Skip to main content
SpringerLink
Log in

Characterization of the level, target sites and inheritance of cytosine methylation in tomato nuclear DNA

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

The tomato nuclear genome was determined to have a G+C content of 37% which is among the lowest reported for any plant species. Non-coding regions have a G+C content even lower (32% average) whereas coding regions are considerably richer in G+C (46%).

5-methyl cytosine was the only modified base detected and on average 23% of the cytosine residues are methylated. Immature tissues and protoplasts have significantly lower levels of cytosine methylation (average 20%) than mature tissues (average 25%). Mature pollen has an intermediate level of methylation (22%). Seeds gave the highest value (27%), suggesting de novo methylation after pollination and during seed development.

Based on isoschizomer studies we estimate 55% of the CpG target sites (detected by Msp I/Hpa II) and 85% of the CpNpG target sites (detected by Bst NI/Eco RI)are methylated. Unmethylated target sites (both CpG and CpNpG) are not randomly distributed throughout the genome, but frequently occur in clusters. These clusters resemble CpG islands recently reported in maize and tobacco.

The low G+C content and high levels of cytosine methylation in tomato may be due to previous transitions of 5mC→T. This is supported by the fact that G+C levels are lowest in non-coding portions of the genome in which selection is relaxed and thus transitions are more likely to be tolerated. This hypothesis is also supported by the general deficiency of methylation target sites in the tomato genome, especially in non-coding regions.

Using methylation isoschizomers and RFLP analysis we have also determined that polymorphism between plants, for cytosine methylation at allelic sites, is common in tomato. Comparing DNA from two tomato species, 20% of the polymorphisms detected by Bst NI/Eco RII could be attributed to differential methylation at the CpNpG target sites. With Msp I/Hpa II, 50% of the polymorphisms were attributable to methylation (CpG and CpNpG sites). Moreover, these polymorphisms were demonstrated to be inherited in a mendelian fashion and to co-segregate with the methylation target site and thus do not represent variation for transacting factors that might be involved in methylation of DNA. The potential role of heritable methylation polymorphism in evolution of gene regulation and in RFLP studies is discussed.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Adams RLP, Burdon RH: Molecular Biology of DNA Methylation. Springer-Verlag, New York, pp. 107–113 (1985).

    Google Scholar 

  2. Alleman M, Freeling M: The Mu transposable elements of maize: Evidence for transposition and copy number regulation during development. Genetics 112: 107–119 (1986).

    PubMed  Google Scholar 

  3. Anteguera F, Bird AP: Unmethylated CpG islands associated with genes in higher plant DNA. EMBO J. 7: 2295–2299 (1989).

    Google Scholar 

  4. Belzile F, Lassner MW, Tong Y, Khush R, Yoder JI: Sexual transmission of transposed Activator elements in transgenic tomatoes. Genetics 123: 181–189 (1989).

    Google Scholar 

  5. Bernatzky R, Tanksley SD: Towards a saturated linkage map in tomato based on isozymes and random cDNA sequences. Genetics 112: 887–898 (1986).

    Google Scholar 

  6. Bernatzky R, Tanksley SD: Methods for detection of single or low copy sequences in tomato on Southern blots. Plant Mol Biol Rep 4: 37–41 (1986).

    Google Scholar 

  7. Bernatzky R, Pichersky E, Malik VS, Tanksley SD: CR1-a dispersed repeated element associated with the Cab-1 locus in tomato. Plant Mol Biol 10: 423–434 (1988).

    Google Scholar 

  8. Bird AP: DNA methylation and the frequency of CpG in animal DNA. Nucl Acids Res 8: 1499–1504 (1980).

    PubMed  Google Scholar 

  9. Bird AP: CpC-rich islands and the function of DNA methylation. Nature 321: 209–213 (1986).

    PubMed  Google Scholar 

  10. Bird CR, Smith CJS, Ray JA, Moureau P, Bevan MW, Bird AS, Hughes S, Morris PC, Grierson D, Schuch W: The tomato polygalacturonase gene and ripening-specific expression in transgenic plants. Plant Mol Biol 11: 651–662 (1989).

    Google Scholar 

  11. Bolden AH, Nalin CM, Ward CA, Poonian MS, Weissbach A: Primary DNA sequence determines sites of maintenance and de novo methylation by mammalian DNA methyltransferases. Mol Cell Biol 6: 1135–1140 (1986).

    PubMed  Google Scholar 

  12. Boudraa M, Perrin P: CpG and TpA frequencies in the plant system. Nucl Acids Res 15: 5729–5737 (1987).

    PubMed  Google Scholar 

  13. Brown WRA, Bird AP: Long-range restriction site mapping of mammalian genomic DNA. Nature 322: 477–481 (1980).

    Google Scholar 

  14. Brown DD, David IB: Specific gene amplification in oocytes. Science 160: 272–280 (1968).

    PubMed  Google Scholar 

  15. Cedar H: DNA methylation and gene activity. Cell 53: 3–4 (1988).

    PubMed  Google Scholar 

  16. Chandler LA, Ghazi H, Jones PA, Boukamp P, Fusanig NE: Allele-specific methylation of the human c-Ha-ras-1 gene. Cell 50: 711–717 (1987).

    PubMed  Google Scholar 

  17. Chu G, Vollrath D, Davis RW: Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science 234: 1582–1585 (1986).

    PubMed  Google Scholar 

  18. Comings DE: Methylation of euchromatic and hetrochromatic DNA. Exptl Cell Res 74: 383–390 (1972).

    PubMed  Google Scholar 

  19. Coulondre C, Miller JH, Farabaugh PJ, Gilbert W: Molecular basis of base substitution hot spots in Escherichia coli. Nature 274: 775–780 (1978).

    PubMed  Google Scholar 

  20. Ergle DR, Katterman FRH: Deoxyribonucleic acid of cotton. Plant Phys 36: 811–815 (1961).

    Google Scholar 

  21. Freifelder PM: Molecular Biology, pp. 938–941. Science Books International, Boston (1983).

    Google Scholar 

  22. Ganal MW, Lapitan NLV, Tanksley SD: A molecular and cytogenetic survey of major repeated DNA sequences in tomato (Lycopersicon esculentum). Mol Gen Genet 213: 262–268 (1988).

    Google Scholar 

  23. Goodfellow PJ, Mondello C, Darling SM, Pym B, Little P, Goodfellow PN: Absence of methylation of a CpG-rich region at the 5′ end of the MIC2 gene on the active X, the inactive X, and the Y chromosomes. Proc Natl Acad Sci USA 85: 5605–5609 (1988).

    PubMed  Google Scholar 

  24. Gruenbaum Y, Naveh-Many T, Cedar H, Razin A: Sequence specificity of methylation in higher plant DNA. Nature 292: 860–863 (1981).

    PubMed  Google Scholar 

  25. Hepburn AG, Clarke LE, Pearson L, White J: The role of cytosine methylation in the control of nopaline synthase gene expression in a plant tumor. J Mol Appl Genet 2: 315–329 (1983).

    PubMed  Google Scholar 

  26. Hepburn AG, Belanger FC, Mattheis JR: DNA methylation in plants. Develop Genet 8: 475–493 (1987).

    Google Scholar 

  27. Hille J, Koornneef M, Ramanna MS, Zabel P: Tomato: a crop species amenable to improvement by cellular and molecular methods. Euphytica 42: 1–23 (1989).

    Google Scholar 

  28. Holdsworth MJ, Bird CR, Ray J, Schuch W, Grierson D: Structure and expression of an ethylene-related mRNA from tomato. Nucl Acids Res 15: 731–739 (1987).

    PubMed  Google Scholar 

  29. Jaenisch R, Schnieke A, Harbers K: Treatment of mice with 5-azacytidine efficiently activates silent retroviral genomes in different tissues. Proc Natl Acad Sci USA 82: 1451–1455 (1985).

    PubMed  Google Scholar 

  30. Jones PA: Gene activation by 5-azacytidine. In: Razin A, Cedar H, Riggs AD (eds) DNA Methylation: Biochemistry and Biological Significance, pp. 237–251. Springer-Verlag, New York (1984).

    Google Scholar 

  31. Kelley DE, Pollok BA, Atchison ML, Perry RP: The coupling between enhancer activity and hypomethylation of K immunoglobulin genes is developmentally regulated. Mol Cell Biol 8: 930–937 (1988).

    PubMed  Google Scholar 

  32. Kiss T, Kiss M, Abel S, Solymosy F: Nucleotide sequence of the 17S–25S spacer region from tomato rDNA. Nucl Acids Res 16: 7179 (1988).

    PubMed  Google Scholar 

  33. Landry BS, Kesseli R, Leung H, Michelmore RW: Comparison of restriction endonucleases and sources of probes for their efficiency in detecting restriction fragment length polymorphisms in lettuce (Lactuca sativa L.). Theor Appl Genet 74: 646–653 (1987).

    Article  Google Scholar 

  34. Lee JS, Brown WE, Graham JS, Pearce G, Fox EA, Breher TW, Ahern KG, Pearson G., Ryan CA: Molecular characterization and phylogenetic studies of a wound-inducible proeteinase inhibitor 1 gene in Lycopersicon species. Proc Natl Acad Sci USA 83: 7277–7281 (1986).

    PubMed  Google Scholar 

  35. Leutwiler LS, Hough-Evans BR, Meyerowitz EM: The DNA of Arabidopsis thaliana. Mol Gen Genet 194: 15–23 (1984).

    Google Scholar 

  36. Marmur J, Doty P: Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5: 109–118 (1961).

    Google Scholar 

  37. Mascarenhas JP: The biochemistry of angiosperm pollen development. Bot Rev 41: 259–314 (1975).

    Google Scholar 

  38. Mascarenhas JP: The male gametophyte of the flowering plant. Plant Cell 1: 657–664 (1989).

    Article  PubMed  Google Scholar 

  39. Mascarenhas JP: Gene activity during pollen development. Ann Rev Plant Phys Plant Mol Biol 41: 317–338 (1990).

    Article  Google Scholar 

  40. Matzke MA, Primig M, Trnovsky J, Matzke AJM: Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. EMBO J 8: 643–649 (1989).

    Google Scholar 

  41. McClelland M: The frequency and distribution of methylated DNA sequences in leguminous plant protein coding genes. J Mol Evol 19: 346–354 (1983).

    PubMed  Google Scholar 

  42. Nelson M, McClelland M: The effect of site-specific methylation on restriction-modification enzymes. Nucl Acads Res (Suppl.) 15: r219-r230 (1987).

    Google Scholar 

  43. Nevers P, Shepherd NS, Saedler H: Plant transposable elements. Adv Bot Res 12: 103–143 (1986).

    Google Scholar 

  44. Ngernprasirtsiri J, Chollet R, Kobayashi H, Sugiyama, Akazawa T: DNA methylation and the differential expression of C4 photosynthesis genes in mesophyll and bundle sheath cells of greening maize leaves. J Biol Chem 264: 8241–8248 (1989).

    PubMed  Google Scholar 

  45. Perl-Treves R, Nacmias B, Aviv D, Zeelon EP, Galun E: Isolation of two cDNA clones from tomato containing two different superoxide dismutase sequences. Plant Mol Biol 11: 609–623 (1988).

    Google Scholar 

  46. Pichersky E, Bernatzky R, Tanksley SD, Cashmore AR: Evidence for selection as a mechanism in the concerted evolution of Lycopersicon esculentum (tomato) genes encoding the small subunit of ribulose-I,5-biphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 83: 3880–3884 (1986).

    PubMed  Google Scholar 

  47. Pichersky E, Hoffman NE, Melik VS, Bernatzky R, Tanksley SD, Szabo L, Cashmore AR: The tomato Cab-4 and Cab-5 genes encode a second type of CAB polypeptides localized in Photosystem II. Plant Mol Biol 9: 109–120 (1987).

    Google Scholar 

  48. Pichersky E, Tanksley SD, Piechulla B, Stayton M, Dunsmuir P: Nucleotide sequence and chromosomal location of Cab-7, the tomato gene encoding the Type II chlorophyll a/b-binding polypeptide of Photosystem I. Plant Mol Biol 11: 69–71 (1988).

    Google Scholar 

  49. Pichersky E, Brock TG, Nguyen D, Hoffman NE, Piechulla B, Tanksley SF, Green BR: A new member of the CAB gene family: structure, expression and chromosomal location of Cab-8, the tomato gene encoding the Type III chlorophyll a/b binding polypeptide of photo-system I. Plant Mol Biol 12: 257–270 (1989).

    Google Scholar 

  50. Proffitt JH, Davie JR, Swinton D, Hattman S: 5-Methylcytosine is not detectable in Saccharomyces cerevisiae. Mol Cell Biol 4: 985–988 (1984).

    PubMed  Google Scholar 

  51. Razin A: DNA methylation patterns: formation and biological functions. In: Razin A, Cedar H, Riggs AD (eds) DNA Methylation: Biochemistry and Biological Significance, pp. 103–154. Springer-Verlag, New York (1984).

    Google Scholar 

  52. Razin A, Cedar H, Riggs AD (eds): DNA Methylation: Biochemistry and Biological Significance. Springer-Verlag, New York (1984).

    Google Scholar 

  53. Razin A, Szyf M, Kafri T, Roll M, Giloh H, Scarpa S, Carotti D, Cantoni GL: Replacement of 5-methylcytosine by cytosine: A possible mechanism for transient DNA demethylation during differentiation. Proc Natl Acad Sci USA 83: 2827–2831 (1986).

    PubMed  Google Scholar 

  54. Rick CM: The tomato. In: King KC (ed) Handbook of Genetics, Vol. 12, pp. 247–280. Plenum, New York (1975).

    Google Scholar 

  55. Rick CM, Yoder J: Classical and molecular genetics of tomato: highlights and perspectives. Ann Rev Genet 22: 281–300 (1988).

    Article  PubMed  Google Scholar 

  56. Schwartz D: Gene-controlled cytosine demethylation in the promoter region of the Ac transposable element in maize. Proc Natl Acad Sci USA 86: 2789–2973 (1989).

    Google Scholar 

  57. Schwartz D, Dennis E: Transposase activity of the Ac controlling element in maize is regulated by its degree of methylation. Mol Gen Genet 205: 476–482 (1986).

    Google Scholar 

  58. Schweizer G, Ganal MW, Ninnemann H, Hemleben V: Species-specific DNA sequences for identification of somatic hybrids between Lycopersicon esculentum and Solanum acaule. Theor Appl Genet 75: 679–684 (1988).

    Article  Google Scholar 

  59. Selker EU: DNA methylation and chromatin structure: a view from below. Trends Biochem Sci 15: 103–107 (1990).

    Article  PubMed  Google Scholar 

  60. Silva AJ, White R: Inheritance of allelic blueprints for methylation patterns. Cell 54: 145–152 (1988).

    Article  PubMed  Google Scholar 

  61. Tanksley SD, Young ND, Paterson AH, Bonierbale MW: RFLP mapping in plant breeding: new tools in an old science. Bio/technology 7: 257–264 (1989).

    Article  Google Scholar 

  62. Toniolo D, Martini G, Migeon BR, Dono R: Expression of the G6PD locus on the human X chromosome is associated with demethylation of three CpG islands within 100 kb of DNA. EMBO J 7: 401–406 (1988).

    PubMed  Google Scholar 

  63. Wagner I, Capesius I: Determination of 5-methycytosine from plant DNA by high-performance liquid chromatography. Biochim Biophys Acta 654: 52–56 (1981).

    PubMed  Google Scholar 

  64. Walbot V, Warren C: Regulation of Mu element copy number in maize lines with an active or inactive Mutator transposable element system. Mol Gen Genet 211: 27–34 (1988).

    PubMed  Google Scholar 

  65. White OR, O'Connell NA: Analysis NA: Analysis of organ-specific DNA methylation of several loci in tomato. Tomato Genet Coop 40: 40–41 (1990).

    Google Scholar 

  66. Wigler M, Levy D, Pencho M: The somatic replication of DNA methylation. Cell 24: 33–40 (1981).

    Article  PubMed  Google Scholar 

  67. Wolf SF, Migeon BR: Studies of X chromosome DNA methylation in normal human cells. Nature 295: 667–671 (1982).

    PubMed  Google Scholar 

  68. Ysiraeli J, Szyt M: Gene methylation patterns and expression. In: Razin R, Cedar H, Riggs AD (eds), DNA Methylation: Biochemistry and Biological Significance, pp. 353–378. Springer-Verlag, New York (1984).

    Google Scholar 

  69. Yisraeli J, Frank D., Razin A, Cedar H: Effect of in vitro DNA methylation on B-globin gene expression. Proc Natl Acad Sci USA 85: 4638–4642 (1988).

    PubMed  Google Scholar 

  70. Zabel P, Meyer D, van deStolpe O, van derZaal B, Ramanna MS, Koornneef M, Krens F, Hille J: Towards the construction of artificial chromosomes for tomato. In: vanVloten-Doting L, Groot GSP, Hall TC (eds) Molecular Form and Function of the Plant Genome, pp. 609–624. Plenum Press, New York (1985).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Plant Breeding and Biometry, Cornell University, 252 Emerson Hall, 14853, Ithaca, NY, USA

    Ramon Messeguer, Martin W. Ganal, John C. Steffens & Steven D. Tanksley

Authors
  1. Ramon Messeguer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Martin W. Ganal
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. John C. Steffens
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Steven D. Tanksley
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Messeguer, R., Ganal, M.W., Steffens, J.C. et al. Characterization of the level, target sites and inheritance of cytosine methylation in tomato nuclear DNA. Plant Mol Biol 16, 753–770 (1991). https://doi.org/10.1007/BF00015069

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00015069

Key words

Search

Navigation