Molecular Breeding

, Volume 13, Issue 2, pp 177–191 | Cite as

Dedifferentiation-mediated changes in transposition behavior make the Activator transposon an ideal tool for functional genomics in rice

  • Ajay Kohli
  • Mark Q. Prynne
  • Berta Miro
  • Andy Pereira
  • Richard M. Twyman
  • Teresa Capell
  • Paul Christou


There is an inverse relationship between the level of cytosine methylation in genomic DNA and the activity of plant transposable elements. Increased transpositional activity is seen during early plant development when genomic methylation patterns are first erased and then reset. Prolonging the period of hypomethylation might therefore result in an increased transposition frequency, which would be useful for rapid genome saturation in transposon-tagged plant lines. We tested this hypothesis using transgenic rice plants containing Activator (Ac) from maize. R1 seeds from an Ac-tagged transgenic rice line were either directly germinated and grown to maturity, or induced to dedifferentiate in vitro, resulting in cell lines that were subsequently regenerated into multiple mature plants. Both populations were then analyzed for the presence, active reinsertion and amplification of Ac. Plants from each population showed excision-reinsertion events to both linked and unlinked sites. However, the frequency of transposition in plants regenerated from cell lines was more than nine-fold greater than that observed in plants germinated directly from seeds. Other aspects of transposon behavior were also markedly affected. For example, we observed a significantly larger proportion of transposition events to unlinked sites in cell line-derived plants. The tendency for Ac to insert into transcribed DNA was not affected by dedifferentiation. The differences in Ac activity coincided with a pronounced reduction in the level of genomic cytosine methylation in dedifferentiated cell cultures. We used the differential transposon behavior induced by dedifferentiation in the cell-line derived population for direct applications in functional genomics and validated the approach by recovering Ac insertions in a number of genes. Our results demonstrate that obtaining multiple Ac insertions is useful for functional annotation of the rice genome.

Activator DNA methylation Functional genomics Rice Tissue culture 


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  1. Bancroft I. and Dean C. 1993. Transposition pattern of the maize element Ds in Arabidopsis thaliana. Genetics 134: 1221–1229.Google Scholar
  2. Bhatt A.M., Lister C., Crawford N. and Dean C. 1998. The transposition frequency of Tag1 elements is increased in transgenic Arabidopsis lines. Plant Cell 10: 427–434.Google Scholar
  3. Brettell R.I.S. and Dennis E.S. 1991. Reactivation of a silent Ac following tissue-culture is associated with heritable alterations in its methylation pattern. Mol. Gen. Genet. 229: 365–372.Google Scholar
  4. Chin H.G., Choe M.S., Lee S.H., Park S.H., Park S.H., Koo J.C. et al. 1999. Molecular analysis of rice plants harboring an Ac/Ds transposable element-mediated gene trapping system. Plant J. 19: 615–623.Google Scholar
  5. Courtial B., Feuerbach F., Eberhard S., Rohmer L., Chiapello H., Camilleri C. and Lucas H. 2001. Tnt1 transposition events are induced by in vitro transformation of Arabidopsis thaliana and transposed copies integrate into genes. Mol. Genet. Genom. 265: 32–42.Google Scholar
  6. DeGreef B. and Jacobs M. 1996. Evidence for Tam3 activity in transgenic Arabidopsis thaliana. In Vitro Cell Dev. Biol. Plant 32: 241–248.Google Scholar
  7. Dooner H.K., Belachew A., Burgess D., Hardings S., Ralston M. and Ralstone E. 1994. Distribution of unlinked receptor-sites for transposed Ac elements from the bz-m2(Ac) allele in maize. Genetics 136: 261–279.Google Scholar
  8. Earp D.J., Lowe B. and Baker B. 1990. Amplification of genomic sequences flanking transposable elements in host and heterologous plants – a tool for transposon tagging and genome characterization. Nucleic Acids Res. 18: 3271–3279.Google Scholar
  9. Edwards K., Johnstone C. and Thompson C. 1991. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res. 19: 1349–1349.Google Scholar
  10. Enoki H., Izawa T., Kawahara M., Komatsu M., Koh S., Kyozuka J. and Shimamoto K. 1999. Ac as a tool for the functional genomics of rice. Plant J. 19: 605–613.Google Scholar
  11. Goff S.A., Ricke D., Lan T.-H., Presting G., Wang R., Dunn M. et al. 2002. A draft sequence of the rice genome (Oryza sativa L. ssp. Japonica). Science 296: 92–100.Google Scholar
  12. Grappin P., Audeon C., Chupeau M.C. and Grandbastien M.A. 1996. Molecular and functional characterization of Slide, an Ac-like autonomous transposable element from tobacco. Mol. Gen. Genet. 252: 386–397.Google Scholar
  13. Greco R., Ouwerkerk P.B.F., Sallaud C., Kohli A., Columbo L., Puigdomenech P. et al. 2001a. Transposon insertional mutagenesis in rice. Plant Physiol. 125: 1175–1177.Google Scholar
  14. Greco R., Ouwerkerk P.B.F., Taal A.J.C., Favalli C., Beguiristain T., Puigdomenech P. et al. 2001b. Early and multiple Ac transpositions in rice suitable for efficient insertional mutagenesis. Plant Mol. Biol. 46: 215–227.Google Scholar
  15. Hamer L., DeZwaan T.M., Montenegro-Chamorro M.V., Frank S.A. and Hamer J.E. 2001. Recent advances in large-scale transposon mutagenesis. Curr. Opin. Chem. Biol. 5: 67–73.Google Scholar
  16. Hirochika H., Sugimoto K., Otsuki Y., Tsugawa H. and Kanda M. 1996. Retrotransposons of rice involved in mutations induced by tissue culture. Proc. Natl. Acad. Sci. USA 93: 7783–7788.Google Scholar
  17. Hirochika H. 1997. Retrotransposons of rice: their regulation and use for genome analysis. Plant Mol. Biol. 35: 231–240.Google Scholar
  18. Hirochika H. 2001. Contribution of the Tos17 retrotransposon to rice functional genomics. Curr. Opin. Plant Biol. 4: 118–122.Google Scholar
  19. Izawa T., Ohnishi T., Nakano T., Ishida N., Enoki H., Hashimoto H. et al. 1997. Transposon tagging in rice. Plant Mol. Biol. 35: 219–229.Google Scholar
  20. Jaligot E., Rival A., Beule T., Dussert S. and Verdeil J. 2000. Somaclonal variation in oil palm (Elaeis guineensis Jacq.): the DNA methylation hypothesis. Plant Cell Rep. 19: 684–690.Google Scholar
  21. Jeon J.S., Lee S., Jung K.H., Jun S.H., Jeong D.H., Lee J. et al. 2000. T-DNA insertional mutagenesis for functional genomics in rice. Plant J. 22: 561–570.Google Scholar
  22. Jeon J.S. and An G.H. 2001. Gene tagging in rice: a high through-put system for functional genomics. Plant Sci. 161: 211–219.Google Scholar
  23. Jones J.D.G., Carland F.C., Lim E., Raltson E. and Dooner H.K. 1990. Preferential transposition of the maize element Activator to linked chromosomal locations in tobacco. Plant Cell 2: 701–707.Google Scholar
  24. Kaeppler S.M. and Phillips R.L. 1993. DNA methylation and tissue culture-induced variation. In Vitro Cell Dev. Biol. Plant 29: 125–130.Google Scholar
  25. Kitamura K., Hashida S., Mikami T. and Kishima Y. 2001. Position effect of the excision frequency of the Antirrhinum transposon Tam3: implications for the degree of position-dependent methylation in the ends of the element. Plant Mol. Biol. 47: 475–490.Google Scholar
  26. Kohli A., Xiong J., Greco R., Christou P. and Pereira A. 2001. Tagged transcriptome display (TTD) in indica rice using Ac transposition. Mol. Genet. Genom. 266: 1–11.Google Scholar
  27. Koprek T., McElroy D., Louwerse J., Williams-Carrier R. and Lemaux P.G. 2000. An efficient method for dispersing Ds elements in the barley genome as a tool for determining gene function. Plant J. 24: 253–263.Google Scholar
  28. Koprek T., Rangel S., McElroy D., Louwerse J.D., Williams-Carrier R.E. and Lemaux P.G. 2001. Transposon-mediated single-copy gene delivery leads to increased transgene expression stability in barley. Plant Physiol. 125: 1354–1362.Google Scholar
  29. Koukalova B., Kuhrova V., Vyskot B., Siroky J. and Bezdek M. 1994. Maintenance of the induced hypomethylated state of tobacco nuclear repetitive DNA sequences in the course of protoplast and plant regeneration. Planta 194: 306–310.Google Scholar
  30. Liu Y.G., Mitsukawa N., Oosumi T. and Whittier R.F. 1995. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J. 8: 457–463.Google Scholar
  31. Maes T., De Keukelerie P. and Gerats T. 1999. Plant tagnology. Trends Plant Sci. 4: 90–96.Google Scholar
  32. Mckenzie N., Wen L.Y. and Dale P.J. 2002. Tissue-culture enhanced transposition of the maize transposable element Dissociation in Brassica oleracea var. ‘Italica’. Theor. Appl. Genet. 105: 23–33.Google Scholar
  33. Miura A., Yonebayashi S., Watanabe K., Toyama T., Shimada H. and Kakutani T. 2001. Mobilization of transposons by a mutation abolishing full DNA methylation in Arabidopsis. Nature 411: 212–214.Google Scholar
  34. Morris P.C., Jessop A. and Altmann T. 1993. Selection for enhanced germinal excision of Ac in transgenic Arabidopsis thaliana. Theor. Appl. Genet. 86: 919–926.Google Scholar
  35. Muller E., Brown P.T.H., Hartke S. and Lorz H. 1990. DNA variation in tissue-culture-derived rice plants. Theor. Appl. Genet 80: 673–679.Google Scholar
  36. Nakagawa Y., Machida C., Machida Y. and Toriyama K. 2000. Frequency and pattern of transposition of the maize transposable element Ds in transgenic rice plants. Plant Cell Physiol. 41: 733–742.Google Scholar
  37. Ozeki Y., Davies E. and Takeda J. 1997. Somatic variation during long term subculturing of plant cells caused by insertion of a transposable element in a phenylalanine ammonia-lyase (PAL) gene. Mol. Gen. Genet. 254: 407–416.Google Scholar
  38. Parinov S. and Sundaresan V. 2000. Functional genomics in Arabidopsis: large-scale insertional mutagenesis complements the genome sequencing project. Curr. Opin. Biotechnol. 11: 157–161.Google Scholar
  39. Peschke V.M. and Phillips R.L. 1991. Activation of the maize transposable element Suppressor-mutator (Spm) in tissue culture. Theor. Appl. Genet. 81: 90–97.Google Scholar
  40. Peschke V.M., Phillips R.L. and Gengenbach B.G. 1987. Discovery of transposable element activity among progeny of tissue culture-derived maize plants. Science 238: 804–807.Google Scholar
  41. Peschke V.M., Phillips R.L. and Gengenbach B.G. 1991. Genetic and molecular analysis of tissue culture-derived Ac elements. Theor. Appl. Genet. 82: 121–129.Google Scholar
  42. Primrose S.B. and Twyman R.M. 2002. Principles of Genome Analysis and Genomics (3rd edn.) Blackwell Science, Oxford UK.Google Scholar
  43. Richards E.J. 1997. DNA methylation and plant development. Trends Genet. 13: 319–323.Google Scholar
  44. Ros F. and Kunze R. 2001. Regulation of Activator/Dissociation transposition by replication and DNA methylation. Genetics 157: 1723–1733.Google Scholar
  45. Schmitt F., Oakeley E.J. and Jost J.P. 1997. Antibiotics induce genome-wide hypermethylation in cultured Nicotiana tabacum plants. J. Biol. Chem. 272: 1534–1540.Google Scholar
  46. Seki M., Ito T., Shibata D. and Shinozaki K. 1999. Regional mutagenesis of specific genes on the CIC5F11/CIC2B9 locus of Arabidopsis thaliana chromosome 5 using the Ac/Ds transposon in combination with the cDNA scanning method. Plant Cell Physiol. 40: 624–639.Google Scholar
  47. Smulders M., Rus-Kortekaas W. and Vosman B. 1995. Tissue culture-induced methylation polymorphisms in repetitive DNA of tomato calli and regenerated plants. Theor. Appl. Genet. 91: 1257–1264.Google Scholar
  48. Sudhakar D., Duc L.T., Bong B.B., Tinjuangjun P., Maqbool S.B., Valdez M. et al. 1998. An efficient rice transformation system utilizing mature seed-derived explants and a portable, inexpensive particle bombardment device. Transgenic Res. 7: 289–294.Google Scholar
  49. Sundaresan V., Springer P., Volpe T., Haward S., Jones J.D.G., Dean C. et al. 1995. Patterns of gene-action in plant development revealed by enhancer trap and gene trap transposable elements. Genes & Dev. 9: 1797–1810.Google Scholar
  50. Takeda S., Sugimoto K., Otsuki H. and 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: 383–393.Google Scholar
  51. Vain P., Worland B., Kohli A., Snape J.W. and Christou P. 1998. Green fluorescent protein (GFP) as a vital screenable marker in rice transformation. Theor. Appl. Genet. 96: 164–169.Google Scholar
  52. Walbot V. 2000. Saturation mutagenesis using maize transposons. Curr. Opin. Plant Biol. 3: 103–107.Google Scholar
  53. Yoder J.A., Walsh C.P. and Bestor T.H. 1997. Cytosine methylation and the ecology of intragenomic parasites. Trends Genet. 13: 335–340.Google Scholar
  54. Yu J., Hu S., Wang J., Wong G. K.-S., Li S., Liu B. et al. 2002. A draft sequence of the rice genome (Oryza sativa L. ssp. Indica). Science 296: 79–92.Google Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Ajay Kohli
    • 1
  • Mark Q. Prynne
    • 2
  • Berta Miro
    • 3
  • Andy Pereira
    • 4
  • Richard M. Twyman
    • 5
  • Teresa Capell
    • 3
  • Paul Christou
    • 3
  1. 1.Norwich Bio-IncubatorNorwichUK
  2. 2.Woodham MortimerLion Seeds Ltd.EssexUK
  3. 3.Fraunhofer Institute for Molecular BiotechnologySchmallenbergGermany
  4. 4.Plant Genomics Unit, Plant Research InternationalWageningenThe Netherlands
  5. 5.Department of BiologyUniversity of YorkHeslingtonUK

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