Skip to main content
SpringerLink
Log in

Silencing of a β-1,3-glucanase transgene is overcome during seed formation

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Expression of a β-1,3-glucanase transgene (gn1) driven by the CaMV 35S promoter is silenced in the T17 homozygous tobacco transgenic line. This silencing process is post-transcriptionally regulated and subject to developmental control. We have examined this phenomenon to investigate the developmental pathways involved in suppression and reactivation of gn1 expression as well as to identify the plant tissues where these processes occur. Analysis of β-1,3-glucanase activity and gene expression have allowed us to determine that suppression of gn1 is a very efficient process reducing the steady-state gn1 mRNA level, simultaneously, in all leaves of the plant. Gene silencing occurs a few weeks after seed germination, and is maintained throughout vegetative growth and floral development. Expression of gn1 is restored in the maturing fruit some time after fertilization. In situ hybridization analyses show that expression of gn1 is restored within the developing seeds in tissues derived from meiotically divided cells. In contrast to the high level of expression found in seedlings obtained from germinated T17 homozygous seeds, the expression of gn1 is not reactivated in plantlets regenerated in vitro from leaf explants of suppressed T17 homozygous plants that is, in plant tissues obtained by mitotic division. Thus, reactivation of gn1 expression specifically occurs along the developmental programme controlling sexual reproduction and likely throughout epigenetic modifications affecting the state of gene expression during meiosis.

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. Assaad FF, Tucker KL, Signer ER: Epigenetic repeat-induced gene silencing (RIGS) in Arabidopsis. Plant Mol Biol 22: 1067-1085 (1993).

    Google Scholar 

  2. Beffa RS, Neuhaus JM, Meins F Jr:Physiological compensation in antisense transformants: Specific induction of an‘ersatz’ glucan endo-1,3-β glucosidase in plants infected with necrotizing viruses. Proc Natl Acad Sci USA 90: 8792-8796 (1993).

    Google Scholar 

  3. Bender J, Fink GR: Epigenetic control of an endogenous gene family is revealed by a novel blue fluorescent mutant of Arabidopsis. Cell 83: 725-734 (1995).

    Google Scholar 

  4. Boerjan W, Bauw G, Van Montagu M, Inzé D: Distinct phenotypes generated by overexpression and suppression of Sadenosyl-L-methionine synthetase reveal developmental patterns of gene silencing in tobacco. Plant Cell 6: 1401-1414 (1994).

    Google Scholar 

  5. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254 (1976).

    Google Scholar 

  6. Brusslan JA, Karlin-Neumann GA, Huan L, Tobin EM: An Arabidopsismutant with a reduced level of cab 140 RNA is a result of cosuppression. Plant Cell 5: 667-677 (1993).

    Google Scholar 

  7. Bucciaglia PA, Smith AG: Cloning and characterization of Tag 1, a tobacco anther β-1,3-glucanase expressed during tetrad dissolution. Plant Mol Biol 24: 903-914 (1994).

    Google Scholar 

  8. Cañas LA, Busscher M, Angenent GC, Beltrán JP, van Tunen AJ: Nuclear localization of the PetuniaMADX box protein FBP1. Plant J 6: 597-604 (1994).

    Google Scholar 

  9. Castresana C, De Carvalho F, Gheysen G, Habets M, Inzé D, Van Montagu M: Tissue-specific and pathogen-induced regulation of a Nicotiana plumbaginifolia β-1,3-glucanase gene. Plant Cell 2: 1131-1143 (1990).

    Google Scholar 

  10. Cox KH, DeLeon DV, Angerer LM, Angerer RC: Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Devel Biol 101: 485-502 (1984).

    Google Scholar 

  11. Cox KH, Goldberg RB: Analysis of plant gene expression. In: Shaw CH (ed) PlantMolecular Biology: A Practical Approach, pp. 1-35. IRL Press, Oxford (1988).

    Google Scholar 

  12. De Carvalho F, Gheysen G, Kushnir S, Van Montagu M, Inzé D, Castresana C: Suppression of β-1,3-glucanase transgene expression in homozygous plants. EMBO J 11: 2595-2602 (1992).

    Google Scholar 

  13. De Carvalho F, Frendo P, Van Montagu M, Cornelissen M: Post-transcriptional cosuppression of β-1,3-glucanase genes does not affect accumulation of transgene nuclearmRNA. Plant Cell 7: 347-358 (1995).

    Google Scholar 

  14. De Loosse M, Alliote T, Gheysen G, Genetello C, Gielen J, Soetaert P, Van Montagu M, Inzé D: Primary structure of a hormonally regulated β-(1,3)-glucanase of Nicotiana plumbaginifolia. Gene 70: 13-23 (1988).

    Google Scholar 

  15. Dehio C, Schell J: Identification of plant genetic loci involvedin a posttranscriptional mechanism for meiotically reversible transgene silencing. Proc Natl Acad Sci USA 91: 5538-5542 (1994).

    Google Scholar 

  16. Elmayan T, Vaucheret H: Expression of single copies of a strong expressed 35S transgene can be silenced posttranscriptionally. Plant J 9: 787-797(1996).

    Google Scholar 

  17. English JJ, Mueller E, Baulcombe DC: Suppression of virus accumulation in transgenic plants exhibiting silencing of nuclear genes. Plant Cell 8: 179-188 (1996).

    Google Scholar 

  18. Flavell RB: Inactivation of gene expression in plants as a consequence of specific sequence duplication. Proc Natl Acad Sci USA 91: 3490-3496 (1994).

    Google Scholar 

  19. Gheysen G, Inzé D, Soetaert P, Van Montagu M, Castresana C: Sequence of a Nicotiana plumbaginifolia β(1,3)-glucanase gene encoding a vacuolar isoform. Nucl Acids Res 18: 6685 (1990).

    Google Scholar 

  20. Goring DR, Thomson L, Rothstein S: Transformation of a partial nopaline synthase gene into tobacco suppresses the expression of a resident wild-type gene. Proc Natl Acad Sci USA 88: 1770-1774 (1991).

    Google Scholar 

  21. Hart CM, Fischer B, Neuhaus JM, Meins F Jr: Regulated inactivation of homologous gene expression in transgenic Nicotiana sylvestrisplants containing a defense-related tobacco chitinase gene. Mol Gen Genet 235: 179-188 (1992).

    Google Scholar 

  22. Hird DL, Worrall D, Hodge R, Smartt S, Paul W, Scott R: The anther-specific protein encoded by the Brassica napusand Arabidopsis thalianaA6 gene displays similarity to β-1,3-glucanases. Plant J 4: 1023-1033 (1993).

    Google Scholar 

  23. Hobbs SLA, Warkentin TD, DeLong CMO: Transgene copy number can be positively or negatively associated with transgene expression. Plant Mol Biol 21: 17-26 (1993).

    Google Scholar 

  24. Ingelbrecht I, Van Houdt H, Van Montagu M, Depicker A: Posttranscriptional silencing of reporter transgenes in tobacco correlates with DNAmethylation. Proc Natl Acad Sci USA 91: 10 502-10 506 (1994).

    Google Scholar 

  25. Jones A, Davies HM, Voelker TA: Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-acp thioesterases. Plant Cell 7: 359-371 (1995).

    Google Scholar 

  26. Jorgensen RA: Cosuppression, flower color patterns, andmetastable gene expression states. Science 268: 686-691 (1995).

    Google Scholar 

  27. Kunz C, Schöb H, Stam M, Kooter JM, Meins FJr: Developmentally regulated silencing and reactivation of tobacco chitinase transgene expression. Plant J 10: 437-450 (1996).

    Google Scholar 

  28. Latham KE, Doherty AS, Scott CD, Schultz RM: Igf2r and Igf2 gene expression in androgenic, gynogenic, and parthenogenetic preimplantation mouse embryos: absence of regulation by genomic imprinting. Genes Devel 8: 290-299 (1994).

    Google Scholar 

  29. Logemann J, Schell J, Willmitzer L: Improved method for the isolation of RNAfrom plant tissues. Anal Biochem 163: 16-20 (1987).

    Google Scholar 

  30. Martienssen RA, Richards EJ: DNAmethylation in eukaryotes. Curr Opin Genet Devel 5: 234-242 (1995).

    Google Scholar 

  31. Matzke AJM, Neuhuber F, Park YD, Ambros PF, Matzke MA:Homology-dependent gene silencing in transgenic plants: epistatic silencing loci contain multiple copies of methylated transgenes. Mol Gen Genet 244: 219-229 (1994).

    Google Scholar 

  32. Matzke MA, Matzke AJM: How and why do plants inactivate homologous (trans)genes? Plant Physiol 107: 679-685 (1995).

    Google Scholar 

  33. 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 

  34. Meyer P: Understanding and controlling transgene expression. Trends Biotechnol 13: 332-337 (1995).

    Google Scholar 

  35. Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473-497 (1962).

    Google Scholar 

  36. Napoli C, Lemieux C, Jorgensen R: Introduction of a chimeric chalcone synthase gene into petunia results in reversible cosuppression of homologous genes in trans. Plant Cell 2: 279-289 (1990).

    Google Scholar 

  37. Ori N, Sessa G, Lotan T, Himmelhoch S, Fluhr R: A major stylar matrix polypeptide (sp41) is a member of the pathogenesis-related proteins superclass: EMBO J 9: 3429-3436 (1990).

    Google Scholar 

  38. Pan SQ, Ye XS, Kuc J: Direct detection of β-1,3-glucanase isozymes on polyacrylamide electrophoresis and isoelectrofocusing gels. Anal Biochem 182: 136-140 (1989).

    Google Scholar 

  39. Pang SZ, Jan FJ, Carney K, Stout J, Tricoli DM, Quemada HD, Gonsalves D: Post-transcriptional transgene silencing and consequent tospovirus resistance in transgenic lettuce are affected by transgene. Plant J 9: 899-909 (1996).

    Google Scholar 

  40. Park YD, Papp I, Moscone EA, Iglesias VA, Vaucheret H, Matzke AJM, Matzke MA: Gene silencing mediated by promoter homology occurs at the level of transcription and results in meiotically heritable alterations in methylation and gene activity. Plant J 9: 183-194 (1996).

    Google Scholar 

  41. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).

    Google Scholar 

  42. Shinshi H, Wenzler H, Neuhaus JM, Felix G, Hofsteenge J, Meins F Jr: Evidence for N-and C-terminal processing of a plant defense-related enzyme: primary structure of tobacco prepro-β-1,3-glucanase. Proc Natl Acad Sci USA 85: 5541-5545 (1988).

    Google Scholar 

  43. Smith CJS, Watson CF, Bird CR, Ray J, Schuch W, Grierson D: Expression of a truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants. Mol Gen Genet 224: 477-481 (1990).

    Google Scholar 

  44. Smith HA, Swaney SL, Parks D, Wernsman EA, Dougherty WG: Transgenic plant virus resistance mediated by untranslatable sense RNAs: expression, regulation, and fate of nonessential RNAs. Plant Cell 6: 1441-1453 (1994).

    Google Scholar 

  45. van Blokland R, van der Geest N, Mol JNM, Kooter JM: Transgene-mediated suppression of chalcone synthase expression in Petunia hybridaresults from an increase in RNA turnover. Plant J 6: 861-877 (1994).

    Google Scholar 

  46. van der Krol AR, Mur LA, Beld M, Mol JNM, Stuitje AR: Flavonoid gene in petunia: Addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2: 291-299 (1990)

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Centro Nacional de Biotecnología, C.S.I.C., Campus, Universidad Autónoma, Cantoblanco, E-28049, Madrid, Spain

    Teresa Balandin & Carmen Castresana

Authors
  1. Teresa Balandin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Carmen Castresana
    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

Balandin, T., Castresana, C. Silencing of a β-1,3-glucanase transgene is overcome during seed formation. Plant Mol Biol 34, 125–137 (1997). https://doi.org/10.1023/A:1005882106266

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1005882106266

Search

Navigation