Antisense RNA in Plants: A Tool for Analysis and Suppression of Gene Function

  • Martin Tabler
Part of the NATO ASI Series book series (NSSA, volume 253)


Expression of antisense genes has become a widely applied technique in plant biotechnology for creating “phenotypic mutants” in which the endogenous target gene is specifically suppressed. The antisense technology is used for various purposes such as silencing or ablating undesired genes. By creating “dominant negative mutants”, gene function can also be assigned to a particular cloned cDNA. Despite the rapidly increasing number of successful applications of antisense genes in plants, the molecular mechanisms in which complementary RNAs down-regulate gene expression is not fully understood.


Transgenic Plant Human Immunodeficiency Virus Type Cucumber Mosaic Virus Tomato Fruit Hammerhead Ribozyme 
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  1. Bird CR, Ray JA, Fletcher JD, Boniwell JM, Bird AS, Teulieres C, Blain I, Bramley PM, Schuch W (1991) Using antisense RNA to study gene function: inhibition of carotenoid biosynthesis in transgenic tomatoes. Biotechnology 9: 635–639.CrossRefGoogle Scholar
  2. Blomberg P, Wagner EG, Nordström K (1990) Control of replication of plasmid R1: the duplex between the antisense RNA, CopA, and its target, CopT, is processed specifically in vivo and in vitro by RNase HI. EMBO J 9: 2331–2340.PubMedGoogle Scholar
  3. Bourque JE, Folk WR (1992) Suppression of gene expression in plant cells utilizing antisense sequences transcribed by RNA polymerase III. Plant Mol Biol 19: 641–647.PubMedCrossRefGoogle Scholar
  4. Bouzayen M, Hamilton A, Picton S, Barton S, Grierson D (1992) Identification of genes for the ethylene-forming enzyme and inhibition of ethylene synthesis in transgenic plants using antisense genes. Biochemical Society Transactions 20: 76–79.PubMedGoogle Scholar
  5. Bramley P, Teulieres C, Blain I, Bird C, Schuch W (1992) Biochemical characterization of transgenic tomato plants in which carotenoid synthesis has been inhibited through the expression of antisense RNA to pTOM5. Plant J 2: 343–349.CrossRefGoogle Scholar
  6. Bruening G (1989) Compilation of self-cleaving sequences from plant virus satellite RNAs and other sources. Methods Enzymol 180: 546–558.PubMedCrossRefGoogle Scholar
  7. Bruening G, Buzayan JM, Hampel A, Gerlach WL (1988) Replication of small satellite RNAs and viroids: possible participation of non-enzymatic reactions. In: Viroids and RNA Recombination, RNA Genetics, Domingo E, Holland JJ, Alquist P (eds), vol II, Retroviruses, CRC Press, Boca Raton, USA, pp 127–145.Google Scholar
  8. Cameron FH, Jennings PA (1989) Specific gene suppression by engineered ribozymes in monkey cells. Proc Natl Acad Sci USA 86: 9139–9143.PubMedCrossRefGoogle Scholar
  9. Cannon M, Platz J, O’Leary M, Sookdeo C, Cannon F (1990) Organ-specific modulation of gene expression in transgenic plants using antisense RNA. Plant Mol Biol 15: 39–47.PubMedCrossRefGoogle Scholar
  10. Cech TR (1987) The chemistry of self-cleaving RNA and RNA enzymes. Science 231: 1532–1539.CrossRefGoogle Scholar
  11. Cesareni G, Helmer-Citterich M, Castagnoli L (1991) Control of ColE1 plasmid replication by antisense RNA.Google Scholar
  12. Chapman S, Kavanagh T, Baulcombe D (1992) Potato virus X as a vector for gene expression in plants. Plant J 2: 549–557.PubMedGoogle Scholar
  13. Choudhary AD, Kessmann H, Lamb CJ, Dixon RA (1990) Stress response in alfalfa (Medicago sativa L.) IV. Expression of defense gene constructs in electroporated suspension cell protoplasts. Plant Cell Rep 9: 42–46.CrossRefGoogle Scholar
  14. Cohen JS (1989) Oligodeoxynucleotides: antisense inhibitors of gene expression, Macmillan/CRC Press, London/Miami.Google Scholar
  15. Cohen JS (1992) Chemically modified oligodeoxynucleotide analogs as regulators of viral and cellular gene expression, in: Gene Regulation: Biology of Antisense RNA and DNA, Erickson RP, Izant JG (eds), Raven Press Ltd, New York, pp 247–259.Google Scholar
  16. Cornelissen M, Vandewiele M (1989) Both RNA level and translation efficiency are reduced by antisense RNA in transgenic tobacco. Nucleic Acids Res 17: 833–843.PubMedCrossRefGoogle Scholar
  17. Cotten M Birnstiel ML (1989) Ribozyme mediated destruction of RNA in vivo. EMBO J 8: 3861–3866.PubMedGoogle Scholar
  18. Cuozzo M, O Connell KM, Kaniewski W, Fang R-X, Chua N-H, Turner NE (1988) Viral protection in transgenic tobacco plants expressing the cucumber mosaic virus coat protein or its antisense RNA. Biotechnology 6: 549–557.CrossRefGoogle Scholar
  19. Day AG, Bejarano ER, Buck KW, Burrell M, Lichtenstein CP (1991) Expression of an antisense viral gene in transgenic tobacco confers resistance to the DNA virus tomato golden mosaic virus. Proc Natl Acad Sci USA 88: 6721–6725.PubMedCrossRefGoogle Scholar
  20. Delauney AJ, Tabaeizadeh Z, Verma DP (1988) A stable bifunctional antisense transcript inhibiting gene expression in transgenic plants. Proc Natl Acad Sci USA 85: 4300–4304.PubMedCrossRefGoogle Scholar
  21. Ecker JR, Davis RW (1986) Inhibition of gene expression in plant cells by expression of antisense RNA. Proc Natl Acad Sci USA 83: 5372–5376.PubMedCrossRefGoogle Scholar
  22. Eckes P (1992) Inhibition of fruit ripening by antisense-RNA technology. Angew Chem Int Ed Engl 31: 175–177.CrossRefGoogle Scholar
  23. Edgington SM (1992) Ribozymes: stop-making sense. Biotechnology 10: 256–262.PubMedCrossRefGoogle Scholar
  24. Eguchi Y, Taleo I, Tomizuwa J (1991) Antisense RNA. Annu Rev Biochem 60: 631–652.PubMedCrossRefGoogle Scholar
  25. Forster AC, Symons RH (1987a) Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites. Cell 49: 211–220.PubMedCrossRefGoogle Scholar
  26. Forster AC, Symons RH (1987b) Self-cleavage of virusoid RNA is performed by the proposed 55-nucleotide active site. Cell 50: 9–16.PubMedCrossRefGoogle Scholar
  27. Gray J, Picton S, Shabbeer J, Schuch W, Grierson D (1992) Molecular biology of fruit ripening and its manipulation with antisense genes. Plant Mol Biol 19: 69–87.PubMedCrossRefGoogle Scholar
  28. Green PJ, Pines O, Inouye M (1986) The role of antisense RNA in gene regulation. Annu Rev Biochem 55: 569–597.PubMedCrossRefGoogle Scholar
  29. Grierson D (1992) Control of ethylene synthesis and ripening by sense and antisense genes in transgenic plants. Proc Roy Soc Edinburgh 99B: 79–88.Google Scholar
  30. Grierson D, Fray RG, Hamilton AJ, Smith CJ, Watson CF (1991) Does co-suppression of sense genes in transgenic plants involve antisense RNA?. Trends Biotechnol 9: 122–123.CrossRefGoogle Scholar
  31. Hamilton AJ, Lycett GM, Grierson D (1990) Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants. Nature 346: 284–287.CrossRefGoogle Scholar
  32. Hamilton AJ, Bouzayen M, Grierson D (1991) Identification of a tomato gene for the ethylene-forming enzyme by expression in yeast. Proc Natl Acad Sci USA 88: 7434–7437.PubMedCrossRefGoogle Scholar
  33. Haseloff J, Gerlach WL (1988) Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature 334: 585–591.PubMedCrossRefGoogle Scholar
  34. Hélène C (1992) Control of gene expression by antisense and antigen oligonucleotide-intercalator conjugates. In: Gene Regulation: Biology of Antisense RNA and DNA, Erickson RP, Izant JG (eds), Raven Press Ltd, New York, pp 109–118.Google Scholar
  35. Hélène C, Toulmé J-J (1990) Specific regulation of gene expression by antisense, sense and antigene nucleic acids. Biochim Biophys Acta 1049: 99–125.PubMedCrossRefGoogle Scholar
  36. Hemenway C, Fang R-X, Kaniewski WK, Chua N-H, Turner NE (1988) Analysis of the mechanism of protection in transgenic plants expressing the potato virus X coat protein or its antisense RNA. EMBO J 7: 1273–1280.PubMedGoogle Scholar
  37. Herschlag D (1991) Implications of ribozyme kinetics for targeting the cleavage of specific RNA molecules in vivo: more isn’t always better. Proc Natl Acad Sci USA 88: 6921–6925.PubMedCrossRefGoogle Scholar
  38. Hobbs SL, Kpodar P, DeLong CM (1990) The effect of T-DNA copy number, position and methylation on reporter gene expression in tobacco transformants. Plant Mol Biol 15: 851–864.PubMedCrossRefGoogle Scholar
  39. Hudson GS, Evans JR, von Caemmerer S, Arvidsson YB, Andrews TJ (1992) Reduction of ribulose-1, 5-bisphosphate carboxylase/oxygenase content by antisense RNA reduces photosynthesis in transgenic tobacco plants. Plant Physiol 98: 294–302.PubMedCrossRefGoogle Scholar
  40. Izant JG, Weintraub H (1984) Inhibition of thymidine kinase gene expression by antisense RNA: a molecular approach to genetic analysis. Cell 36: 1007–1015.PubMedCrossRefGoogle Scholar
  41. Jorgensen R (1990) Altered gene expression in plants due to trans interactions between homologous genes. Trends Biotechnol 8: 340–344.PubMedCrossRefGoogle Scholar
  42. Jorgensen R (1991) Beyond antisense-How do transgenes interact with homologous plant genes?. Trends Biotechnol 9: 266–261.CrossRefGoogle Scholar
  43. Kawchuk LM, Martin RR, McPherson J (1991) Sense and antisense RNA-mediated resistance to potato leafroll virus in russet burbank potato plants. Mol Plant Microbe Interaction 4: 247–253.CrossRefGoogle Scholar
  44. Kimelman D (1992) Regulation of eukaryotic gene expression by natural antisense transcripts. In: Gene Regulation: Biology of Antisense RNA and DNA, Erickson RP, Izant JG (eds), Raven Press Ltd, New York, pp 1–10.Google Scholar
  45. Knutzon DS, Thompson GA, Radke SE, Johnson WB, Knauf VC, Kridl JC (1992) Modification of Brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene. Proc Natl Acad Sci USA 89: 2624–2628.PubMedCrossRefGoogle Scholar
  46. Kramer M, Sanders RA, Sheehy RE, Melis M, Kuehn M, Hiatt UR (1990) Field Evaluation of Tomatoes with reduced polygalacturonase by antisense RNA. Horticultural Biotechnology, Wiley-Liss, Inc, pp 347-355.Google Scholar
  47. Krystal GW (1992) Regulation of eukaryotic gene expression by naturally occurring antisense RNA. In: Gene Regulation: Biology of Antisense RNA and DNA, Erickson RP, Izant JG (eds), Raven Press Ltd, New York, pp 11–20.Google Scholar
  48. Matzke MA, Matzke AJ (1991) Differential inactivation and methylation of a transgene in plants by two suppressor loci containing homologous sequences. Plant Mol Biol 16: 821–830.PubMedCrossRefGoogle Scholar
  49. Matzke MA, Primig M, Trnovsky J, Matzke AJ (1989) Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. EMBO J 8: 643–649.PubMedGoogle Scholar
  50. McGurl B, Pearce G, Orozco-Cardenas M, Ryan CA (1992) Structure, expression and antisense inhibition of the systemic precursor gene. Science 255: 1570–1573.PubMedCrossRefGoogle Scholar
  51. Mol JN, van der Krol AR, van Tunen AJ, van Blokland R, de Lange P, Stuitje AR (1990) Regulation of plant gene expression by antisense RNA. FEBS Lett 268: 427–430.PubMedCrossRefGoogle Scholar
  52. Mol J, van Blokland R, Kooter J (1991) More about co-suppression. Trends Biotechnol 9: 182–183.CrossRefGoogle Scholar
  53. Müller-Röber B, Sonnewald U, Willmitzer L (1992) Inhibition of the ADP-glucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes. EMBO J 11: 1229–1236.PubMedGoogle Scholar
  54. Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2: 279–289.PubMedGoogle Scholar
  55. Oeller PW, Min-Wong L, Taylor LP, Pike DA, Theologis A (1991) Reversible inhibition of tomato fruit senescene by antisense RNA. Science 254: 437–439.PubMedCrossRefGoogle Scholar
  56. Offringa R, van den Elzen PJ, Hooykaas PJ (1992) Gene targeting in plants using the Agrobacterium vector system. Transgenic Res 1: 114–123.CrossRefGoogle Scholar
  57. Penarrubia L, Aguilar M, Margossian L, Fischer RL (1992) An antisense gene stimulates ethylene hormone production during tomato fruit ripening. The Plant Cell 4: 681–687.PubMedGoogle Scholar
  58. Persson C, Wagner EG, Nordström K (1988) Control of replication of plasmid R1: kinetics of in vitro interaction between the antisense RNA, CopA, and its target, CopT. EMBO J 7: 3279–3288.PubMedGoogle Scholar
  59. Powell PA, Stark DM, Sanders PR, Beachy RN (1989) Protection against tobacco mosaic virus in transgenic plants that express tobacco mosaic virus antisense RNA. Proc Natl Acad Sci USA 86: 6949–6952.PubMedCrossRefGoogle Scholar
  60. Quick WP, Schurr U, Scheibe R, Schulze E-D, Rodermel SR, Bogorad L, Stitt M (1991) Decreased ribulose-1, 5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with “antisense” rbcS. I. Impact on photosynthesis in ambient growth conditions. Planta 183: 542–554.CrossRefGoogle Scholar
  61. Rezian MA, Skene, KG, Ellis JG (1988) Anti-sense RNAs of cucumber mosaic virus in transgenic plants assessed for control of the virus. Plant Mol Biol 11: 463–471.CrossRefGoogle Scholar
  62. Rittner K, Sczakiel G (1991) Identification and analysis of antisense RNA target regions of the human immunodeficiency vims type 1. Nucleic Acids Res 19: 1421–1426.PubMedCrossRefGoogle Scholar
  63. Robert LS, Donaldson PA, Ladaique C, Altosaar I, Amison PG, Fabijanski SF (1989) Antisense RNA inhibition of α-glucuronidase gene expression in transgenic tobacco plants. Plant Mol Biol 13: 399–409.PubMedCrossRefGoogle Scholar
  64. Rodermel SR, Abbott MS, Bogorad L (1988) Nuclear-organelle interactions: nuclear antisense gene inhibits ribulose bisphosphate carboxylase enzyme levels in transformed tobacco plants. Cell 55: 673–681.PubMedCrossRefGoogle Scholar
  65. Rogers JC (1988) RNA complementary to α-amylase mRNA in barley. Plant Mol Biol 11: 125–138.CrossRefGoogle Scholar
  66. Rothstein SJ, Lagrimini LM (1989) Silencing gene expression in plants. Oxford surveys of Plant Molecular and Cell Biology 6: 221–246.Google Scholar
  67. Rothstein SJ, DiMaio J, Strand M, Rice D (1987) Stable and heritable inhibition of the expression of nopaline synthase in tobacco expressing antisense RNA. Proc Natl Acad Sci USA 84: 8439–8443.PubMedCrossRefGoogle Scholar
  68. Sandler SJ, Stayton M, Townsend JA, Ralston ML, Bedbrook JR, Dunsmuir P (1988) Inhibition of gene expression in transformed plants by antisense RNA. Plant Mol Biol 11: 301–310.CrossRefGoogle Scholar
  69. Sarver N, Cantin EM, Chang PS, Zaia JA, Ladne PA, Stephens DA, Rossi JJ (1990) Ribozymes as potential anti-HIV-1 therapeutic agents. Science 247: 1222–1225.PubMedCrossRefGoogle Scholar
  70. Saxena SK, Ackerman EJ (1990) Ribozymes correctly cleave a model substrate and endogenous RNA in vivo. J Biol Chem 265: 17106–17109.PubMedGoogle Scholar
  71. Scanlon KJ, Jiao L, Funato T, Wang W, Tone T, Rossi JJ, Kashani-Sabet M (1991) Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein. Proc Natl Acad Sci USA 88: 10591–10595.PubMedCrossRefGoogle Scholar
  72. Schuch W, Bird CR, Ray J, Smith CJ, Watson CF, Morris PC, Gray JE, Arnold C, Seymour GB, Tucker GA, Grierson D (1989) Control and manipulation of gene expression during tomato fruit ripening. Plant Mol Biol 13: 303–311.PubMedCrossRefGoogle Scholar
  73. Sheehy RE, Kramer M, Hiatt WR (1988) Reduction of polygalacturonase activity in tomato fruit by antisense RNA. Proc Natl Acad Sci USA 85: 8805–8809.PubMedCrossRefGoogle Scholar
  74. Simons RW, Kleckner N (1988) Biological regulation by antisense RNA in prokaryotes. Annu Rev Genet 22: 567–600.PubMedCrossRefGoogle Scholar
  75. Sioud M, Drlica K (1991) Prevention of human immunodeficiency virus type 1 integrase expression in Escherichia coli by a ribozyme. Proc Natl Acad Sci USA 88: 7303–7307.PubMedCrossRefGoogle Scholar
  76. Smith CJ, Watson CF, Ray J, Bird CR, Morris PC, Schuch W, Grierson D (1988) Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes. Nature 334: 724–726.CrossRefGoogle Scholar
  77. Smith CJ, Watson CF, Morris PC, Bird CR, Seymour GB, Gray JE, Arnold C, Tucker A, Schuch W, Harding S, Grierson D (1990a) Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes. Plant Mol Biol 14: 369–379.PubMedCrossRefGoogle Scholar
  78. Smith CJ, Watson CF, Bird CR, Ray J, Schuch W, Grierson D (1990b) Expression of a truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants. Mol Gen Genet 224: 477–481.PubMedCrossRefGoogle Scholar
  79. Spanu P, Reinhardt D, Boiler T (1991) Analysis and cloning of the ethylene-form ing enzyme from tomato by functional expression of its mRNA in Xenopus laevis oocytes. EMBO J 10: 2007–2013.PubMedGoogle Scholar
  80. Steinecke P, Herget T, Schreier PH (1992) Expression of a chimeric ribozyme gene results in endonucleolytic cleavage of target mRNA and a concomitant reduction of gene expression in vivo. EMBO J 11: 1525–1530.PubMedGoogle Scholar
  81. Stitt M, Quick WP, Schurr U, Schulze E-D, Rodermel SR, Bogorad L (1991) Decreased ribulose-1, 5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with “antisense” rbcS. II. Flux-control coefficients for photosynthesis in varying light, CO2, and air humidity. Planta 183: 555–556.Google Scholar
  82. Stockhaus J, Höfer M, Renger G, Westhoff P, Wydrzynski T, Willmitzer L (1990) Anti-sense RNA efficiently inhibits formation of the 10kd polypeptide of photosystem II in transgenic potato plants: analysis of the role of the 10 kd protein. EMBO J 9: 3013–3021.PubMedGoogle Scholar
  83. Symons RH (1989) Self-cleavage of RNA in the replication of small pathogens of plants and animals. Trends Biochem Sci 14: 445–450.PubMedCrossRefGoogle Scholar
  84. Symons RH (1990) The fascination of low molecular weight pathogenic RNAs. Sem Virology 1: 75–81.Google Scholar
  85. Tabler M, Tsagris M (1990) Viroid replication mechanisms. In: Recognition and Response in Plant-virus Interactions, Fraser RS (ed), NATO ASI Series, vol. H41: 185-205.Google Scholar
  86. Tabler M, Tsagris M (1991) Catalytic antisense RNAs produced by incorporating ribozyme cassettes into cDNA. Gene 108: 175–183.PubMedCrossRefGoogle Scholar
  87. Taylor JE, Tucker GA, Lasslett Y, Smith CJ, Arnold CM, Watson CF, Schuch W, Grierson D, Roberts JA (1990) Polygalacturonase expression during leaf abscission of normal and transgenic tomato plants. Planta 183: 133–138.Google Scholar
  88. Tieman DM, Harriman RW, Ramamohan G, Handa AK (1992) An antisense pectin methylesterase gene alters pectin chemistry and soluble solids in tomato fruit Plant Cell 4: 667–679.PubMedGoogle Scholar
  89. Uhlmann E, Peyman A (1990) Antisense oligonucleotides: a new therapeutic principle. Chem Rev 90: 543–584.CrossRefGoogle Scholar
  90. Van der Krol AR, Lenting PE, Veenstra J, van der Meer IM, Koes RE, Gerats AG, Mol JN, Stuitje AR (1988a) An anti-sense chalcone synthase gene in transgenic plants inhibits flower pigmentation. Nature 333: 866–869.CrossRefGoogle Scholar
  91. Van der Krol AR, Mol JN, Stuitje AR (1988b) Modulation of eukaryotic gene expression by complementary RNA or DNA sequences. BioTechniques 6: 958–976.PubMedGoogle Scholar
  92. Van der Krol AR, Mol JN, Stuitje AR (1988c) Antisense genes in plants: an overview. Gene 72: 45–50.PubMedCrossRefGoogle Scholar
  93. Van der Krol AR, Mur LA, de Lange P, Gerats AG, Mol JN, Stuitje AR (1990a) Antisense chalcone synthase genes in petunia: visualization of variable transgene expression. Mol Gen Genet 220: 204–212.CrossRefGoogle Scholar
  94. Van der Krol AR, Mur LA, de Lange P, Mol JN, Stuitje AR (1990b) Inhibition of flower pigmentation by antisense CHS genes: promoter and minimal sequence requirements for the antisense effect. Plant Mol Biol 14: 457–466.PubMedCrossRefGoogle Scholar
  95. Van der Krol AR, Mur LA, Beld M, Mol JN, Stuitje AR (1990c) Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2: 291–299.PubMedGoogle Scholar
  96. Van der Meer IM, Stam ME, van Tunen AJ, Mol JN, Stuitje AR (1992) Antisense inhibition of flavonoid biosynthesis in petunia anthers results in male sterility. Plant Cell 4: 253–262.PubMedGoogle Scholar
  97. Visser RG, Somhorst I, Kuipers GJ, Ruys NJ, Feenstra WJ, Jacobsen E (1991) Inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs. Mol Gen Genet 225: 289–296.PubMedCrossRefGoogle Scholar
  98. Wickstrom E (1992) Antisense DNA therapeutics neutral analogs and their stereochemistry. In: Gene Regulation: Biology of Antisense RNA and DNA, Erickson RP, Izant JG (eds), Raven Press Ltd, New York, pp 119–132.Google Scholar

Copyright information

© Springer Science+Business Media New York 1993

Authors and Affiliations

  • Martin Tabler
    • 1
  1. 1.Institute of Molecular Biology and BiotechnologyFoundation for Research and TechnologyHeraklionGreece

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