Abstract Among several factors affecting radiation sensitivity, genome size has received limited attention during the last 50 years since research at Brookhaven National Laboratory (USA) and other locations demonstrated substantial differences in radiation sensitivities, e.g. between tree species with large (e.g. conifers such as pines) versus small (e.g. dicots such as oaks) genome sizes. Taking advantage of the wide range of genome sizes among species, we investigated radiation sensitivity which we define in this study as DNA damage (break frequency) measured with the alkaline comet assay in isolated nuclei exposed to X-rays. As a starting point, we considered two possible explanations for the high radiation sensitivity of plants with large genome sizes: (i) inherently higher sensitivity of larger genomes and/or (ii) impaired DNA repair. In terms of genome size effects, experiments exposing isolated nuclei from six different plant species to X-rays, varying in genome sizes from 2.6 to 19.2 Gbp, showed that larger genomes are more sensitive to DNA damage by a relationship approximating the cube-root of the nuclear volume; e.g. a 10-fold increase in genome size increases sensitivity by about 2-fold. With regard to DNA repair, two conifer species, Sawara cypress (Chamaecyparis pisifera, 8.9 Gbp genome size) and Scots pine (Pinus sylvestris, 20 Gbp genome size), both effectively repaired DNA damage within 50 and 70 min, respectively, after acute X-ray exposures. Both species also showed delayed repair of double-strand DNA breaks, as we previously showed with Arabidopsis thaliana and Lolium multiflorum. Introduction One of the important factors determining the radiation sensitivities of organisms is their ability to repair DNA damage effectively. DNA repair has been an active area of research for more than 50 years (1–3). On the other hand, studies of the effect of genome size on radiation sensitivity are much more limited even though this factor has been recognized for many years (4–8). A classic series of studies of the effect of genome size on radiation sensitivity involved research at Brookhaven National Laboratory and other locations during 1960s on relative effects of gamma radiation on different species. Seeds (4), seedlings (5,6) and mature trees (7) were tested. It was concluded (4–6) that differences in radiation sensitivities could be explained based on genome sizes. However, an alternative explanation in terms of differences in DNA repair was not evaluated. Therefore, we considered 2 possible explanations for the high radiation sensitivity of plants with large genomes: (i) inherently higher sensitivity of larger genomes and/or (ii) impaired DNA repair. The assumption underlying the present research was that the comet assay can be used with plants (9) to show that genome size can affect radiation sensitivity (break frequency) which we define in this report as DNA damage (breaks, % tail DNA) measured with the alkaline comet assay in isolated nuclei exposed to X-rays. In addition, we investigate whether differences in DNA repair capacities might also be involved. Materials and Methods Leaf samples of the following diploid species were purchased locally: spinach (Spinacia oleracea, 12.6 Gbp haploid genome size), celery (Apium graveolens, 14.1 Gbp), pea (Pisum sativum, 3.9 Gbp) and lettuce (Lactuca sativa, 2.6 Gbp). Samples of the following diploid conifer species (10) were collected on the campus of the Norwegian University of Life Sciences at Aas, Norway: Norway spruce (Picea abies, 19.2 Gbp), Scots pine (Pinus sylvestris, 20 Gbp) and Sawara cypress (Chamaecyparis pisifera, 8.9 Gbp). Methods for X-irradiation as well as isolation and embedding of nuclei have been described (11). Times for DNA unwinding were increased to at least 30 min and electrophoresis time was increased to 30 min to compensate for large genome sizes. Analysis of comets was performed as described with 50 comets scored per sample. DNA damage was measured as % tail DNA with the Comet Assay IV image analysis program (Perceptive Instruments). % Tail DNA is related to the number of breaks per 109daltons DNA; i.e. break frequency. As recommended (12), significance was also confirmed using the t-test and P values were calculated. Results Sensitivity depends on genome size An earlier study with Arabidopsis thaliana (11) used X-ray exposures to demonstrate that DNA repair consisted of an initial rapid phase followed by a delayed phase when double-strand breaks were repaired. The rapid initial phase of repair could be detected with the comet assay after exposures corresponding to 5 and 10 Gy but not after a 2 Gy exposure because the rapid phase in these plants was so fast that the initial repair phase was completed within the time taken to isolate and embed nuclei. Keeping this in mind, we decided to isolate nuclei from different species and embed nuclei in agarose before X-ray irradiation. In this way, we expected to be able to determine inherent sensitivities for DNA damage in nuclei independent of differences in DNA repair effectiveness. Figure 1 shows % tail values after 1 Gy X-irradiation for isolated nuclei representing six different plant species. Fig. 1. View largeDownload slide Effect of genome size on DNA damage (% tail) caused by an acute X-ray exposure (1 Gy) of isolated nuclei. Symbols are as follows: open circles, lettuce; closed circles, pea; open squares, Sawara cypress; solid squares, spinach; open triangles, celery; closed triangles, Scots pine. Each point is the mean % tail DNA value for 50 comets. Line A is the linear regression of the data, while line B corresponds to the cube-root of nuclear volumes, assuming that lettuce has a nuclear radius corresponding to 1.0 (r axis). Fig. 1. View largeDownload slide Effect of genome size on DNA damage (% tail) caused by an acute X-ray exposure (1 Gy) of isolated nuclei. Symbols are as follows: open circles, lettuce; closed circles, pea; open squares, Sawara cypress; solid squares, spinach; open triangles, celery; closed triangles, Scots pine. Each point is the mean % tail DNA value for 50 comets. Line A is the linear regression of the data, while line B corresponds to the cube-root of nuclear volumes, assuming that lettuce has a nuclear radius corresponding to 1.0 (r axis). Conifers effectively repair DNA after X-irradiation As shown in Figure 2, we were only able to follow the delayed phase of repair in Scots pine and Sawara cypress, again, presumably because the early rapid phase was too fast to be detected with the comet assay. Nevertheless, Figure 2 shows that both conifers effectively repaired DNA damage. Fig. 2. View largeDownload slide Repair of DNA damage after X-ray exposures of conifers. Upper panel shows time course of DNA repair after a 5 Gy exposure of Sawara cypress (Chamaecyparis pisifera), while the lower panel shows repair after a 1 Gy exposure of Scots pine (Pinus sylvestris). Each point is the mean % tail DNA value for 50 comets. Different symbols indicate results obtained in different, independent experiments. Control values shown are the means of control values for the different experiments reported in the figure. Fig. 2. View largeDownload slide Repair of DNA damage after X-ray exposures of conifers. Upper panel shows time course of DNA repair after a 5 Gy exposure of Sawara cypress (Chamaecyparis pisifera), while the lower panel shows repair after a 1 Gy exposure of Scots pine (Pinus sylvestris). Each point is the mean % tail DNA value for 50 comets. Different symbols indicate results obtained in different, independent experiments. Control values shown are the means of control values for the different experiments reported in the figure. Discussion Our results confirm earlier studies, showing a relationship between genome size and radiation sensitivity. These earlier studies used intact cells and scored for survival, using mammalian cells in vitro (14), seeds (4), seedlings (5,6) and mature trees (7). On the other hand, our experiments analyzed DNA damage caused by X-irradiation in isolated nuclei from plant species with different genome sizes, varying from 2.6 to 19.2 Gbp. By using isolated nuclei, we could measure the inherent sensitivity of nuclear DNA, independent of possible differences in DNA repair. As far as the relationship between genome size and radiation sensitivity in G0 leaf cells (15) is concerned, Bowen (4), Sparrow and Miksche (5) and Baetcke et al. (6), all tried to relate their data on DNA contents per cell (nuclear volumes) versus LD50 for radiation damage by calculating linear regressions. When we calculated a linear regression for % tail DNA damage versus genome size, we obtained y = 0.27x + 2.72, r2 = 0.69. Given df = 10 and SE of the slope equal to 0.06, a slope value of 0.27 gives P values that are <0.001 for both slope equals 0 (null hypothesis for no effect of genome size on % tail DNA) and slope equals 1 (null hypothesis for linear relationship between genome size and % tail DNA). Rather than a linear relationship between genome size and radiosensitivity, an alternative explanation of the results is that the probability that a given photon of radiation will cause a break in DNA, directly or indirectly via production of reactive oxygen species (16), increases in relation to the distance travelled through a cell nucleus. For example, if genome size increases by 10-fold (v = ¾ π r3), the radius of the sphere increases by the cube-root of 10 which corresponds to about 2-fold (Figure 1). Kohn et al. (17) X-irradiated (2.5 Gy) mouse leukemia cells in growth medium, then isolated DNA and fractionated it through a filter. Based on the lengths of eluted strands, they calculated that 2.5 Gy exposure causes approximately 450 breaks per Gbp of DNA. For larger nuclei containing more DNA, as is the case with many conifers (10), one would expect a higher frequency of breaks because a longer flight path increases the probability that any given photon will cause a break in DNA, perhaps because reactive oxygen free radicals produced by photons would be more likely to reach a DNA target within a large nuclear mass. Our study also involved a direct evaluation of the possible role of DNA repair in explaining differences in radiation sensitivity. Both Scots pine and Sawara cypress were shown to be at least as effective as Arabidopsis (11) in repairing DNA damage caused by X-ray exposures. It was further shown that the repair process in these conifers as in Arabidopsis as well as Lolium (11) involved a delayed phase, presumably because the expression of repair enzymes (14) has to be induced. Finally, inherent differences in radiosensitivity between organisms with different genome sizes should be kept in mind when evaluating results with reference plants and animals (18). References 1. Jeggo, P. A., Pearl, L. H. and Carr, A. M. ( 2016) DNA repair, genome stability and cancer: a historical perspective. Nat. Rev. Cancer , 16, 35– 42. Google Scholar CrossRef Search ADS PubMed 2. Brookes, P. and Lawley, P. D. ( 1960) The reaction of mustard gas with nucleic acids in vitro and in vivo. Biochem. J ., 77, 478– 484. Google Scholar CrossRef Search ADS PubMed 3. Friedberg, E. C. ( 2008) A brief history of the DNA repair field. Cell Res ., 18, 3– 7. Google Scholar CrossRef Search ADS PubMed 4. Bowen, H. J. M. ( 1961) Radiosensitivity of higher plants, and correlations with cell weight and DNA content. Radiat. Bot ., 1, 223– 228. Google Scholar CrossRef Search ADS 5. Sparrow, A. H. and Miksche, J. P. ( 1961) Correlation of nuclear volume and DNA content with higher plant tolerance to chronic radiation. Science , 134, 282– 283. Google Scholar CrossRef Search ADS PubMed 6. Baetcke, K. P., Sparrow, A. H., Nauman, C. H. and Schwemmer, S. S. ( 1967) The relationship of DNA content to nuclear and chromosome volumes and to radiosensitivity (LD50). Proc. Natl. Acad. Sci. U. S. A ., 58, 533– 540. Google Scholar CrossRef Search ADS PubMed 7. Woodwell, G. M.( 1962) Effects of ionizing radiation on terrestrial ecosystems. Science , 138, 572– 577. Google Scholar CrossRef Search ADS PubMed 8. Hall, E. R. and Giaccia, A. J. ( 2006) Radiobiology for the Radiologist : 6th edition. Lippincott Williams and Wilkins Publishing , Philadelphia; ISBN 0-7817-4151-3. 9. Santos, C. L. M., Pourrut, B. and Ferreira de Oliveira, J. M. P. ( 2015) The use of comet assay in plant toxicology: recent advances. Front. Genet , 6, 53– 70. Google Scholar CrossRef Search ADS 10. Mackay, J., Dean, J. F. D., Plomion, C.et al. ( 2012) Towards decoding the conifer giga-genome. Plant Mol. Biol ., 80, 555– 569. Google Scholar CrossRef Search ADS PubMed 11. Einset, J. and Collins, A. R. ( 2015) DNA repair after X-irradiation: lessons from plants. Mutagenesis , 30, 45– 50. Google Scholar CrossRef Search ADS PubMed 12. Lovell, D. P. and Omori, T. ( 2008) Statistical issues in the use of the comet assay. Mutagenesis , 23, 171– 182. Google Scholar CrossRef Search ADS PubMed 13. Puck, T. T. ( 1959) Quantitative studies on mammalian cells in vitro. Rev. Mod. Phys ., 31, 433– 448. Google Scholar CrossRef Search ADS 14. Lafarge, S. and Montané, M. H. ( 2003) Characterization of Arabidopsis thaliana ortholog of the human breast cancer susceptibility gene 1: AtBRCA1, strongly induced by gamma rays. Nucleic Acids Res ., 31, 1148– 1155. Google Scholar CrossRef Search ADS PubMed 15. Esau, K. ( 1965) Plant Anatomy . John Wiley & Sons, Inc., New York. ISBN 0-471-24455-4. 16. Coderre, J. ( 2004) 22.55J Principles of Radiation Interactions . Fall 2004. Massachusetts Institute of Technology: MIT OpenCourseWare. https://ocw.mit.edu (accessed September 28, 2017). 17. Kohn, K. W., Erickson, L. C., Ewig, R. A. G. and Friedman, C. A. ( 1976) Fractionation of DNA from mammalian cells by alkaline elution. Biochemistry , 15, 4629– 4637. Google Scholar CrossRef Search ADS PubMed 18. ICRP( 2008) Environmental protection—the concept and use of reference animals and plants. ICRP Publication 108. Ann. ICRP , 38, 25– 35. © The Author(s) 2017. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: firstname.lastname@example.org.
Mutagenesis – Oxford University Press
Published: Jan 1, 2018
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