml和2ml微量离心管(不含DNA酶)(Corning,USA)
  6. 15ml和50ml离心管(不含DNA酶)(Corning,USA)
  7. 铝箔(任何标准品)
  8. 玻璃幻灯片(任何标准品)
  9. 封面(任何标准品)
  10. 1-4周龄的水稻(“Oryza sativa”)IR64)幼苗(见“程序”部分开头的注释)
  11. 乙醇(Sigma-Aldrich,目录号:24102)
    注意:本产品已停产。
  12. ProLong ®使用DAPI的金防晕装置(Thermo Fisher Scientific,Invitrogen TM,目录号:P36931)
  13. DeadEnd TM 荧光TUNEL系统(Promega,目录号:G3250)
    注意:*如果DeadEnd TM 正在使用荧光TUNEL系统(Promega,目录号:G3250),则化学品/标有星号(*)的试剂不需要采购。见下文注2。
  14. 氯化钠(NaCl)(AMRESCO,目录号:0241)
  15. 氯化钾(KCl)(AMRESCO,目录号:0395)
  16. 磷酸氢二钠(Na 2 HPO 4)(AMRESCO,目录号:0404)
  17. 磷酸二氢钾(KH 2 PO 4)(AMRESCO,目录号:0781)
  18. 多聚甲醛粉(Sigma-Aldrich,目录号:158127)
  19. 柠檬酸一水合物(Sigma-Aldrich,目录号:C1909)
  20. 柠檬酸三钠二水合物(Sigma-Aldrich,目录号:S1804)
  21. Triton X-100(Sigma-Aldrich,目录号:T8787)
  22. 三羟甲基氨基甲烷(Sigma-Aldrich,目录号:252859)
  23. 二甲胂酸钠*(Sigma-Aldrich,目录号:C4945)
  24. 氯化钴(II)六水合物*(Sigma-Aldrich,目录号:C8661)
  25. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:10708984001)
    制造商:Roche Diagnostics,目录号:10708984001。
  26. 牛血清白蛋白(BSA)(AMRESCO,目录号:0332)
  27. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:03620)
  28. 荧光素-12-dUTP *(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0101)
  29. dATP *(Promega,目录号:U1202)
  30. 末端脱氧核苷酸转移酶,重组(rTdT)*(Promega,目录号:M1871)*
  31. 柠檬酸钠
  32. 盐酸(HCl)
  33. 碘化丙啶(PI)(Sigma-Aldrich,目录号:81845)
  34. 无DNA酶蛋白酶K(Thermo Fisher Scientific,Invitrogen TM,目录号:AM2544)
  35. 1x磷酸缓冲盐水(PBS),pH 7.4(参见食谱)
  36. 固定缓冲液(见配方)
  37. 100 mM柠檬酸溶液(见食谱)
  38. 100mM柠檬酸三钠二水合物溶液(参见食谱)
  39. 100mM柠檬酸钠缓冲液,pH6.0(参见食谱)
  40. 渗透溶液(见配方)
  41. 蛋白酶K(参见食谱)
  42. 平衡缓冲器(见配方)
  43. 核苷酸混合物(参见食谱)
  44. TUNEL反应混合物(参见食谱)
  45. 20x盐水 - 柠檬酸钠(SSC)缓冲液(见配方)
  46. 2x盐水 - 柠檬酸钠(SSC)缓冲液(参见食谱)
  47. 碘化丙啶储备溶液(1μg/μl)(见配方)

设备

  1. 0.2-2μl移液器(Gilson,PIPETMAN Classic ®)
  2. 2-200μl移液器(Gilson,PIPETMAN Classic ®)
  3. 100-1,000μl移液器(Gilson,PIPETMAN Classic ®)
  4. 体积为20毫升,50毫升和100毫升的玻璃烧杯(德国Schott Duran)
  5. 试剂瓶,杯体积为50毫升,100毫升,500毫升和1,000毫升(任何标准品)
  6. 实验室通风柜(任何标准品)
  7. 磁力搅拌器(Genetix Brand,India)
  8. 水浴(任何标准品)
  9. 共聚焦显微镜(尼康,型号:尼康A1R)

软件

  1. Image J标准版本1.46r( http://imagej.net/mbf/
  2. NIS Elements AR(Nikon,Japan)或用于共焦显微镜图像采集和分析的可比软件

程序

注意:已经描述了下面提到的步骤,用于在从1-4周龄的水稻(Oryza sativa cv.IR64)幼苗的根组织中获得的样品中进行原位TUNEL测定。对于大于4周的水稻植物的根组织,或对于其他草本植物物种的根组织,可以相应地调整容器和试剂的体积。

  1. 种子发芽,生长条件和样品处理
    种子可以在水培系统中或在蛭石中或在固体培养基上发芽。对于水稻,建议的生长条件是28±2℃和16小时/ 8小时光周期(Tripathi等人,2015)。在所需天数(例如15天)生长之后,通过使用剃刀刀片切割将根组织从幼苗中分离出来。

  2. 固定术
    1. 从植物中固定根部样品,其中DNA片段化和PCD必须通过在4℃下将固定缓冲液中的根孵育16小时来评估。确保根部完全浸入固定液中。固定可以在50ml管中进行,其盖子正确关闭。
      注意:固定缓冲液含有多聚甲醛 - 支气管,眼睛和皮肤刺激物。请参阅“食谱”部分中关于固定缓冲液的准备和处理的注意事项。
    2. 用无水乙醇洗涤固定的根(通过加入无水乙醇,温和振荡10-15秒),室温下洗涤两次。洗涤后,在4℃下将根样品在70%乙醇中培养24小时。解决方案(见注1)。
    3. 将样品在1x PBS(见食谱)中孵育20分钟。取出溶液,用新鲜的PBS重复此步骤三次,彻底清除痕量的乙醇
    1. 从PBS溶液取出根样品,并将样品在100毫升柠檬酸钠缓冲液(pH 6.0)中(见食谱)在新鲜管中室温孵育15分钟。
    2. 根据根组织的厚度将受试根样品浸入柠檬酸钠缓冲液至微波(350W)持续30-60秒。为了使样品进行微波辐射,根部样品可以放置在200ml微波安全玻璃烧杯中。这些烧杯中柠檬酸盐缓冲液的体积应足以确保样品完全浸泡。对于从1-2周龄水稻幼苗获得的根部样品,通常足够的缓冲液体积为100ml。通过加入等体积的蒸馏水快速冷却样品。
    3. 取出溶液,并将渗透溶液(见食谱)中的样品在37℃静置30分钟,并在间歇摇晃的水浴中。
    4. 为了消化蛋白质,加入不含DNA酶的蛋白酶K(参见食谱)至终浓度为20μg/ ml,然后在间歇摇动的水浴中于37℃温育30分钟。
    5. 取出溶液,并在室温下用1x PBS洗涤根部样品三次
  3. TUNEL反应
    1. 对于原位 TUNEL测定,使用锋利的剃刀刀片和玻璃板从根尖切割约1厘米长度的根部样品。在微量离心管(2 ml)中收集切割根尖,每个样品一根管。现在可以在这些管中进行TUNEL反应。
      注意:切割根部样品时,应避免机械损坏,因为剃须刀应该锋利。
    2. 准备TUNEL反应混合物(参见食谱),并向每个微量离心管中加入110μl混合物(见下文注2)。反应应在37℃下进行1小时。微量离心管应用铝箔覆盖,以避免曝光。
      注意:步骤C2应该在C1之后立即执行。
    3. 通过加入1ml 2ml盐酸柠檬酸钠(2x SSC)缓冲液(pH 7.0,参见食谱)停止反应。
    4. 通过加入100μl的1μg/ ml碘化丙啶溶液(最终浓度,参见食谱)去除2x SSC溶液并用碘化丙锭染色根尖,并在37℃下孵育10分钟。取出PI溶液,用1x PBS洗涤三次。
      注意:微量离心管应用铝箔覆盖,以避免暴露在光照下。
    5. 使用Antifade安装试剂将根尖装载到载玻片上。对于这项研究,我们已经使用ProLong ® Gold Antifade试剂与DAPI(Thermo Fisher Scientific,USA)。
    6. 在共聚焦显微镜下,使用405nm(对于DAPI)和488nm-(对于PI和荧光素) - 适用,以及基于相应染料的发射范围的通道设置可视化载玻片。代表性的图像如图1所示

      图1.显示盐胁迫处理的水稻根组织进行TUNEL测定以评估原位DNA片段化的显微照片。 根据上述方案,将来自15天龄水稻幼苗的根组织进行盐胁迫(200mM NaCl)60小时,然后进行TUNEL测定。 F:荧光素(绿色),PI:碘化丙啶(红色),DAPI:4',6-二脒基-2-苯基吲哚(蓝色)。将样品安装在载体(ProLong®®>>>>>>> D D D D D D D D D。。。。。on on on on on on on on on on on。。。。。。。。。。。。。。。。。) NIS Elements AR软件(Nikon,Japan)用于获取和处理图像。 DAPI和PI用于染色DNA(损伤和未损伤)。荧光素荧光(绿色)是由于在TUNEL反应期间引入荧光素-12-dUTP,并且对应于游离DNA末端的数目。刻度棒=100μm。

数据分析

在TUNEL反应期间掺入标记的dUTP理论上与游离DNA末端的频率(其主要是由于DNA断裂而产生)的频率成正比,因此,将指示DNA断裂的程度,其又可以提供粗略的估计的PCD。通过使用诸如ImageJ( http://imagej.net/mbf/ )或NIS Elements(Nikon,Japan)。 ImageJ的文档部分介绍了ImageJ的荧光强度分析,可以在URL中找到: https://imagej.nih.gov/ij/docs/ 。 选择投资回报率和分析图像强度的途径之一是Analyze>工具>投资回报率管理器,通过其可以访问投资回报率管理器。可以选择选择工具,并且可以通过绘图来选择ROI。接下来,为了分析强度,在ROI管理器窗口中单击“添加[t]”,然后单击“度量”,这将给出最大值,最小值和平均强度值以及ROI的面积。有关图像处理和分析的详细说明可以在ImageJ文档部分和教程和示例部分( https://imagej.nih.gov/ij/docs/examples/index.html )。或者,当根部的纵向切片用于TUNEL测定时,可以通过计数显示荧光素荧光的细胞数和所定义的ROI中的细胞总数来确定TUNEL阳性细胞的频率。不考虑所选择的方法,显微镜的捕获设置和ROI的面积应保持与要比较的样品相同。在研究不同植物的胁迫适应性时,通过分析荧光强度对DNA片段化程度的相对评估通常用于目的。

笔记

  1. 在缓冲液或乙醇中培养全根样本应在适量的烧杯中进行。在孵化期间,烧杯/管子应正确覆盖铝箔。应仔细进行洗涤或孵育后的溶液的倾析/去除,以免干扰根部样品,并立即进行下一步骤,以避免样品干燥。
  2. 为了进行TUNEL反应,对于我们最近的研究(Tripathi等人,2016),我们使用平衡缓冲液,核苷酸混合物(含荧光素-12-dUTP)和重组末端脱氧核苷酸转移酶(rTdT)随附的 DeadEnd TM 荧光TUNEL系统(来自美国Promega)。然而,使用试剂(分子生物学级)制备的缓冲液和反应混合物,以及单独采用的末端脱氧核苷酸转移酶(TdT)酶和荧光标记的dUTP也可用于进行TUNEL反应。缓冲液的制备和反应混合物的组成如下。

食谱

  1. 1x磷酸缓冲盐水(PBS),pH 7.4(1,000ml) 8克NaCl
    0.2克KCl
    1.44g Na 2 HPO 4
    0.24g KH 2 PO 4
    溶解在800毫升去离子水中,并用去离子水将体积调至1,000毫升。在室温下存放
  2. 固定缓冲液(4%多聚甲醛[在1×PBS中制备,pH 7.4])
    称取20g多聚甲醛,加入1×PBS以使体积达到500ml。为了正确溶解,将封闭的瓶子在65°C的水浴中加热1.5-2小时。溶液可以储存在4°C 注意:可以注意到,多聚甲醛是相当危险的,是支气管,眼睛和皮肤刺激物,因此应在通风柜中称重。使用后的固定缓冲液应通过在危险废物容器中排空而丢弃。
  3. 100 mM柠檬酸溶液
    2.10克柠檬酸一水合物在100ml去离子水中。溶液可以储存在4°C
  4. 100mM柠檬酸三钠二水合物溶液
    2.94克柠檬酸三钠二水合物在100ml去离子水中的溶液。溶液可以储存在4°C
  5. 100mM柠檬酸钠缓冲液(100ml),pH6.0
    混合11.5ml 100mM柠檬酸溶液和88.5ml 100mM柠檬酸三钠二水合物溶液。确保所得溶液的pH值为6.0。溶液可以储存在4°C
  6. 渗透溶液
    0.1%Triton X-100在100mM柠檬酸钠缓冲液中,pH6.0。解决方案应该是新鲜的
  7. 蛋白酶K
    在10mM Tris-Cl,pH7.5中制备20mg / ml。酶溶液应储存在-20°C
  8. 平衡缓冲器*
    200mM二聚氰酸钠(pH6.5)
    25mM Tris-Cl(pH6.5)
    2.5 mM氯化钴
    200μM二硫苏糖醇
    250μg/ ml牛血清白蛋白 解决方案应该是新鲜的
  9. 核苷酸混合*
    10mM Tris-HCl(pH7.5)
    1 mM EDTA
    60μM荧光素-12-dUTP
    100μMdATP
    混合物应保存在-20°C
    注意:如果正在使用Fluorometric TUNEL系统(美国Promega)的DeadEnd TM ,则用星号(*)不需要准备,因为试剂盒提供了类似的试剂。见上文注2。
  10. TUNEL反应混合物(每次反应)
    100μl平衡缓冲液
    10μl核苷酸混合物
    rTdT(Promega,USA)的60U(2μl)
    组合应该是新鲜准备的
  11. 20x盐水 - 柠檬酸钠(SSC)缓冲液(100ml),pH7.0。
    3 M(17.54 g)氯化钠(NaCl) 0.3M(8.82g)柠檬酸钠
    溶于80ml去离子水中;用盐酸(HCl)将pH调节至7.0,用去离子水将最终体积调至100ml。溶液可以在室温下储存
  12. 2x盐水 - 柠檬酸钠(SSC)缓冲液(100ml) 10 ml 20x SSC,pH 7.0
    90 ml去离子水
  13. 碘化丙啶储备溶液(1 mg / ml)
    将10毫克碘化丙啶溶于10ml 1x PBS中 注意:该溶液要在4℃下在黑暗中储存。此外,碘化丙啶可能是致癌的。避免接触皮肤,并在处理溶液时穿上适当的防护服。碘化丙啶溶液在使用后应通过在危险废物容器中排空而丢弃。

致谢

我们实验室的研究得到了生物科技部(DBT),科技部,印度政府和ICGEB的资助。 AKT一直是印度政府DBT的高级研究奖学金的获得者,非常感谢。本文提出的方案详细描述了在我们最近的研究(Tripathi等人)中用于TUNEL测定以评估各种应激条件下根组织中的DNA断裂和程序性细胞死亡的更广泛的方法>,2016)刊登在“植物生理学”杂志上。

参考

  1. Bray,CM和West,CE(2005)。 DNA植物修复机制:用于维持基因组完整性的关键传感器和效应器。新的Phytol 168(3):511-528。
  2. Gavrieli,Y.,Sherman,Y.and Ben-Sasson,SA(1992)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/1400587 “target =”_ blank“>通过核DNA断裂的特异性标记原位鉴定程序性细胞死亡。 J Cell Biol 119(3):493- 501.
  3. Kapoor,D.,Sharma,R.,Handa,N.,Kaur,H.,Rattan,A.,Yadav,P.,Gautam,V.,Kaur,R.,and Bhardwaj,R。(2015) ; 非生物胁迫下植物中的氧化还原体内平衡:电子载体,能量代谢介质和蛋白质硫醇的作用。前沿环境科学 3:13.
  4. Kwon,YI,Abe,K.,Endo,M.,Osakabe,K.,Ohtsuki,N.,Nishizawa-Yokoi,A.,Tagiri,A.,Saika,H.and Toki,S。(2013) ; DNA复制阻滞导致分生组织中同源重组和细胞死亡的增强的水稻OsRecQl4突变体。 BMC Plant Biol 13:62.
  5. Manova,V.和Gruszka,D。(2015)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/26557130”target =“_ blank” >植物中的DNA损伤和修复 - 从模型到作物。前植物科学 6:885.
  6. Phan,HA,Iacuone,S.,Li,SF and Parish,RW(2011)。  MYB80转录因子是拟南芥花粉发育和调节绒毡层程序性细胞死亡所必需的。植物细胞23 (6):2209-2224。
  7. Santos,C.,Pourrut,B.,and Ferreira de Oliveira,J。(2015)。  在植物毒理学中使用彗星测定:最近的进展。 6:216.
  8. Smerdon,MJ(1991)。  DNA修复和角色的染色质结构。 Curr Opin Cell Biol 3(3):422-428。
  9. Tripathi,AK,Pareek,A.和Singla-Pareek,SL(2016)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/27342307” target =“_ blank”> NAP家族组蛋白伴侣在非生物胁迫反应和适应中的作用。植物生理学171(4):2854-2868。
  10. Tripathi,AK,Singh K.,Pareek,A. and Singla-Pareek,SL(2015)。  拟南芥中的组蛋白伴侣和水稻:全基因组鉴定,系统发育,结构和转录调控。 BMC Plant Biol 15:42.
  11. Wang,Y.,Xu,C.,Du,LQ,Cao,J.,Liu,JX,Su,X.,Zhao,H.,Fan,FY,Wang,B.,Katsube,T.,Fan,SJ和Liu,Q.(2013)。评估用于评估由电离辐射诱导的DNA损伤的剂量 - 反应关系的彗星测定。 Int J Mol Sci 14(11):22449-22461。
  12. Yang,ZT,Wang,MJ,Sun,L.,Lu,SJ,Bi,DL,Sun,L.,Song,ZT,Zhang,SS,Zhou,SF and Liu,JX(2014) =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/24675811”target =“_ blank”>膜相关转录因子NAC089控制ER-应激诱导的程序性细胞死亡植物。 PLoS Genet 10(3):e1004243。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Tripathi, A. K., Pareek, A. and Singla-Pareek, S. L. (2017). TUNEL Assay to Assess Extent of DNA Fragmentation and Programmed Cell Death in Root Cells under Various Stress Conditions. Bio-protocol 7(16): e2502. DOI: 10.21769/BioProtoc.2502.
  2. Tripathi, A. K., Pareek, A. and Singla-Pareek, S. L. (2016). A NAP-family histone chaperone functions in abiotic stress response and adaptation. Plant Physiol 171(4): 2854-2868.
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