Advertisement

Plant Molecular Biology

, Volume 66, Issue 5, pp 463–473 | Cite as

Hypomethylation and transcriptional reactivation of retrotransposon-like sequences in ddm1 transgenic plants of Brassica rapa

  • Ryo Fujimoto
  • Taku Sasaki
  • Hisashi Inoue
  • Takeshi Nishio
Article

Abstract

DNA methylation and histone modification play important roles in regulating gene expression. The DDM1 gene in Arabidopsis thaliana (AtDDM1) is required for the maintenance of DNA methylation level and histone H3 methylation pattern. We isolated DDM1 homologs of Brassica rapa, BrDDM1a and BrDDM1b, which have 84.4% and 84.1% deduced amino acid sequence identities with AtDDM1, respectively. Both the BrDDM1a and BrDDM1b genes were found to be expressed in vegetative and reproductive tissues. B. rapa ddm1-RNAi transgenic plants with reduced levels of BrDDM1a/BrDDM1b expression showed genome-wide and non-tissue-specific demethylation. These results suggest that the BrDDM1a and BrDDM1b genes are orthologs of AtDDM1 and are required for the maintenance of DNA methylation as is AtDDM1. Despite genome-wide demethylation, developmental abnormalities were not found in the ddm1-RNAi transgenic plants. Dominance relationships of SP11/SCR alleles, the determinant of pollen recognition specificity in Brassica self-incompatibility, in S heterozygotes in B. rapa were not influenced by the low level of the BrDDM1 expression. Transcriptional reactivation of retrotransposon-like sequences observed in the ddm1-RNAi transgenic plants indicates that BrDDM1a and BrDDM1b participate in silencing of retrotransposons. Hypomethylation states of the ddm1-RNAi transgenic plants were inherited by plants of the next generation even by plants which had lost the RNAi construct by segregation. Remethylation was observed in a few progenies. Efficiencies of remethylation in the progenies without the RNAi construct were different between 18S rDNA, BoSTF12a/15a, and BrTto1 sequences.

Keywords

DDM1 DNA methylation Retrotransposon Epigenetics RNAi 

Abbreviations

CMT3

Chromomethylase 3

DDM1

Decrease in DNA methylation 1

GUS

β-Glucuronidase

LTR

Long terminal repeat

Lsh

Lymphocyte-specific helicase

NosT

Nopaline synthase gene

MET1

Methyltransferase 1

rDNA

Ribosomal DNA

RNAi

RNA interference

SCR

S-locus cysteine-rich

SP11

S-locus protein 11

SSC

Saline-sodium citrate

STF

S-locus retrotransposon family

Tto1

Tobacco retrotransposon 1

WT

Wild type

Notes

Acknowledgements

We are grateful to Dr. T. Kakutani for his helpful suggestions on this manuscript, Dr. Y. Sato for his technical advice, and Dr. Y. Kuginuki for providing a doubled haploid line.

Supplementary material

11103_2007_9285_MOESM1_ESM.TIF.tif
Supplementary Fig. 1. Alignment of the amino acid sequences of BrDDM1a, BrDDM1b, and AtDDM1 (AAD28303). Boxes outlined by a dotted line and a solid line indicate the SNF2 family N-terminal domain and the helicase-conserved C-terminal domain, respectively, both of which were identified by P fam analysis. (TIF 4850 kb)
11103_2007_9285_MOESM2_ESM.TIF.tif
Supplementary Fig. 2. Analysis of DNA methylation levels of repetitive sequences and retrotransposon-like sequences in the transgenic plants by Southern blot analysis using methylation-sensitive and methylation-insensitive restriction enzymes. (A) After electrophoresis, genomic DNAs of T0-2 and WT digested with Msp I (M) and Hpa II (H) were hybridized with the probes of the promoter region of 25S rDNA, the coding region of 5S rDNA, and putative centromeric repeat. (B) Genomic DNAs of T0-2 and WT digested with Hind III/Msp I (M) and Hind III/Hpa II (H) were probed with gag regions of BoSTF7a and BoSTF12b, an rvt region of BrSTF60a, and an MuDR region of MuDR transposon. (TIF 4497 kb)
11103_2007_9285_MOESM3_ESM.TIF.tif
Supplementary Fig. 3. Analysis of DNA methylation levels in genomic DNAs extracted from various tissues of T0-2 by Southern blot analysis. DNAs digested with Msp I (M) and Hpa II (H) were probed with the promoter region of 18S rDNA. L, leaves; FB, flower buds; ST, stamens; PI, pistils. 0-1, 1-3, 3-5, and 5-8 indicate the lengths of flower buds (mm). (TIF 4669 kb)
11103_2007_9285_MOESM4_ESM.TIF.tif
Supplementary Fig. 4. Southern-blot analysis of genomic DNAs of T0 plants. After electrophoresis, genomic DNAs digested with Eco RI were hybridized with the probes of rvt regions of BoSTF12a/15a and BrSTF60a, rnaseH region of BrTto1, and MuDR region of MuDR transposon. (TIF 4316 kb)
11103_2007_9285_MOESM5_ESM.TIF.tif
Supplementary Fig. 5. Analysis of DNA methylation and transcripts of the transposon-like sequences in the T1 plants derived from T0-2. (A, B) Southern blot analysis of genomic DNAs of the T1 and F1 plants of T0-2. Genomic DNAs digested with Hind III/Msp I and Hind III/Hpa II were probed with an rvt region of BoSTF12a/15a (A) and an rnaseH region of BrTto1 (B). (C) Detection of transcripts in leaves of BoSTF12a/15a and BrTto1. The actin gene was used as a positive control. RNAi+ and RNAi- indicate plants having the RNAi construct and those without the RNAi construct, respectively. (TIF 4533 kb)
11103_2007_9285_MOESM6_ESM.doc (32 kb)
(DOC 32 kb)

References

  1. Bartee L, Malagnac F, Bender J (2001) Arabidopsis cmt3 chromomethylase mutations block non-CG methylation and silencing of an endogenous gene. Genes Dev 15:1753–1758PubMedCrossRefGoogle Scholar
  2. Cao X, Jacobsen SE (2002) Locus-specific control of asymmetric and CpNpG methylation by the DRM and CMT3 methyltransferase genes. Proc Natl Acad Sci USA 99:16491–16498PubMedCrossRefGoogle Scholar
  3. Chan SWL, Henderson IR, Jacobsen SE (2005) Gardening the genome: DNA methylation in Arabidopsis thaliana. Nat Rev Genet 6:351–360PubMedCrossRefGoogle Scholar
  4. 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
  5. Dennis K, Fan T, Geiman T, Yan Q, Muegge K (2001) Lsh, a member of the SNF2 family, is required for genome-wide methylation. Genes Dev 15:2940–2944PubMedCrossRefGoogle Scholar
  6. Finnegan EJ, Peacock WJ, Dennis ES (1996) Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development. Proc Natl Acad Sci USA 93:8449–8454PubMedCrossRefGoogle Scholar
  7. Foss HM, Roberts CJ, Claeys KM, Selker EU (1993) Abnormal chromosome behavior in Neurospora mutants defective in DNA methylation. Science 262:1737–1741PubMedCrossRefGoogle Scholar
  8. Freitag M, Hickey PC, Khlafallah TK, Read ND, Selker EU (2004) HP1 is essential for DNA methylation in Neurospora. Mol Cell 13:427–434PubMedCrossRefGoogle Scholar
  9. Fujimoto R, Okazaki K, Fukai E, Kusaba M, Nishio T (2006a) Comparison of the genome structure of the self-incompatibility (S) locus in interspecific pairs of S haplotypes. Genetics 173:1157–1167PubMedCrossRefGoogle Scholar
  10. Fujimoto R, Sasaki T, Nishio T (2006b) Characterization of DNA methyltransferase genes in Brassica rapa. Genes Genet Syst 81:235–242PubMedCrossRefGoogle Scholar
  11. Fujimoto R, Takuno S, Sasaki T, Nishio T (2008) The pattern of amplification and differentiation of Ty1-copia and Ty3-gypsy retrotransposons in Brassicaceae species. Genes Genet Syst (in press)Google Scholar
  12. Gendrel AV, Lippman Z, Yordan C, Colot V, Martienssen RA (2002) Dependence of heterochromatic histone H3 methylation patterns on the Arabidopsis gene DDM1. Science 297:1871–1873PubMedCrossRefGoogle Scholar
  13. Grandbastien MA (1998) Activation of plant retrotransposons under stress conditions. Trend Plant Sci 3:181–187CrossRefGoogle Scholar
  14. Hirochika H, Okamoto H, Kakutani T (2000) Silencing of retrotransposons in Arabidopsis and reactivation by the ddm1 mutation. Plant Cell 12:357–368PubMedCrossRefGoogle Scholar
  15. Jeddeloh JA, Stokes TL, Richards EJ (1999) Maintenance of genomic methylation requires a SWI2/SNF2-like protein. Nat Genet 22:94–97PubMedCrossRefGoogle Scholar
  16. Johnston JS, Pepper AE, Hall AE, Chen ZJ, Hodnett G, Drabek J, Lopez R, Price HJ (2005) Evolution of genome size in Brassicaceae. Ann Bot 95:229–235PubMedCrossRefGoogle Scholar
  17. Jones JD, Shlumukov L, Carland F, English J, Scofield SR, Bishop GJ, Harrison K (1992) Effective vectors for transformation, expression of heterologous genes, and assaying transposon excision in transgenic plants. Transgenic Res 1:285–297PubMedCrossRefGoogle Scholar
  18. Kakutani T (1997) Genetic characterization of late-flowering traits induced by DNA hypomethylation mutation in Arabidopsis thaliana. Plant J 12:1447–1451PubMedCrossRefGoogle Scholar
  19. Kakutani T, Jeddeloh JA, Richards EJ (1995) Characterization of an Arabidopsis thaliana DNA hypomethylation mutant. Nucleic Acids Res 23:130–137PubMedCrossRefGoogle Scholar
  20. Kakutani T, Jeddeloh JA, Flowers SK, Munakata K, Richards EJ (1996) Developmental abnormalities and epimutations associated with DNA hypomethylation mutations. Proc Natl Acad Sci USA 93:12406–12411PubMedCrossRefGoogle Scholar
  21. 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
  22. Kouzminova E, Selker EU (2001) dim-2 encodes a DNA methyltransferase responsible for all known cytosine methylation in Neurospora. EMBO J 20:4309–4323PubMedCrossRefGoogle Scholar
  23. Kusaba M, Dwyer K, Hendershot J, Vrebalov J, Nasrallah JB, Nasrallah ME (2001) Self-incompatibility in the genus Arabidopsis: characterization of the S locus in the outcrossing A. lyrata and its autogamous relative A. thaliana. Plant Cell 13:627–643PubMedCrossRefGoogle Scholar
  24. Li E, Bestor TH, Jaenisch R (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69:915–926PubMedCrossRefGoogle Scholar
  25. Lindroth AM, Cao X, Jackson JP, Zilberman D, McCallum CM, Henikoff S, Jacobsen SE (2001) Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation. Science 292:2077–2080PubMedCrossRefGoogle Scholar
  26. Lippman Z, Gendrel AV, Black M, Vaughn MW, Dedhia N, McCombie WR, Lavine K, Mittal V, May B, Kasschau KD, Carrington JC, Doerge RW, Colot V, Martienssen R (2004) Role of transposable elements in heterochromatin and epigenetic control. Nature 430:471–476PubMedCrossRefGoogle Scholar
  27. Lukens LN, Pires JC, Leon E, Vogelzang R, Oslach L, Osborn T (2006) Patterns of sequence loss and cytosine methylation within a population of newly resynthesized Brassica napus allopolyploids. Plant Physiol 140:336–348PubMedCrossRefGoogle Scholar
  28. Manning K, Tor M, Poole M, Hong Y, Thompson AJ, King GJ, Giovannoni JJ, Seymour GB (2006) A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening. Nat Genet 38:948–952PubMedCrossRefGoogle Scholar
  29. Miura A, Yonebayashi S, Watanabe K, Toyama T, Shimada H, Kakutani T (2001) Mobilization of transposons by a mutation abolishing full DNA methylation in Arabidopsis. Nature 411:212–214PubMedCrossRefGoogle Scholar
  30. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325PubMedCrossRefGoogle Scholar
  31. Nasrallah ME, Liu P, Nasrallah JB (2002) Generation of self-incompatible Arabidopsis thaliana by transfer of two S locus genes from A. lyrata. Science 297:247–249PubMedCrossRefGoogle Scholar
  32. Okano M, Bell DW, Haber DA, Li E (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99:247–257PubMedCrossRefGoogle Scholar
  33. Paulin R, Grigg GW, Davey MW, Piper AA (1998) Urea improves efficiency of bisulphate-mediated sequencing of 5′-methylcytosine in genomic DNA. Nucleic Acids Res 26:5009–5010PubMedCrossRefGoogle Scholar
  34. Pouteau S, Huttner E, Grandbastien MA, Caboche M (1991) Specific expression of the tobacco Tnt1 retrotransposon in protoplasts. EMBO J 10:1911–1918PubMedGoogle Scholar
  35. Ronemus MJ, Galbiati M, Ticknor C, Chen J, Dellaporta SL (1996) Demethylation-induced developmental pleiotropy in Arabidopsis. Science 273:654–657PubMedCrossRefGoogle Scholar
  36. Sato Y, Fujimoto R, Toriyama K, Nishio T (2003) Commonality of self-recognition specificity of S haplotypes between Brassica oleracea and Brassica rapa. Plant Mol Biol 52:617–626PubMedCrossRefGoogle Scholar
  37. Shiba H, Kakizaki T, Iwano M, Tarutani Y, Watanabe M, Isogai A, Takayama S (2006) Dominance relationships between self-incompatibility alleles controlled by DNA methylation. Nat Genet 38:297–299PubMedCrossRefGoogle Scholar
  38. Singer T, Yordan C, Martienssen RA (2001) Robertson’s Mutator transposons in A. thaliana are regulated by the chromatin-remodeling gene Decrease in DNA Methylation (DDM1). Genes Dev 15:591–602PubMedCrossRefGoogle Scholar
  39. 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 6:791–802PubMedCrossRefGoogle Scholar
  40. Stokes TL, Kunkel BN, Richards EJ (2002) Epigenetic variation in Arabidopsis disease resistance. Genes Dev 16:171–182PubMedCrossRefGoogle Scholar
  41. Takeda S, Sugimoto K, Otsuki H, Hirochika H (1999) A 13-bp cis-regulatory element in the LTR promoter of the tobacco retrotransposon Tto1 is involved in responsiveness to tissue culture, wounding, methyl jasmonate and fungal elicitors. Plant J 18:383–393PubMedCrossRefGoogle Scholar
  42. Tamaru H, Selker EU (2001) A histone H3 methyltransferase controls DNA methylation in Neurospora crassa. Nature 414:277–283PubMedCrossRefGoogle Scholar
  43. Vongs A, Kakutani T, Martienssen RA, Richards EJ (1993) Arabidopsis thaliana DNA methylation mutants. Science 260:1926–1928PubMedCrossRefGoogle Scholar
  44. Waterhouse PM, Helliwell CA (2003) Exploring plant genomes by RNA-induced gene silencing. Nat Rev Genet 4:29–38PubMedCrossRefGoogle Scholar
  45. Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjgesdijk PA, Robinson SP, Gleave AP, Green AG, Waterhouse PM (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27:581–590PubMedCrossRefGoogle Scholar
  46. Xiao W, Custard KD, Brown RC, Lemmon BE, Harada JJ, Goldberg RB, Fischer RL (2006) DNA methylation is critical for Arabidopsis embryogenesis and seed viability. Plant Cell 18:805–814PubMedCrossRefGoogle Scholar
  47. Zhang X, Wessler SR (2004) Genome-wide comparative analysis of the transposable elements in the related species Arabidopsis thaliana and Brassica oleracea. Proc Natl Acad Sci USA 101:5589–5594PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Ryo Fujimoto
    • 1
    • 2
  • Taku Sasaki
    • 1
  • Hisashi Inoue
    • 1
  • Takeshi Nishio
    • 1
  1. 1.Graduate School of Agricultural ScienceTohoku UniversitySendaiJapan
  2. 2.National Institute of GeneticsMishimaJapan

Personalised recommendations