Theoretical and Applied Genetics

, Volume 111, Issue 1, pp 136–149

DNA hypomethylation in 5-azacytidine-induced early-flowering lines of flax

  • M. A. Fieldes
  • S. M. Schaeffer
  • M. J. Krech
  • J. C. L. Brown
Original Paper


HPLC analysis was used to examine the cytosine methylation of total DNA extracted from four early-flowering lines that were induced by treating germinating seeds of flax (Linum usitatissimum) with the DNA demethylating agent 5-azacytidine. In the normal lines that gave rise to the induced early-flowering lines, flowering usually begins approximately 50 days after sowing. The early-flowering lines flower 7–13 days earlier than normal. The normal level of cytosine methylation was approximately 14% of the cytosines and 2.7% of the nucleosides. In the early-flowering lines, these levels were 6.2% lower than normal in DNA from the terminal leaf clusters of 14-day-old seedlings and 9.7% lower than normal in DNA from the cotyledons and immature shoot buds of 4-day-old seedlings. This hypomethylation was seen in lines that were five to nine generations beyond the treatment generation. The level of hypomethylation was similar in three of the four early-flowering lines, but was not as low in the fourth line, which flowers early but not quite as early as the other three lines. Unexpectedly, the degree of hypomethylation seen in segregant lines, derived by selecting for the early-flowering phenotype in the F2 and F3 generations of out-crosses, was similar to that seen in the early-flowering lines. Analysis of the methylation levels in segregating generations of out-crosses between early-flowering and normal lines demonstrated a decrease in methylation level during the selection of early-flowering segregants. The results suggest an association between hypomethylation and the early-flowering phenotype, and that the hypomethylated regions may not be randomly distributed throughout the genome of the early-flowering lines.


  1. Alleman M, Doctor J (2000) Genomic imprinting in plants: observations and evolutionary implications. Plant Mol Biol 43:147–161CrossRefPubMedGoogle Scholar
  2. Amado L, Abranches R, Neves N, Viegas W (1997) Development-dependent inheritance of 5-azacytidine-induced epimutations in triticale: analysis of rDNA expression patterns. Chromosome Res 5:445–450CrossRefPubMedGoogle Scholar
  3. Amyot LM (1997) Characterization of 5-azacytidine-induced early flowering lines in flax. MSc Thesis. Department of Biology, University of Waterloo, WaterlooGoogle Scholar
  4. Bastow R, Mylne JS, Lister C, Lippman Z, Martienssen RA, Dean C (2004) Vernalization requires epigenetic silencing by histone methylation. Nature 427:164–167CrossRefPubMedGoogle Scholar
  5. Burn JE, Bagnall DJ, Metzger JD, Dennis ES, Peacock WJ (1993) DNA methylation, vernalization, and the initiation of flowering. Proc Natl Acad Sci USA 90:287–291PubMedGoogle Scholar
  6. Chen ZJ, Pikaard CS (1997) Epigenetic silencing of RNA polymerase I transcription: a role for DNA methylation and histone modification in nucleolar dominance. Genes Dev 11:2124–2136PubMedGoogle Scholar
  7. Conklin KF, Groudine M (1984) Chromatin structure and gene expression. In: Razin A, Cedar H, Riggs AD (eds) DNA methylation biochemistry and biological significance. Springer, Berlin Heidelberg New York, pp 293–352Google Scholar
  8. Cui H, Fedoroff NV (2002) Inducible DNA demethylation mediated by the maize suppressor-mutator transposon-encoded TnpA protein. Plant Cell 14:1–17CrossRefPubMedGoogle Scholar
  9. Cullis CA (1981) DNA sequence organization in the flax genome. Biochim Biophys Acta 652:1–15PubMedGoogle Scholar
  10. Cullis CA, Swami S, Song Y (1999) RAPD polymorphisms detected among the flax genotrophs. Plant Mol Biol 41:795–800CrossRefPubMedGoogle Scholar
  11. Dawson RMC, Elliott DC, Elliott WH, Jones KM (eds) (1969) Data for biochemical research, 2nd edn. Oxford University Press, LondonGoogle Scholar
  12. Durrant A (1971) Induction and growth of flax genotrophs. Heredity 27:277–298Google Scholar
  13. Fieldes MA (1994) Heritable effects of 5-azacytidine treatments on the growth and development of flax (Linum usitatissimum) genotrophs and genotypes. Genome 37:1–11Google Scholar
  14. Fieldes MA, Amyot LM (1999a) Epigenetic control of early flowering in flax lines induced by 5-azacytidine applied to germinating seed. J Hered 90:199–206CrossRefGoogle Scholar
  15. Fieldes MA, Amyot LM (1999b) Evaluating the potential of using 5-azacytidine as an epimutagen. Can J Bot 77:1617–1622CrossRefGoogle Scholar
  16. Fieldes MA, Harvey CG (2004) Differences in developmental programming and node number at flowering in the 5-azacytidine-induced, early-flowering flax lines and their controls. Int J Plant Sci 165:695–706CrossRefGoogle Scholar
  17. Finnegan EJ, Peacock WJ, Dennis ES (1996) Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development. Proc Natl Acad Sci USA 93:8449–8454CrossRefPubMedGoogle Scholar
  18. Finnegan EJ, Peacock WJ, Dennis ES (1998) DNA methylation and the promotion of flowering by vernalization. Proc Natl Acad Sci USA 95:5824–5829CrossRefPubMedGoogle Scholar
  19. Follmann H, Balzer H-J, Schleicher R (1990) Biosynthesis and distribution of methylcytosine in wheat DNA. How different are plant DNA methyltransferases? In: Clawson GA, Willis DB, Weissbach A, Jones PA (eds) Nucleic acid methylation. Liss, New York, pp 199–210Google Scholar
  20. Furner IJ, Sheikh MA, Collett CE (1998) Gene silencing and homology-dependent gene silencing in Arabidopsis: genetic modifiers and DNA methylation. Genetics 149:651–662PubMedGoogle Scholar
  21. Galaud J-P, Gaspar T, Boyer N (1993) Effect of anti-DNA methylation drugs on growth, level of methylated DNA, peroxidase activity and ethylene production of Bryonia dioica internodes. Physiol Plant 87:528–534CrossRefGoogle Scholar
  22. Gendall AR, Levy YY, Wilson A, Dean C (2001) The VERNALIZATION 2 gene mediates the epigenetic regulation of vernalization in Arabidopsis. Cell 107:525–535CrossRefPubMedGoogle Scholar
  23. Genger RK, Peacock WJ, Dennis ES, Finnegan EJ (2003) Opposing effects of reduced DNA methylation on flowering time in Arabidopsis thaliana. Planta 216:461–466PubMedGoogle Scholar
  24. Glyn MCP, Egertová M, Gazdova B, Kovarik A, Bezdek M, Leitch AR (1997) The influence of 5-azacytidine on the condensation of the short arm of rye chromosome 1R in Triticum aestivum L. root tip meristematic nuclei. Chromosoma 106:485–492CrossRefPubMedGoogle Scholar
  25. Heslop-Harrison JS (1990) Gene expression and parental dominance in hybrid plants. Development [1990 Suppl]:21–28Google Scholar
  26. Hoekenga OA, Muszynski MG, Cone KC (2000) Developmental patterns of chromatin structure and DNA methylation responsible for epigenetic expression of a maize regulatory gene. Genetics 155:1889–1902PubMedGoogle Scholar
  27. Horváth E, Szalai G, Janda T, Páldi E, Rácz I, Lásztity D (2002) Effect of vernalisation and azacytidine on the DNA methylation level in wheat (Triticum aestivum L. cv. Mv 15). Proceedings of the Seventh Hungarian Congress on Plant Physiology, vol 46, pp 35–36Google Scholar
  28. Houchins K, O’Dell M, Flavell RB, Gustafson JP (1997) Cytosine methylation and nucleolar dominance in cereal hybrids. Mol Gen Genet 255:294–301CrossRefPubMedGoogle Scholar
  29. Jablonka E, Lamb MJ (1989) The inheritance of acquired epigenetic variations. J Theor Biol 139:69–83PubMedGoogle Scholar
  30. Jacobsen SE, Meyerowitz EM (1997) Hypermethylated SUPERMAN epigenetic alleles in Arabidopsis. Science 277:1100–1103CrossRefPubMedGoogle Scholar
  31. Jacobsen SE, Sakai H, Finnegan EJ, Cao X, Meyerowitz EM (2000) Ectopic hypermethylation of flower-specific genes in Arabidopsis. Curr Biol 10:179–186CrossRefPubMedGoogle Scholar
  32. Jaligot E, Rival A, Beulé T, Dussert S, Verdeil J-L (2000) Somoclonal variation in oil palm (Elaeis guineensis Jacq.): the DNA methylation hypothesis. Plant Cell Rep 19:684–690CrossRefGoogle Scholar
  33. Jaligot E, Beulé T, Baurens F-C Billotte N, Rival A (2004) Search for methylation-sensitive amplification polymorphisms associated with the “mantled” variant phenotype in oil palm (Elaeis guineensis Jacq.). Genome 47:224–228CrossRefPubMedGoogle Scholar
  34. Jones PA (1984) Gene activation by 5-azacytidine. In: Razin A, Cedar H, Riggs AD (eds) DNA methylation biochemistry and biological significance. Springer, Berlin Heidelberg New York, pp 165–188Google Scholar
  35. Kakutani T (1997) Genetic characterization of late-flowering traits induced by DNA hypomethylation mutation in Arabidopsis thaliana. Plant J 12:1447–1451CrossRefPubMedGoogle Scholar
  36. Kakutani T, Jeddeloh JA, Flowers SK, Munakata K, Richards EJ (1996) Developmental abnormalities and epimutations associated with DNA hypomethylation mutations. Proc Natl Sci USA 93:12406–12411CrossRefGoogle Scholar
  37. Kakutani T, Munakata K, Richards EJ, Hirochika H (1999) Meiotically and mitotically stable inheritance of DNA hypomethylation induced by ddm1 mutation of Arabidopsis thaliana. Genetics 151:831–838PubMedGoogle Scholar
  38. Kankel MW, Ramsey DE, Stokes TL, Flowers SK, Haag JR, Jeddeloh JA, Riddle NC, Verbsky ML, Richards EJ (2003) Arabidopsis MET1 cytosine methyltransferase mutants. Genetics 163:1109–1122PubMedGoogle Scholar
  39. King GJ (1995) Morphological development in Brassica oleracea is modulated by in vivo treatment with 5-azacytidine. J Hort Sci 70:333–342Google Scholar
  40. Kovarik A, Koukalova B, Lim KY, Matyasek R, Lichtenstein CP, Leitch AR, Bezdek M (2000) Comparative analysis of DNA methylation in tobacco heterochromatic sequences. Chromosome Res 8:527–541CrossRefPubMedGoogle Scholar
  41. Leutwiler LS, Hough-Evans BR, Meyerowitz EM (1984) The DNA of Arabidopsis thaliana. Mol Gen Genet 194:15–23CrossRefGoogle Scholar
  42. Li G, Hall TC, Holmes-Davis R (2002) Plant chromatin: development and gene control. BioEssays 24:234–243CrossRefPubMedGoogle Scholar
  43. LoSchiavo F, Pitto L, Giuliano G, Torti G, Nuti-Ronchi V, Orselli S, Terzi M (1989) DNA methylation of embryogenic carrot cell cultures and its variations as caused by mutation, differentiation, hormones and hypomethylating drugs. Theor Appl Genet 77:325–331CrossRefGoogle Scholar
  44. Matassi G, Melis R, Kuo KC, Macaya G, Gehrke CW, Bernardi G (1992) Large-scale methylation patterns in the nuclear genomes of plants. Gene 122:239–245CrossRefPubMedGoogle Scholar
  45. Matzke MA, Matzke AJM (1998) Epigenetic silencing of plant transgenes as a consequence of diverse cellular defence responses. Cell Mol Life Sci 54:94–103CrossRefPubMedGoogle Scholar
  46. Messeguer R, Ganal MW, Steffens JC, Tanksley SD (1991) Characterization of the level, target site and inheritance of cytosine methylation in tomato nuclear DNA. Plant Mol Biol 16:753–770CrossRefPubMedGoogle Scholar
  47. Mittelsten Scheid O, Probst AV, Afsar K, Paszkowski J (2002) Two regulatory levels of transcriptional gene silencing in Arabidopsis. Proc Natl Sci USA 99:13659–13662CrossRefGoogle Scholar
  48. Miura A, Yonebayashi S, Watanabe K, Toyama T, Shimada H, Kakutani T (2001) Mobilization of transposons by a mutant abolishing full DNA methylation in Arabidopsis. Nature 411:212–214CrossRefPubMedGoogle Scholar
  49. Richards EJ (1997) DNA methylation and plant development. Trends Genet 13:319–323CrossRefPubMedGoogle Scholar
  50. Ronemus MJ, Galbiati M, Ticknor C, Chen J, Dellaporta SL (1996) Demethylation-induced developmental pleiotropy in Arabidopsis. Science 273:654–657PubMedGoogle Scholar
  51. Sano H, Kamada I, Youssefian S, Katsumi M, Wabiko H (1990) A single treatment of rice seedlings with 5-azacytidine induces heritable dwarfism and undermethylation of genomic DNA. Mol Gen Genet 220:441–447CrossRefGoogle Scholar
  52. Santi DV, Garrett CE, Barr PJ (1983) On the mechanism of inhibition of DNA-cytosine methyltransferases by cytosine analogs. Cell 33:9–10CrossRefPubMedGoogle Scholar
  53. Santos D, Fevereiro P (2002) Loss of DNA methylation affects somatic embryogenesis in Medicago truncatula. Plant Cell Tissue Org Cult 70:155–161CrossRefGoogle Scholar
  54. Sheldon CC, Finnegan EJ, Rouse DT, Tadege M, Bagnall DJ, Helliwell CA, Peacock WJ, Dennis ES (2000a) The control of flowering by vernalization. Curr Opin Plant Biol 3:418–422CrossRefPubMedGoogle Scholar
  55. Sheldon CC, Rouse DT, Finnegan EJ, Peacock WJ, Dennis ES (2000b) The molecular basis of vernalization: The central role of FLOWERING LOCUS C (FLC). Proc Natl Acad Sci USA 97:3753–3758CrossRefPubMedGoogle Scholar
  56. Sober HA (ed) (1970) Handbook of biochemistry selected data for molecular biology, 2nd edn. Chemical Rubber, ClevelandGoogle Scholar
  57. Sokal RR, Rohlf FJ (1981) Biometry, 2nd edn. Freeman, San FranciscoGoogle Scholar
  58. Soppe WJJ, Jacobsen SE, Alonso-Blanco C, Jackson JP, Kakutani T, Koornneef M, Peeters AJM (2000) The late flowering phenotype of fwa mutants is caused by gain-of-function epigenetic alleles of a homeodomain gene. Mol Cell 6:791–802PubMedGoogle Scholar
  59. Steimer A, Schöb H, Grossniklaus U (2004) Epigenetic control of plant development: new layers of complexity. Curr Opin Plant Biol 7:11–19CrossRefPubMedGoogle Scholar
  60. Stokes TL, Kunkel BN, Richards EJ (2002) Epigenetic variation in Arabidopsis disease resistance. Genes Dev 16:171–182CrossRefPubMedGoogle Scholar
  61. Tariq M, Saze H, Probst AV, Lichota J, Habu Y, Paszkowski J (2003) Erasure of CpG methylation in Arabidopsis alters patterns of histone methylation in heterochromatin. Proc Natl Acad Sci USA 100:8823–8827CrossRefPubMedGoogle Scholar
  62. Tatra GS, Miranda J, Chinnappa CC, Reid DM (2000) Effect of light quality and 5-azacytidine on genomic methylation and stem elongation in two ecotypes of Stellaria longipes. Physiol Plant 109:313–321CrossRefGoogle Scholar
  63. Timmis JN, Ingle J (1973) Environmentally induced changes in rRNA gene redundancy. Nature 244:235–236PubMedGoogle Scholar
  64. Vanyushin BF, Belozerskii AN (1959) Nucleotide composition of deoxyribonucleic acid in higher plants (in Russian). Dokl Akad Nauk SSSR 129:944–946Google Scholar
  65. Vongs A, Kakutani T, Martienssen RA, Richards EJ (1993) Arabidopsis thaliana DNA methylation mutants. Science 260:1926–1928PubMedGoogle Scholar
  66. Vyskot B, Araya A, Veuskens J, Negrutiu I, Mouras A (1993) DNA methylation of sex chromosomes in a dioecious plant, Melandrium album. Mol Gen Genet 239:219–224PubMedGoogle Scholar
  67. Vyskot B, Koukalova B, Kovarik A, Sachambula L, Reynolds D, Bezdek M (1995) Meiotic transmission of a hypomethylated repetitive DNA family in tobacco. Theor Appl Genet 91:659–664CrossRefGoogle Scholar
  68. Zluvova J, Janousek B, Vyskot B (2001) Immunohistochemical study of DNA methylation dynamics during plant development. J Exp Bot 52:2265–2273CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • M. A. Fieldes
    • 1
  • S. M. Schaeffer
    • 1
  • M. J. Krech
    • 2
  • J. C. L. Brown
    • 1
  1. 1.Department of BiologyWilfrid Laurier UniversityWaterlooCanada
  2. 2.Department of ChemistryWilfrid Laurier UniversityWaterlooCanada

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