Skip to main content
SpringerLink
Log in

Identification of SNPs in nicotine biosynthesis related genes by targeted re-sequencing of TILLING population and germplasm with varying nicotine levels in tobacco

  • Published:
Euphytica Aims and scope Submit manuscript

Abstract

Nicotine is an important plant metabolite having high commercial value due to wide applications as insecticide and therapeutic agent apart from its parasympathomimetic properties. In addition, nicotine biosynthesis serves as model pathway to study the genetic regulation of secondary metabolites in plants. In this study, we report the identification of genetic variations in terms of SNPs in the nicotine biosynthesis related genes. Targeted re-sequencing had identified a total of 50 natural variations across 19 genes in the nicotine biosynthesis related genes. In addition, mutations (n = 32) were also identified in TILLING mutant population of tobacco. A non-synonymous SNP in PMT2 gene leads to conversion of H25Q in PMT2 protein. The change results in formation of a highly conserved N terminal repeat motif in PMT2 protein. Germplasm regression analysis revealed that this particular SNP contributes 3 % of total variation observed for nicotine content. It was also observed that all the germplasm lines with the minor allele had nicotine content higher than the commercial cultivar Kanchan (K326). Functional characterisation of the natural/induced variations identified in this study would be of use to understand the genetic regulation of secondary metabolites and for development of pre-breeding lines with varied content of nicotine.

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.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

SNP:

Single nucleotide polymorphism

TILLING:

Targeting induced local lesions in genomes

References

  • Baldwin IT (1996) Methyl jasmonate-induced nicotine production in Nicotiana attenuata: inducing defenses in the field without wounding. Entomol Exp Appl 80:213–220

    Article  CAS  Google Scholar 

  • Biostoff S, Reinhard N, Reva V, Brandt W, Drager B (2009) Evolution of putrescine N-methyl transferase from spermidine synthase demanded alterations in substrate binding. FEBS Lett 583:3367–3374

    Article  Google Scholar 

  • Booker CJ, Bedmutha R, Vogel T, Gloor A, Xu R, Ferrante L, Yeung KC, Scott IM, Conn KL, Berruti F, Briens C (2010) Experimental investigations into the insecticidal, fungicidal, and bactericidal properties of pyrolysis bio-oil from tobacco leaves using a fluidized bed pilot plant. Ind Eng Chem Res 49:10074–10079

    Article  CAS  Google Scholar 

  • Bortolotti C, Cordeiro A, Alcázar R, Borrell A, Culiañez-Macià FA, Tiburcio AF, Altabella T (2004) Localization of arginine decarboxylase in tobacco plants. Physiol Plant 120:84–92

    Article  CAS  PubMed  Google Scholar 

  • Dawson RF (1941) The localisation of the nicotine synthetic mechanism in the tobacco plant. Science 94:396–397

    Article  CAS  PubMed  Google Scholar 

  • De Sutter V, Vanderhaeghen R, Tilleman S, Lammertyn F, Vanhoutte I, Karimi M, Inzé D, Goossens A, Hilson P (2005) Exploration of jasmonate signaling via automated and standardized transient expression assays in tobacco cells. Plant J 44:1065–1076

    Article  PubMed  Google Scholar 

  • Dewey RE, Xie J (2013) Molecular genetics of alkaloid biosynthesis in Nicotiana tabacum. Phytochemistry 94:10–27

    Article  CAS  PubMed  Google Scholar 

  • Hashimoto T, Tamaki K, Suzuki K, Yamada Y (1998a) Molecular cloning of plant spermidine synthases. Plant Cell Physiol 39:73–79

    Article  CAS  PubMed  Google Scholar 

  • Hashimoto T, Shoji T, Mihara T, Oguri H, Tamaki K, Suzuki K, Yamada Y (1998b) Intraspecific variability of the tandem repeats in Nicotiana putrescine N methyltransferases. Plant Mol Biol 37:25–37

    Article  CAS  PubMed  Google Scholar 

  • Hasimoto T, Yamada Y (1994) Alkaloid biosynthesis–molecular aspects. Annu Rev Plant Physiol Plant Mol Biol 45:257–285

    Article  Google Scholar 

  • Hibi N, Higashiguchi S, Hashimoto T, Yamada Y (1994) Gene expression in tobacco low-nicotine mutants. Plant Cell 6:723–735

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Hildreth SB, Gehman EA, Yang H, Lu RH, Ritesh KC, Harich KC, Yu S, Lin J, Sandoe JL, Okumoto S, Murphy AS, Jelesko JG (2011) Tobacco nicotine uptake permease (NUP1) affects alkaloid metabolism. Proc Natl Acad Sci USA 108:18179–18184

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Julio E, Laporte F, Reis S, Rothan C, Dorlhac de Borne F (2008) Reducing the content of nornicotine in tobacco via targeted mutation breeding. Mol Breed 21:369–381

    Article  CAS  Google Scholar 

  • Kajikawa M, Shoji T, Kato A, Hashimoto T (2011) Vacuole-localized berberine bridge enzyme-like proteins are required for a late step of nicotine biosynthesis in tobacco. Plant Physiol 155:2010–2022

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Katoh A, Shoji T, Hashimoto T (2007) Molecular cloning of N-methylputrescine Oxidase from tobacco. Plant Cell Physiol 48:550–554

    Article  CAS  PubMed  Google Scholar 

  • Kidd SK, Melillo AA, Lu RH, Reed DG, Kuno N, Uchida K, Furuya M, Jelesko JG (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 60:699–716

    Article  CAS  PubMed  Google Scholar 

  • Li D, Lewis R, Jack A, Dewey R, Bowen S, Miller RD (2011) Development of CAPS and dCAPS markers for CYP82E4, CYP82E5v2 and CYP82E10 gene mutants reducing nicotine to nornicotine conversion in tobacco. Mol Breed 29:589–599

    Article  Google Scholar 

  • Loke P, Sim TS (1999) Glutamine-230 influences enzyme solubility but not catalysis in Streptomyces clavuligerus isopenicillin N synthase. FEMS Microbiol Lett 173:439–443

    Article  CAS  PubMed  Google Scholar 

  • Masouleh AK, Waters DL, Reinke RF, Henry RJ (2011) Discovery of polymorphisms in starch related genes in rice germplasm by amplification of pooled DNA and deeply parallel sequencing. Plant Biotechnol J 9:1074–1085

    Article  Google Scholar 

  • Morita M, Shitan N, Sawada K, Van Montagu MC, Inzé D, Rischer H, Goossens A, Oksman-Caldentey KM, Moriyama Y, Yazaki K (2009) Vacuolar transport of nicotine is mediated by a multidrug and toxic compound extrusion (MATE) transporter in Nicotiana tabacum. Proc Natl Acad Sci USA 106:2447–2452

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Powledge TM (2004) Nicotine as therapy. PLoS Biol 2:e404

    Article  PubMed Central  PubMed  Google Scholar 

  • Reddy TV, Dwivedi S, Sharma NK (2012) Development of TILLING by sequencing platform towards enhanced leaf yield in tobacco. Ind Crops Prod 40:324–335

    Article  CAS  Google Scholar 

  • Riechers DE, Timko MP (1999) Structure and expression of the gene family encoding putrescine N-methyltransferase in Nicotiana tabacum: new clues to the evolutionary origin of cultivated tobacco. Plant Mol Biol 41:387–401

    Article  CAS  PubMed  Google Scholar 

  • Roeder S, Dreschler K, Wirtz M, Cristescu SM, van Harren FJ, Hell R, Piechulla B (2009) SAM levels, gene expression of SAM synthetase, methionine synthase and ACC oxidase, and ethylene emission from N. suaveolens flowers. Plant Mol Biol 70:535–546

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ryan SM, Cane KA, DeBoer KD, Sinclair SJ, Brimblecombe R, Hamill JD (2012) Structure and expression of the quinolinate phosphoribosyltransferase (QPT) gene family in Nicotiana. Plant Sci 188–189:102–110

    Article  PubMed  Google Scholar 

  • Shoji T, Ogawa T, Hashimoto T (2008) Jasmonate-induced nicotine formation in tobacco is mediated by tobacco COI1 and JAZ genes. Plant Cell Physiol 49:1003–1012

    Article  CAS  PubMed  Google Scholar 

  • Shoji T, Inai K, Yazaki Y, Sato Y, Takase H, Shitan N, Yazaki K, Goto Y, Toyooka K, Matsuoka K, Hashimoto T (2009) Multidrug and toxic compound extrusion-type transporters implicated in vacuolar sequestration of nicotine in tobacco roots. Plant Physiol 149:708–718

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Shoji T, Kajikawa M, Hashimoto T (2010) Clustered transcription factor genes regulate nicotine biosynthesis in tobacco. Plant Cell 22:3390–3409

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Sierro N, Battey JN, Ouadi S, Bakaher N, Bovet L, Willig A, Goepfert S, Peitsch MC, Ivanov NV (2014) The tobacco genome sequence and its comparison with those of tomato and potato. Nat Commun 5:3833

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Steppuhn A, Gase K, Krock B, Halitschke R, Baldwin IT (2004) Nicotine’s defensive function in nature. PLoS Biol 2:1074–1080

    Article  CAS  Google Scholar 

  • Tiburcio AF, Galston AW (1986) Arginine decarboxylase as the source of putrescine for tobacco alkaloids. Phytochemistry 25:107–110

    Article  CAS  PubMed  Google Scholar 

  • Todd AT, Liu E, Polvi SL, Pammett RT, Page JE (2010) A functional genomics screen identifies diverse transcription factors that regulate alkaloid biosynthesis in Nicotiana benthamiana. Plant J 62:589–600

    Article  CAS  PubMed  Google Scholar 

  • Valleau WD (1949) Breeding low-nicotine tobacco. J Agric Res 78:171–181

    CAS  Google Scholar 

  • Wang J, Sheehan M, Brookman H, Timko MP (2000) Characterization of cDNAs differentially expressed in roots of tobacco (Nicotiana tabacum cv Burley 21) during the early stages of alkaloid biosynthesis. Plant Sci 158:19–32

    Article  CAS  PubMed  Google Scholar 

  • Wi SJ, Kim WT, Park KY (2006) Overexpression of carnation S-adenosylmethionine decarboxylase gene generates a broad-spectrum tolerance to abiotic stresses in transgenic tobacco plants. Plant Cell Rep 25:1111–1121

    Article  CAS  PubMed  Google Scholar 

  • Xu B, Sheehan MJ, Timko MP (2004) Differential induction of ornithine decarboxylase (ODC) gene family members in transgenic tobacco (Nicotiana tabacum L. cv. Bright Yellow 2) cell suspensions by methyl-jasmonate treatment. Plant Growth Regul 44:101–116

    Article  CAS  Google Scholar 

  • Zhang HB, Bokowiec MT, Rushton PJ, Han SC, Timko MP (2012) Tobacco transcription factors NtMYC2a and NtMYC2b form nuclear complexes with the NtJAZ1 repressor and regulate multiple jasmonate-inducible steps in nicotine biosynthesis. Mol Plant 5:73–84

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Authors would like to acknowledge Dr. Lakshmanan and Mr. Ramaswamy for critical reviewing of the project. Acknowledgements are duly rendered to Drs Samresh Dwivedi, Gurumurthy, Mani, Eswar Reddy, and Chakravarthi for suggestions during the course of research work and critical comments on manuscript. The technical help rendered by Mrs. Philomine Beseant in PCR amplification is greatly acknowledged. Authors wish to thank Mr.Uma Mahesh for estimation of nicotine content.

Author information

Authors and Affiliations

  1. Corporate R&D (Agriscience), ITC Life Sciences and Technology Centre, ITC Limited, No. 3, I Main, I Phase, Peenya Industrial Area, Bangalore, 560058, India

    Thamalampudi Venkata Reddy & G. V. S. Saiprasad

Authors
  1. Thamalampudi Venkata Reddy
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. G. V. S. Saiprasad
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Thamalampudi Venkata Reddy.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 46 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Reddy, T.V., Saiprasad, G.V.S. Identification of SNPs in nicotine biosynthesis related genes by targeted re-sequencing of TILLING population and germplasm with varying nicotine levels in tobacco. Euphytica 203, 659–671 (2015). https://doi.org/10.1007/s10681-014-1300-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10681-014-1300-4

Keywords

Search

Navigation