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Molecular Breeding

, Volume 21, Issue 3, pp 369–381 | Cite as

Reducing the content of nornicotine in tobacco via targeted mutation breeding

  • Emilie Julio
  • Frédéric Laporte
  • Stéphanie Reis
  • Christophe Rothan
  • François Dorlhac de Borne
Article

Abstract

Cultivated tobacco produces secondary alkaloids involved in the formation of nitrosamines with health concerns. The recent identification of target genes in nicotine and nornicotine biosynthetic pathways now allows biotechnological approaches for their control. We demonstrate here that mutation breeding can be used as an alternative to genetically modified (GM) plants for generating nornicotine-free tobacco. Ten alleles of the NtabCYP82E4 gene (nicotine N-demethylase) were identified by screening 1,311 M2 families of tobacco ethylmethane sulphonate (EMS) mutants. Alkaloid analysis indicated that the nornicotine contents of homozygous M2 plants carrying nonsense or missense alleles of NtabCYP82E4 were very low or near-null. Backcrossing with tobacco elite varieties yielded BC1 plants phenotypically undistinguishable from parental lines. This major objective of tobacco breeders in the last few decades could be reached in a period of less than 1.5 years, including the creation of highly mutagenised tobacco mutant collections and the detection of mutated alleles using a simple and versatile detection technology (capillary electrophoresis-single strand conformation polymorphism, CE-SSCP) accessible to most breeding companies and crop species.

Keywords

CE-SSCP Mutagenesis Nicotiana tabacum Nornicotine 

Notes

Acknowledgements

The authors are grateful to Prof. Avi Levy (Weizmann Institute, IL) for the critical reading of the manuscript and helpful suggestions. We thank Béatrice Denoyes-Rothan for help with the statistical analyses. Special thanks go to members of the Altadis Research Group (Bergerac, France) for their excellent assistance with the plant culture and alkaloid analysis.

References

  1. Adams KL, Wendel JF (2005) Polyploidy and genome evolution in plants. Curr Opin Plant Biol 8:135–141PubMedCrossRefGoogle Scholar
  2. Bannwarth S, Procaccio V, Paquis-Flucklinger V (2006) Rapid identification of unknown heteroplasmic mutations across the entire human mitochondrial genome with mismatch-specific Surveyor Nuclease. Nat Protoc 1:2037–2047PubMedCrossRefGoogle Scholar
  3. Bjørheim J, Ekstrøm PO (2005) Review of denaturant capillary electrophoresis in DNA variation analysis. Electrophoresis 26:2520–2530PubMedCrossRefGoogle Scholar
  4. Bottley A, Xia GM, Koebner RMD (2006) Homoeologous gene silencing in hexaploid wheat. Plant J 47:897–906PubMedCrossRefGoogle Scholar
  5. Bush LP, Cui M, Shi H et al (2001) Formation of tobacco-specific nitrosamines in air-cured tobacco. Rec Adv Tob Sci 27:23–46Google Scholar
  6. Caldwell DG, McCallum N, Shaw P et al (2004) A structured mutant population for forward and reverse genetics in Barley (Hordeum vulgare L.). Plant J 40:143–150PubMedCrossRefGoogle Scholar
  7. Chintapakorn Y, Hamill JD (2003) Antisense-mediated down-regulation of putrescine N-methyltransferase activity in transgenic Nicotiana tabacum L. can lead to elevated levels of anatabine at the expense of nicotine. Plant Mol Biol 53:87–105PubMedCrossRefGoogle Scholar
  8. Colbert T, Till BJ, Tompa R et al (2001) High-throughput screening for induced point mutations. Plant Physiol 126:480–484PubMedCrossRefGoogle Scholar
  9. Comai L, Henikoff S (2006) TILLING: practical single-nucleotide mutation discovery. Plant J 45:684–694PubMedCrossRefGoogle Scholar
  10. Davies H, Dicks E, Stephens P et al (2006) High throughput DNA sequence variant detection by conformation sensitive capillary electrophoresis and automated peak comparison. Genomics 87:427–432PubMedCrossRefGoogle Scholar
  11. de Roton C, Wiernik A, Wahlberg I et al (2005) Factors influencing the formation of tobacco-specific nitrosamines in French air-cured tobaccos in trials and at the farm level. Beitr Tabakforsch Int 21:305–320Google Scholar
  12. Delon R, Poisson C, Bardon JC et al (1999) Les nicotianées en collection à l’Institut du Tabac, 3rd edn. Annales du Tabac, SEITA, ParisGoogle Scholar
  13. Dickerson TJ, Janda KD (2002) A previously undescribed chemical link between smoking and metabolic disease. Proc Natl Acad Sci USA 99:15084–15088PubMedCrossRefGoogle Scholar
  14. Doi K, Doi H, Noiri E et al (2004) High-throughput single nucleotide polymorphism typing by fluorescent single-strand conformation polymorphism analysis with capillary electrophoresis. Electrophoresis 25:833–838PubMedCrossRefGoogle Scholar
  15. Ellis LA, Taylor CF, Taylor GR (2000) A comparison of fluorescent SSCP and denaturing HPLC for high throughput mutation scanning. Hum Mutat 15:556–564PubMedCrossRefGoogle Scholar
  16. Fayeulle JP, de Salles de Hys L, Duméry B et al (1992) La nornicotine chez les variétés industrielles de tabac. I: état des connaissances, suivi du caractère et efforts visant à son élimination par sélection. Annales du Tabac, SEITA, Bergerac, France, Sect 2–24Google Scholar
  17. Fernie AR, Tadmor Y, Zamir D (2006) Natural genetic variation for improving crop quality. Curr Opin Plant Biol 9:196–202PubMedCrossRefGoogle Scholar
  18. Gavilano LB, Coleman NP, Burnley LE et al (2006) Genetic engineering of Nicotiana tabacum for reduced nornicotine content. J Agric Food Chem 54:9071–9078PubMedCrossRefGoogle Scholar
  19. Gavilano LB, Coleman NP, Bowen SW et al (2007) Functional analysis of nicotine demethylase genes reveals insights into the evolution of modern tobacco. J Biol Chem 282:249–256PubMedCrossRefGoogle Scholar
  20. Greene EA, Codomo CA, Taylor NE et al (2003) Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics 164:731–740PubMedGoogle Scholar
  21. Gu Z, Steinmetz LM, Gu X et al (2003) Role of duplicate genes in genetic robustness against null mutations. Nature 421:63–66PubMedCrossRefGoogle Scholar
  22. Hashimoto T, Yamada Y (1994) Alkaloid biogenesis: molecular aspects. Annu Rev Plant Physiol Plant Mol Biol 45:257–285Google Scholar
  23. Hecht SS (2003) Tobacco carcinogens, their biomarkers and tobacco-induced cancer. Nat Rev Cancer 3:733–744PubMedCrossRefGoogle Scholar
  24. Henikoff S, Comai L (2003) Single-nucleotide mutations for plant functional genomics. Annu Rev Plant Biol 54:375–401PubMedCrossRefGoogle Scholar
  25. Hoffmann D, Djordjevic MV, Hoffmann I (1997) The changing cigarette. Prev Med 26:427–434PubMedCrossRefGoogle Scholar
  26. Kashkush K, Feldman M, Levy AA (2002) Gene loss, silencing and activation in a newly synthesized wheat allotetraploid. Genetics 160:1651–1659PubMedGoogle Scholar
  27. Kidd SK, Melillo AA, Lu R-H et al (2006) The A and B loci in tobacco regulate a network of stress response genes, few of which are associated with nicotine biosynthesis. Plant Mol Biol 60:699–716PubMedCrossRefGoogle Scholar
  28. Koornneef M, Dellaert LWM, van den Veen JH (1982) EMS- and radiation-induced mutation frequencies at individual loci in Arabidopsis thaliana (L.) Heynh. Mutat Res 93:109–123PubMedGoogle Scholar
  29. Madlung A, Comai L (2004) The effect of stress on genome regulation and structure. Ann Bot 94:481–495PubMedCrossRefGoogle Scholar
  30. McCallum CM, Comai L, Greene EA et al (2000) Targeted screening for induced mutations. Nat Biotechnol 18:455–457PubMedCrossRefGoogle Scholar
  31. McGinnis K, Murphy N, Carlson AR et al (2007) Assessing the efficiency of RNA interference for maize functional genomics. Plant Physiol 143:1441–1451PubMedCrossRefGoogle Scholar
  32. Ng PC, Henikoff S (2003) SIFT: predicting amino acid changes that affect protein function. Nucleic Acid Res 31:3812–3814PubMedCrossRefGoogle Scholar
  33. Shaked H, Kashkush K, Ozkan H et al (2001) Sequence elimination and cytosine methylation are rapid and reproducible responses of the genome to wide hybridization and allopolyploidy in wheat. Plant Cell 13:1749–1759PubMedCrossRefGoogle Scholar
  34. Shi H, Kalengamaliro NE, Krauss MR et al (2003) Stimulation of nicotine demethylation by NaHCO3 treatment using greenhouse-grown burley tobacco. J Agric Food Chem 51:7679–7683PubMedCrossRefGoogle Scholar
  35. Siminszky B, Gavilano L, Bowen SW et al (2005) Conversion of nicotine to nornicotine in Nicotiana tabacum is mediated by CYP82E4, a cytochrome P450 monooxygenase. Proc Natl Acad Sci USA 102:14919–14924PubMedCrossRefGoogle Scholar
  36. Slade AJ, Fuerstenberg SI, Loeffler D et al (2005) A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING. Nat Biotechnol 23:75–81PubMedCrossRefGoogle Scholar
  37. Stephens RL, Weybrew JA (1959) Isatin: a color reagent for nornicotine. Tobacco Sci 3:48–51Google Scholar
  38. Tanksley SD, McCouch SR (1997) Seed banks and molecular maps: unlocking genetic potential from the wild. Science 277:1063–1066PubMedCrossRefGoogle Scholar
  39. Till BJ, Reynolds SH, Greene EA et al (2003) Large-scale discovery of induced point mutations with high-throughput TILLING. Genome Res 13:524–530PubMedCrossRefGoogle Scholar
  40. Wang J, Sheehan M, Brookman H et al (2000) Characterization of cDNAs differentially expressed in roots of tobacco (Nicotiana tabacum cv Burley 21) during the early stages of alkaloid biosynthesis. Plant Sci 158:19–32PubMedCrossRefGoogle Scholar
  41. Xu D, Shen Y, Chappell J et al (2007) Biochemical and molecular characterizations of nicotine demethylase in tobacco. Physiol Plant 129:307–319CrossRefGoogle Scholar
  42. Yeung AT, Hattangadi D, Blakesley L et al (2005) Enzymatic mutation detection technologies. Biotechniques 38:749–758PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Emilie Julio
    • 1
  • Frédéric Laporte
    • 2
  • Stéphanie Reis
    • 1
  • Christophe Rothan
    • 2
  • François Dorlhac de Borne
    • 1
  1. 1.Altadis—Institut du TabacBergeracFrance
  2. 2.INRA-UMR 619 Biologie du Fruit, IBVI, INRA & Universités Bordeaux 1 & 2Villenave d’OrnonFrance

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