Molecular Genetics and Genomics

, Volume 285, Issue 3, pp 225–236 | Cite as

Inhibition of SAH-hydrolase activity during seed germination leads to deregulation of flowering genes and altered flower morphology in tobacco

  • Jaroslav FulnečekEmail author
  • Roman Matyášek
  • Ivan Votruba
  • Antonín Holý
  • Kateřina Křížová
  • Aleš Kovařík
Original Paper


Developmental processes are closely connected to certain states of epigenetic information which, among others, rely on methylation of chromatin. S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are key cofactors of enzymes catalyzing DNA and histone methylation. To study the consequences of altered SAH/SAM levels on plant development we applied 9-(S)-(2,3-dihydroxypropyl)-adenine (DHPA), an inhibitor of SAH-hydrolase, on tobacco seeds during a short phase of germination period (6 days). The transient drug treatment induced: (1) dosage-dependent global DNA hypomethylation mitotically transmitted to adult plants; (2) pleiotropic developmental defects including decreased apical dominance, altered leaf and flower symmetry, flower whorl malformations and reduced fertility; (3) dramatic upregulation of floral organ identity genes NTDEF, NTGLO and NAG1 in leaves. We conclude that temporal SAH-hydrolase inhibition deregulated floral genes expression probably via chromatin methylation changes. The data further show that plants might be particularly sensitive to accurate setting of SAH/SAM levels during critical developmental periods.


DNA methylation Nicotiana tabacum Plant development S-adenosyl-l-homocysteine hydrolase inhibitor DHPA MADS box genes DEFICIENS/APETALA 3 GLOBOSA/PISTILLATA PLENA/AGAMOUS FLORICAULA/LEAFY 



We thank Dr. Jiří Široký for the help with pollen microscopy. The scintillation method to detect uptake and transport of DHPA was introduced by Dr. Richard Tykva at the Institute of Organic Chemistry and Biochemistry AS CR, v.v.i. and tested in tobacco plants at the Institute of Experimental Botany AS CR, v.v.i. in Prague. We further thank an anonymous referee for the idea to analyze floral gene expression. This research was funded by the Grant Agency of the Czech Republic (206/09/1751, P501/10/0208, P501/11/P667) and the Academy of Sciences of the Czech Republic (AVOZ50040507 and AVOZ50040702).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

438_2011_601_MOESM1_ESM.pdf (2.5 mb)
Supplementary material 1 (PDF 2536 kb)


  1. Barbes C, Sanchez J, Yebra MJ, Robertgero M, Hardisson C (1990) Effects of sinefungin and S-adenosylhomocysteine on DNA and protein methyltransferases from Streptomyces and other bacteria. FEMS Microbiol Lett 69:239–244CrossRefGoogle Scholar
  2. Bastow R, Mylne JS, Lister C, Lippman Z, Martienssen RA, Dean C (2004) Vernalization requires epigenetic silencing of FLC by histone methylation. Nature 427:164–167CrossRefPubMedGoogle Scholar
  3. Baubec T, Dinh HQ, Pecinka A, Rakic B, Rozhon W, Wohlrab B, von Haeseler A, Mittelsten Scheid O (2010) Cooperation of multiple chromatin modifications can generate unanticipated stability of epigenetic states in Arabidopsis. Plant Cell 22:34–47CrossRefPubMedGoogle Scholar
  4. Benes K, Holy A, Melichar O (1984) The effect of 9-(2,3-dihydroxypropyl)adenine (DHPA) on seedling roots of Vicia faba L. in comparison with adenine, adenosine and some cytokinins. Biol Plant 26:144–150CrossRefGoogle Scholar
  5. Bradley D, Carpenter R, Sommer H, Hartley N, Coen E (1993) Complementary floral homeotic phenotypes result from opposite orientations of a transposon at the plena locus of Antirrhinum. Cell 72:85–95CrossRefPubMedGoogle Scholar
  6. Brzezinski K, Bujacz G, Jaskolski M (2008) Purification, crystallization and preliminary crystallographic studies of plant S-adenosyl-l-homocysteine hydrolase (Lupinus luteus). Acta Crystallogr Sect F Struct Biol Cryst Commun 64:671–673CrossRefPubMedGoogle Scholar
  7. Cantoni GL (1986) The centrality of S-adenosylhomocysteinase in the regulation of the biological utilization of S-adenosylmethionine. In: Borchardt RT, Creveling CR, Ueland PM (eds) Biological methylation and drug design. Humana Press, Clifton, NJ, pp 227–238Google Scholar
  8. Cao X, Springer NM, Muszynski MG, Phillips RL, Kaeppler S, Jacobsen SE (2000) Conserved plant genes with similarity to mammalian de novo DNA methyltransferases. Proc Natl Acad Sci USA 97:4979–4984CrossRefPubMedGoogle Scholar
  9. Comai L (2000) Genetic and epigenetic interactions in allopolyploid plants. Plant Mol Biol 43:387–399CrossRefPubMedGoogle Scholar
  10. Davies B, Di Rosa A, Eneva T, Saedler H, Sommer H (1996) Alteration of tobacco floral organ identity by expression of combinations of Antirrhinum MADS-box genes. Plant J 10:663–677CrossRefPubMedGoogle Scholar
  11. Dragun M, Rada B, Holy A (1983) Transport of antiviral agent 9-(S)-(2,3-dihydroxypropyl) adenine to animal cells. Acta Virol 27:119–129PubMedGoogle Scholar
  12. Estruch JJ, Granell A, Hansen G, Prinsen E, Redig P, Vanonckelen H, Schwarzsommer Z, Sommer H, Spena A (1993) Floral development and expression of floral homeotic genes are influenced by cytokinins. Plant J 4:379–384CrossRefPubMedGoogle Scholar
  13. Fieldes MA, Schaeffer SM, Krech MJ, Brown JC (2005) DNA hypomethylation in 5-azacytidine-induced early-flowering lines of flax. Theor Appl Genet 111:136–149CrossRefPubMedGoogle Scholar
  14. Finnegan EJ, Kovac KA (2000) Plant DNA methyltransferases. Plant Mol Biol 43:189–201CrossRefPubMedGoogle Scholar
  15. 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
  16. Fojtova M, Kovarik A, Votruba I, Holy A (1998) Evaluation of the impact of S-adenosylhomocysteine metabolic pools on cytosine methylation of the tobacco genome. Eur J Biochem 252:347–352CrossRefPubMedGoogle Scholar
  17. Fojtova M, Kovarik A, Matyasek R (2001) Cytosine methylation of plastid genome in higher plants. Fact or artefact? Plant Sci 160:585–593CrossRefPubMedGoogle Scholar
  18. Fulnecek J, Kovarik A (2007) Low abundant spacer 5S rRNA transcripts are frequently polyadenylated in Nicotiana. Mol Genet Genomics 278:565–573CrossRefPubMedGoogle Scholar
  19. Fulnecek J, Matyasek R, Kovarik A, Bezdek M (1998) Mapping of 5-methylcytosine residues in Nicotiana tabacum 5S rRNA genes by genomic sequencing. Mol Gen Genet 259:133–141CrossRefPubMedGoogle Scholar
  20. Fulnecek J, Lim KY, Leitch AR, Kovarik A, Matyasek R (2002) Evolution and structure of 5S rDNA loci in allotetraploid Nicotiana tabacum and its putative parental species. Heredity 88:19–25CrossRefPubMedGoogle Scholar
  21. Gallardo K, Job C, Groot SP, Puype M, Demol H, Vandekerckhove J, Job D (2002) Importance of methionine biosynthesis for Arabidopsis seed germination and seedling growth. Physiol Plant 116:238–247CrossRefPubMedGoogle Scholar
  22. 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
  23. Goodspeed TH (1954) The genus Nicotiana. Waltham, Massachusetts, USAGoogle Scholar
  24. Hansen G, Estruch JJ, Sommer H, Spena A (1993) NTGLO: a tobacco homologue of the GLOBOSA floral homeotic gene of Antirrhinum majus: cDNA sequence and expression pattern. Mol Gen Genet 239:310–312PubMedGoogle Scholar
  25. Henikoff S, Comai L (1998) A DNA methyltransferase homolog with a chromodomain exists in multiple polymorphic forms in Arabidopsis. Genetics 149:307–318PubMedGoogle Scholar
  26. Holy A (1975) Aliphatic analogues of nucleosides, nucleotides and oligonucleotides. Collect Czech Chem C 40:187–214Google Scholar
  27. Holy A (2005) Synthesis of acyclic analogs of adenosine. Curr Protoc Nucleic Acid Chem 14.1.1–14.1.21Google Scholar
  28. Jackson JP, Lindroth AM, Cao X, Jacobsen SE (2002) Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature 416:556–560CrossRefPubMedGoogle Scholar
  29. Jacobsen SE, Meyerowitz EM (1997) Hypermethylated SUPERMAN epigenetic alleles in Arabidopsis. Science 277:1100–1103CrossRefPubMedGoogle Scholar
  30. 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
  31. Jordan ND, West JP, Bottley A, Sheikh M, Furner I (2007) Transcript profiling of the hypomethylated hog1 mutant of Arabidopsis. Plant Mol Biol 65:571–586CrossRefPubMedGoogle Scholar
  32. Kashkush K, Feldman M, Levy AA (2003) Transcriptional activation of retrotransposons alters the expression of adjacent genes in wheat. Nat Genet 33:102–106CrossRefPubMedGoogle Scholar
  33. Kelly AJ, Bonnlander MB, Meeks-Wagner DR (1995) NFL, the tobacco homolog of FLORICAULA and LEAFY, is transcriptionally expressed in both vegetative and floral meristems. Plant Cell 7:225–234CrossRefPubMedGoogle Scholar
  34. Kempin SA, Mandel MA, Yanofsky MF (1993) Conversion of perianth into reproductive organs by ectopic expression of the tobacco floral homeotic gene NAG1. Plant Physiol 103:1041–1046CrossRefPubMedGoogle Scholar
  35. Koltunow AM, Truettner J, Cox KH, Wallroth M, Goldberg RB (1990) Different temporal and spatial gene expression patterns occur during anther development. Plant Cell 2:1201–1224CrossRefPubMedGoogle Scholar
  36. Koukalova B, Reich J, Matyasek R, Kuhrova V, Bezdek M (1989) A BamHI family of highly repeated DNA sequences of Nicotiana tabacum. Theor Appl Genet 78:77–80CrossRefGoogle Scholar
  37. Koukalova B, Votruba I, Fojtova M, Holy A, Kovarik A (2002) Hypomethylation of CNG targets induced with dihydroxypropyladenine is rapidly reversed in the course of mitotic cell division in tobacco. Theor Appl Genet 105:796–801CrossRefPubMedGoogle Scholar
  38. Kovarik A, Koukalova B, Holy A, Bezdek M (1994) Sequence-specific hypomethylation of the tobacco genome induced with dihydroxypropyladenine, ethionine and 5-azacytidine. FEBS Lett 353:309–311CrossRefPubMedGoogle Scholar
  39. Kovarik A, Koukalova B, Lim KY, Matyasek R, Lichtenstein CP, Leitch AR, Bezdek M (2000a) Comparative analysis of DNA methylation in tobacco heterochromatic sequences. Chromosome Res 8:527–541CrossRefPubMedGoogle Scholar
  40. Kovarik A, Van Houdt H, Holy A, Depicker A (2000b) Drug-induced hypomethylation of a posttranscriptionally silenced transgene locus of tobacco leads to partial release of silencing. FEBS Lett 467:47–51CrossRefPubMedGoogle Scholar
  41. Krizova K, Fojtova M, Depicker A, Kovarik A (2009) Cell culture-induced gradual and frequent epigenetic reprogramming of invertedly repeated tobacco transgene epialleles. Plant Physiol 149:1493–1504CrossRefPubMedGoogle Scholar
  42. Law JA, Jacobsen SE (2010) Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 11:204–220CrossRefPubMedGoogle Scholar
  43. Lindroth AM, Cao X, Jackson JP, Zilberman D, McCallum CM, Henikoff S, Jacobsen SE (2001) Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation. Science 292:2077–2080CrossRefPubMedGoogle Scholar
  44. Lindroth AM, Shultis D, Jasencakova Z, Fuchs J, Johnson L, Schubert D, Patnaik D, Pradhan S, Goodrich J, Schubert I, Jenuwein T, Khorasanizadeh S, Jacobsen SE (2004) Dual histone H3 methylation marks at lysines 9 and 27 required for interaction with CHROMOMETHYLASE3. EMBO J 23:4286–4296CrossRefPubMedGoogle Scholar
  45. Liu Z, Mara C (2010) Regulatory mechanisms for floral homeotic gene expression. Semin Cell Dev Biol 21:80–86CrossRefPubMedGoogle Scholar
  46. Lunerova-Bedrichova J, Bleys A, Fojtova M, Khaitova L, Depicker A, Kovarik A (2008) Trans-generation inheritance of methylation patterns in a tobacco transgene following a post-transcriptional silencing event. Plant J 54:1049–1062CrossRefPubMedGoogle Scholar
  47. Ma H, Yanofsky MF, Meyerowitz EM (1991) AGL1-AGL6, an Arabidopsis gene family with similarity to floral homeotic and transcription factor genes. Genes Dev 5:484–495CrossRefPubMedGoogle Scholar
  48. Madlung A, Masuelli RW, Watson B, Reynolds SH, Davison J, Comai L (2002) Remodeling of DNA methylation and phenotypic and transcriptional changes in synthetic Arabidopsis allotetraploids. Plant Physiol 129:733–746CrossRefPubMedGoogle Scholar
  49. Malagnac F, Bartee L, Bender J (2002) An Arabidopsis SET domain protein required for maintenance but not establishment of DNA methylation. EMBO J 21:6842–6852CrossRefPubMedGoogle Scholar
  50. Masuta C, Tanaka H, Uehara K, Kuwata S, Koiwai A, Noma M (1995) Broad resistance to plant viruses in transgenic plants conferred by antisense inhibition of a host gene essential in S-adenosylmethionine-dependent transmethylation reactions. Proc Natl Acad Sci USA 92:6117–6121CrossRefPubMedGoogle Scholar
  51. Mathieu O, Reinders J, Caikovski M, Smathajitt C, Paszkowski J (2007) Transgenerational stability of the Arabidopsis epigenome is coordinated by CG methylation. Cell 130:851–862CrossRefPubMedGoogle Scholar
  52. Matzke M, Kanno T, Daxinger L, Huettel B, Matzke AJ (2009) RNA-mediated chromatin-based silencing in plants. Curr Opin Cell Biol 21:367–376CrossRefPubMedGoogle Scholar
  53. Melayah D, Bonnivard E, Chalhoub B, Audeon C, Grandbastien MA (2001) The mobility of the tobacco Tnt1 retrotransposon correlates with its transcriptional activation by fungal factors. Plant J 28:159–168CrossRefPubMedGoogle Scholar
  54. Mirouze M, Reinders J, Bucher E, Nishimura T, Schneeberger K, Ossowski S, Cao J, Weigel D, Paszkowski J, Mathieu O (2009) Selective epigenetic control of retrotransposition in Arabidopsis. Nature 461:427–430CrossRefPubMedGoogle Scholar
  55. Mull L, Ebbs ML, Bender J (2006) A histone methylation-dependent DNA methylation pathway is uniquely impaired by deficiency in Arabidopsis S-adenosylhomocysteine hydrolase. Genetics 174:1161–1171CrossRefPubMedGoogle Scholar
  56. Papa CM, Springer NM, Muszynski MG, Meeley R, Kaeppler SM (2001) Maize chromomethylase Zea methyltransferase2 is required for CpNpG methylation. Plant Cell 13:1919–1928CrossRefPubMedGoogle Scholar
  57. Parenicova L, de Folter S, Kieffer M, Horner DS, Favalli C, Busscher J, Cook HE, Ingram RM, Kater MM, Davies B, Angenent GC, Colombo L (2003) Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis: new openings to the MADS world. Plant Cell 15:1538–1551CrossRefPubMedGoogle Scholar
  58. Petit M, Guidat C, Daniel J, Denis E, Montoriol E, Bui QT, Lim KY, Kovarik A, Leitch AR, Grandbastien MA, Mhiri C (2010) Mobilization of retrotransposons in synthetic allotetraploid tobacco. New Phytol 186:135–147CrossRefPubMedGoogle Scholar
  59. Richards EJ (1997) DNA methylation and plant development. Trends Genet 13:319–323CrossRefPubMedGoogle Scholar
  60. Rocha PS, Sheikh M, Melchiorre R, Fagard M, Boutet S, Loach R, Moffatt B, Wagner C, Vaucheret H, Furner I (2005) The Arabidopsis HOMOLOGY-DEPENDENT GENE SILENCING1 gene codes for an S-adenosyl-l-homocysteine hydrolase required for DNA methylation-dependent gene silencing. Plant Cell 17:404–417CrossRefPubMedGoogle Scholar
  61. Ronemus MJ, Galbiati M, Ticknor C, Chen J, Dellaporta SL (1996) Demethylation-induced developmental pleiotropy in Arabidopsis. Science 273:654–657CrossRefPubMedGoogle Scholar
  62. Rushton PJ, Bokowiec MT, Laudeman TW, Brannock JF, Chen X, Timko MP (2008) TOBFAC: the database of tobacco transcription factors. BMC Bioinform 9:53CrossRefGoogle Scholar
  63. Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW (1984) Ribosomal DNA spacer-length polymorphisms in barley: mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci USA 81:8014–8018CrossRefPubMedGoogle Scholar
  64. Sebestova L, Votruba I, Holy A (1984) Studies on S-adenosyl-l-homocysteine hydrolase.11. S-adenosyl-l-homocysteine hydrolase from Nicotiana tabacum L: isolation and properties. Collect Czech Chem Commun 49:1543–1551CrossRefGoogle Scholar
  65. Tanaka H, Masuta C, Uehara K, Kataoka J, Koiwai A, Noma M (1997) Morphological changes and hypomethylation of DNA in transgenic tobacco expressing antisense RNA of the S-adenosyl-l-homocysteine hydrolase gene. Plant Mol Biol 35:981–986CrossRefPubMedGoogle Scholar
  66. Teixeira FK, Heredia F, Sarazin A, Roudier F, Boccara M, Ciaudo C, Cruaud C, Poulain J, Berdasco M, Fraga MF, Voinnet O, Wincker P, Esteller M, Colot V (2009) A role for RNAi in the selective correction of DNA methylation defects. Science 323:1600–1604CrossRefPubMedGoogle Scholar
  67. Votruba I, Holy A, De Clercq E (1983) Metabolism of the broad-spectrum antiviral agent, 9-(S)-(2,3-dihydroxypropyl) adenine, in different cell lines. Acta Virol 27:273–276PubMedGoogle Scholar
  68. 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
  69. Yuiko I, Takeshi S, Kiyotoshi T (2010) Flowering and dwarfism induced by DNA demethylation in Pharbitis nil. Physiol Plant 139:118–127CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Jaroslav Fulneček
    • 1
    Email author
  • Roman Matyášek
    • 1
  • Ivan Votruba
    • 2
  • Antonín Holý
    • 2
  • Kateřina Křížová
    • 1
  • Aleš Kovařík
    • 1
  1. 1.Institute of BiophysicsAcademy of Sciences of the Czech RepublicBrnoCzech Republic
  2. 2.Institute of Organic Chemistry and BiochemistryAcademy of Sciences of the Czech RepublicPraha 6Czech Republic

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