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CYP82E4-mediated nicotine to nornicotine conversion in tobacco is regulated by a senescence-specific signaling pathway

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Abstract

Nicotine to nornicotine conversion in tobacco (Nicotiana tabacum L.) is regulated by an unstable converter locus which in its activated state gives rise to a high nornicotine, low nicotine phenotype in the senescing leaves. In plants that carry the high nornicotine trait, nicotine conversion is primarily catalyzed by a cytochrome P450 protein, designated CYP82E4 whose transcription is strongly upregulated during leaf senescence. To further investigate the regulation of CYP82E4 expression, we examined the spatiotemporal distribution and the stress- and signaling molecule-elicited expression patterns of CYP82E4 using alkaloid analysis and a fusion construct between the 2.2 kb upstream regulatory region of CYP82E4 and the β-glucurodinase (GUS) gene. Histochemical and fluorometric analyses of GUS expression revealed that the CYP82E4 promoter confers high levels of expression in the senescing leaves and flowers, and in the green stems of young and mature plants, but only very low activity was detected in the roots. In the leaves, GUS activity was strongly correlated with the progression of senescence. Treatments of leaf tissue with various signaling molecules including abscisic acid, ethylene, jasmonic acid, salicylic acid and yeast extract; and stresses, such as drought, wounding and tobacco mosaic virus infection did not enhance nicotine conversion or GUS activity in the green leaves, but an increase in CYP82E4 expression was observed in response to ethylene- or tobacco mosaic virus-induced senescence. These results suggest that the expression of CYP82E4 is senescence-specific in the leaves and the use of the CYP82E4 promoter could provide a valuable tool for regulating gene expression in the senescing leaves.

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Abbreviations

ABA:

Abscisic acid

GUS:

β-Glucurodinase

JA:

Jasmonic acid

MUG:

4-Methylumbelliferyl β-d-glucuronide

P450:

Cytochrome P450

SAG:

Senescence-associated gene

TMV:

Tobacco mosaic virus

References

  • Baldwin IT, Ohnmeiss TE (1993) Alkaloidal responses to damage in Nicotiana native to North America. J Chem Ecol 19:1143–1153

    Article  CAS  Google Scholar 

  • Baldwin IT, Schmelz EA, Ohnmeiss TE (1994) Wound-induced changes in root and shoot jasmonic acid pools correlate with induced nicotine synthesis in Nicotiana sylvestris Spegazzini and Comes. J Chem Ecol 20:2139–2157

    Article  CAS  Google Scholar 

  • Becker W, Apel K (1993) Differences in gene-expression between natural and artificially induced leaf senescence. Planta 189:74–79

    Article  CAS  Google Scholar 

  • Beyene G, Foyer CH, Kunert KJ (2006) Two new cysteine proteinases with specific expression patterns in mature and senescent tobacco (Nicotiana tabacum L.) leaves. J Exp Bot 57:1431–1443

    Article  PubMed  CAS  Google Scholar 

  • Buchanan-Wollaston V (1994) Isolation of cDNA clones for genes that are expressed during leaf senescence in Brassica napus – identification of a gene encoding a senescence-specific metallothionein-like protein. Plant Physiol 105:839–846

    Article  PubMed  CAS  Google Scholar 

  • Buchanan-Wollaston V (1997) The molecular biology of leaf senescence. J Exp Bot 48:181–199

    Article  Google Scholar 

  • Butt A, Mousley C, Morris K, Beynon J, Can C, Holub E, Greenberg JT, Buchanan-Wollaston V (1998) Differential expression of a senescence-enhanced metallothionein gene in Arabidopsis in response to isolates of Peronospora parasitica and Pseudomonas syringae. Plant J 16:209–221

    Article  PubMed  CAS  Google Scholar 

  • Chakrabarti M, Meekins KM, Gavilano LB, Siminszky B (2007) Inactivation of the cytochrome P450 gene CYP82E2 by degenerative mutations was a key event in the evolution of the alkaloid profile of modern tobacco. New Phytol 175:565–574

    Article  PubMed  CAS  Google Scholar 

  • Chernikova T, Robinson J, Lee E, Mulchi C (2000) Ozone tolerance and antioxidant enzyme activity in soybean cultivars. Photosynth Res 64:15–26

    Article  PubMed  CAS  Google Scholar 

  • Dawson RF (1942) Accumulation of nicotine in reciprocal grafts of tomato and tobacco. Am J Bot 29:66–71

    Article  CAS  Google Scholar 

  • Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5:199–206

    Article  PubMed  CAS  Google Scholar 

  • Fannin FF, Bush LP (1992) Nicotine demethylation in Nicotiana. Med Sci Res 380:33–41

    Google Scholar 

  • Fukuda H (2000) Programmed cell death of tracheary elements as a paradigm in plants. Plant Mol Biol 44:245–253

    Article  PubMed  CAS  Google Scholar 

  • Gan S, Amasino RM (1995) Inhibition of leaf senescence by autoregulated production of cytokinin. Science 270:1986–1988

    Article  PubMed  CAS  Google Scholar 

  • Gan SS, Amasino RM (1997) Making sense of senescence – molecular genetic regulation and manipulation of leaf senescence. Plant Physiol 113:313–319

    PubMed  CAS  Google Scholar 

  • Gavilano L, Siminszky B (2007) Isolation and characterization of the cytochrome P450 gene CYP82E5v2 that mediates nicotine to nornicotine conversion in the green leaves of tobacco. Plant Cell Physiol 48:1567–1574

    Article  PubMed  CAS  Google Scholar 

  • Gavilano LB, Coleman LP, Burnley L, Bowman ML, Kalengamaliro NE, Hayes A, Bush LP, Siminszky B (2006) Genetic engineering of Nicotiana tabacum (L.) for reduced nornicotine content. J Agric Food Chem 54:9071–9078

    Article  PubMed  CAS  Google Scholar 

  • Gavilano LB, Coleman NP, Bowen SW, Siminszky B (2007) Functional analysis of nicotine demethylase genes reveals insights into the evolution of modern tobacco. J Biol Chem 282:249–256

    Article  PubMed  CAS  Google Scholar 

  • Grbic V, Bleecker AB (1995) Ethylene regulates the timing of leaf senescence in Arabidopsis. Plant J 8:595–602

    Article  CAS  Google Scholar 

  • Halitschke R, Baldwin IT (2003) Antisense LOX expression increases herbivore performance by decreasing defense responses and inhibiting growth-related transcriptional reorganization in Nicotiana attenuata. Plant J 36:794–807

    Article  PubMed  CAS  Google Scholar 

  • Hashimoto T, Yamada Y (1994) Alkaloid biogenesis – molecular aspects. Annu Rev Plant Phys 45:257–285

    CAS  Google Scholar 

  • Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res 27:297–300

    Article  PubMed  CAS  Google Scholar 

  • Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general-method for transferring genes into plants. Science 227:1229–1231

    Article  CAS  Google Scholar 

  • Kimura Y, Tosa Y, Shimada S, Sogo R, Kusaba M, Sunaga T, Betsuyaku S, Eto Y, Nakayashiki H, Mayama S (2001) OARE-1, a Ty1-copia retrotransposon in oat activated by abiotic and biotic stresses. Plant Cell Physiol 42:1345–1354

    Article  PubMed  CAS  Google Scholar 

  • Koltunow AM, Truettner J, Cox KH, Wallroth M, Goldberg RB (1990) Different temporal and spatial gene expression patterns occur during anther development. Plant Cell 2:1201–1224

    Article  PubMed  CAS  Google Scholar 

  • Latchman DS (1998) Eukaryotic transcription factors. Academic Press, San Diego

    Google Scholar 

  • Lim PO, Kim HJ, Gil Nam H (2007) Leaf senescence. Annu Rev Plant Biol 58:115–136

    Article  PubMed  CAS  Google Scholar 

  • Lohman KN, Gan SS, John MC, Amasino RM (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana. Physiol Plant 92:322–328

    Article  CAS  Google Scholar 

  • Mann TJ, Weybrew JA, Matzinger DF, Hall JL (1964) Inheritance of the conversion of nicotine to nornicotine in varieties of Nicotiana tabacum L. and related amphidiploids. Crop Sci 4:349–353

    Article  CAS  Google Scholar 

  • Miao Y, Laun T, Zimmermann P, Zentgraf U (2004) Targets of the WRKY53 transcription factor and its role during leaf senescence in Arabidopsis. Plant Mol Biol 55:853–867

    PubMed  CAS  Google Scholar 

  • Pasquali G, Erven ASW, Ouwerkerk PBF, Menke FLH, Memelink J (1999) The promoter of the strictosidine synthase gene from periwinkle confers elicitor-inducible expression in transgenic tobacco and binds nuclear factors GT-1 and GBF. Plant Mol Biol 39:1299–1310

    Article  PubMed  CAS  Google Scholar 

  • Riah O, Dousset JC, Courriere P, Baziard-Mouysset G, Ecalle R (1997) Synthesis of cotinine and cotinine N-oxide: evaluation of their interaction with nicotine in the insecticidal activity. Nat Prod Lett 11:37–45

    CAS  Google Scholar 

  • Saitoh F, Noma M, Kawashima N (1985) The alkaloid contents of 60 Nicotiana species. Phytochemistry 24:477–480

    Article  CAS  Google Scholar 

  • Samuel G (1931) Some experiments on inoculating methods with plant viruses, and on local lesions. Ann Appl Biol 18:494–507

    Article  Google Scholar 

  • Shi HZ, Kalengamaliro NE, Krauss MR, Hempfling WP, Gadani F (2003) Stimulation of nicotine demethylation by NaHCO3 treatment using greenhouse-grown burley tobacco. J Agric Food Chem 51:7679–7683

    Article  PubMed  CAS  Google Scholar 

  • Siegler EH, Bowen CV (1946) Toxicity of nicotine, nornicotine, and anabasine to codling moth larvae. J Econ Entomol 39:673–674

    CAS  Google Scholar 

  • Siminszky B, Gavilano L, Bowen SW, Dewey RE (2005) Conversion of nicotine to nornicotine in Nicotiana tabacum is mediated by CYP82E4, a cytochrome P450 monooxygenase. Proc Natl Acad Sci USA 102:14919–14924

    Article  PubMed  CAS  Google Scholar 

  • Sisson VA, Severson RF (1990) Alkaloid composition of the Nicotiana species. Beitr Tabakforsch 14:327–339

    CAS  Google Scholar 

  • Szabo E, Thelen A, Petersen M (1999) Fungal elicitor preparations and methyl jasmonate enhance rosmarinic acid accumulation in suspension cultures of Coleus blumei. Plant Cell Rep 18:485–489

    Article  CAS  Google Scholar 

  • Thurston R, Smith WT, Cooper BP (1966) Alkaloid secretion by trichomes of Nicotiana species and resistance to aphids. Entomol Exp Appl 9:428–432

    Article  CAS  Google Scholar 

  • Weaver LM, Gan SS, Quirino B, Amasino RM (1998) A comparison of the expression patterns of several senescence-associated genes in response to stress and hormone treatment. Plant Mol Biol 37:455–469

    Article  PubMed  CAS  Google Scholar 

  • Weinzierl ROJ (1999) Mechanisms of gene expression. Imperial College Press, London

    Google Scholar 

  • Wernsman EA, Matzinger DF (1968) Time and site of nicotine conversion in tobacco. Tob Sci 12:226–228

    CAS  Google Scholar 

  • Whitham S, Dineshkumar SP, Choi D, Hehl R, Corr C, Baker B (1994) The product of the tobacco mosaic-virus resistance gene-N: similarity to toll and the interleukin-1 receptor. Cell 78:1101–1115

    Article  PubMed  CAS  Google Scholar 

  • Xu D, Shen Y, Chappell J, Cui M, Nielsen M (2007) Biochemical and molecular characterizations of nicotine demethylase in tobacco. Physiol Plant 129:307–319

    Article  CAS  Google Scholar 

  • Yamamoto I, Soeda Y, Kamimura H, Yamamoto R (1968) Studies on nicotinoids as an insecticide .7. Cholinesterase inhibition by nicotinoids and pyridylalkylamines – its significance to mode of action. Agric Biol Chem 32:1341–1348

    CAS  Google Scholar 

  • Yang PZ, Chen CH, Wang ZP, Fan BF, Chen ZX (1999) A pathogen- and salicylic acid-induced WRKY DNA-binding activity recognizes the elicitor response element of the tobacco class I chitinase gene promoter. Plant J 18:141–149

    Article  CAS  Google Scholar 

  • Ye ZH, Droste DL (1996) Isolation and characterization of cDNAs encoding xylogenesis-associated and wounding-induced ribonucleases in Zinnia elegans. Plant Mol Biol 30:697–709

    Article  PubMed  CAS  Google Scholar 

  • Ye ZH, Varner JE (1996) Induction of cysteine and serine proteases during xylogenesis in Zinnia elegans. Plant Mol Biol 30:1233–1246

    Article  PubMed  CAS  Google Scholar 

  • Yin SH, Mei L, Newman J, Back K, Chappell J (1997) Regulation of sesquiterpene cyclase gene expression – characterization of an elicitor- and pathogen-inducible promoter. Plant Physiol 115:437–451

    Article  PubMed  CAS  Google Scholar 

  • Yoshida S (2003) Molecular regulation of leaf senescence. Curr Opin Plant Biol 6:79–84

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Earl Wernsman for the DH98-325-5 and DH98-325-6 tobacco seed. We are grateful to Lowell Bush and Xiaolong Li for alkaloid analysis. We also thank Robert Geneve and Sharyn Perry for help with microscopy, Sharon Kester for ethylene analysis and Steve Lommel for screening the tobacco BAC library. This work was supported in part by Philip Morris USA.

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Authors and Affiliations

  1. Department of Plant and Soil Sciences, University of Kentucky, 409 Plant Sci Bldg, 1405 Veterans Drive, Lexington, KY, 40546-0312, USA

    Manohar Chakrabarti, Nicholas P. Coleman, Karen M. Meekins & Balazs Siminszky

  2. Department of Crop Science, North Carolina State University, Raleigh, NC, 27695, USA

    Steven W. Bowen & Ralph E. Dewey

Authors
  1. Manohar Chakrabarti
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  2. Steven W. Bowen
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  3. Nicholas P. Coleman
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  4. Karen M. Meekins
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  5. Ralph E. Dewey
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  6. Balazs Siminszky
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Correspondence to Balazs Siminszky.

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Chakrabarti, M., Bowen, S.W., Coleman, N.P. et al. CYP82E4-mediated nicotine to nornicotine conversion in tobacco is regulated by a senescence-specific signaling pathway. Plant Mol Biol 66, 415–427 (2008). https://doi.org/10.1007/s11103-007-9280-6

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  • DOI: https://doi.org/10.1007/s11103-007-9280-6

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