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Reactive oxygen species regulate alkaloid metabolism in undifferentiated N. tabacum cells

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Abstract

Plants produce an immense number of natural products and undifferentiated cells from various plant tissues have long been considered an ideal source for their synthesis. However, undifferentiated plant cells often either lose their biosynthetic capacity over time or exhibit immediate repression of the required pathways once dedifferentiated. In this study, freshly prepared callus tissue was employed to further investigate the regulation of a natural product pathway in undifferentiated tobacco cells. Putrescine N-methyltransferase (PMT) is a pathway-specific enzyme required in nicotinic alkaloid production in Nicotiana species. Callus derived from transgenic Nicotiana tabacum plants harboring PMT promoter–GUS fusions were used to study factors that influence PMT expression. Under normal callus growth conditions in the presence of light and auxin, PMT promoter activity was strongly repressed. Conversely, dark conditions and the absence of auxin were found to upregulate PMT promoter activity, with light being dominant to the repressive effects of auxin. Since reactive oxygen species (ROS) are known by-products of photosynthesis and have been implicated in signaling, their involvement was investigated in transgenic callus by treatment with the ROS scavenger, dimethylthiourea, or catalase. Under highly repressive conditions for alkaloid synthesis, including normal culture conditions in the light, both ROS scavengers resulted in significant induction of PMT promoter activity. Moreover, treatment of callus with catalase resulted in the upregulation of PMT promoter activity and alkaloid accumulation in this tissue. These results suggest that ROS impact the regulation of the alkaloid pathway in undifferentiated cells and have implications for regulation of the pathway in other plant tissues.

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References

  • Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399

    Article  PubMed  CAS  Google Scholar 

  • Baldwin IT (1989) Mechanism of damage-induced alkaloid production in wild tobacco. J Chem Ecol 15:1661–1680

    Article  CAS  Google Scholar 

  • Baldwin IT (1996) Allometric limits to the induced accumulation of nicotine in native tobacco. Plant Species Biol 11:107–114

    Article  Google Scholar 

  • Baldwin IT (1998) Jasmonate-induced responses are costly but benefit plants under attack in native populations. PNAS 95:8113–8118

    Article  PubMed  CAS  Google Scholar 

  • Baldwin IT, Hamilton W III (2000) Jasmonate-induced responses of Nicotiana sylvestris results in fitness costs due to impaired competitive ability for nitrogen. J Chem Ecol 26:915–952

    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 

  • Baldwin IT, Schmelz EA, Zhang Z-P (1996) Effects of octadecanoid metabolites and inhibitors on induced nicotine accumulation in Nicotiana sylvestris. J Chem Ecol 22:61–74

    Article  CAS  Google Scholar 

  • Baldwin IT, Zhang Z-P, Diab N, Ohnmeiss TE, McCloud ES, Lynds GY, Schmelz EA (1997) Quantification, correlations and manipulation of wound-induced changes in jasmonic acid and nicotine in Nicotiana sylvestris. Planta 201:397–404

    Article  CAS  Google Scholar 

  • Baldwin IT, Gorham D, Schmelz EA, Lewandowski CA, Lynds GY (1998) Allocation of nitrogen to an inducible defense and seed production in Nicotiana attenuata. Oecologia (Berlin) 115:541–552

    Article  Google Scholar 

  • Bartholomeusz TA, Bhogal RK, Molinie R, Felpin FX, Mathe-Allainmat M, Meier AC, Drager B, Lebreton J, Roscher A, Robins RJ, Mesnard F (2005) Nicotine demethylation in Nicotiana cell suspension cultures: N′-formylnornicotine is not involved. Phytochemistry 66:2432–2440

    Article  PubMed  CAS  Google Scholar 

  • Broeckling CD, Huhman DV, Farag MA, Smith JT, May GD, Mendes P, Dixon RA, Sumner LW (2005) Metabolic profiling of Medicago truncatula cell cultures reveals the effects of biotic and abiotic elicitors on metabolism. J Exp Bot 56:323–336

    Article  PubMed  CAS  Google Scholar 

  • Casano LM, Martin M, Sabater B (2001) Hydrogen peroxide mediates the induction of chloroplastic Ndh complex under photooxidative stress in barley. Plant Physiol 125:1450–1458

    Article  PubMed  CAS  Google Scholar 

  • 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 53:87–105

    CAS  Google Scholar 

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

    Article  PubMed  Google Scholar 

  • Desikan R, Reynolds A, Hancock JT, Neill SJ (1998) Harpin and hydrogen peroxide both initiate programmed cell death but have differential effects on defence gene expression in Arabidopsis suspension cultures. Biochem J 330(Pt 1):115–120

    PubMed  CAS  Google Scholar 

  • Feth F, Wagner R, Wagner KG (1986) Regulation in tobacco callus of enzyme activities of the nicotine pathway. I. The route ornithine to methylpyrroline. Planta 168:402–407

    Article  CAS  Google Scholar 

  • Finkel T (1998) Oxygen radicals and signaling. Curt Opin Cell Biol 10:248–253

    Article  CAS  Google Scholar 

  • Fritze K, Walden R (1995) Gene activation by T-DNA tagging. In: Methods in molecular biology. Humana Press, Clifton, pp 281–294

    Google Scholar 

  • Gattu M, Terry AV Jr, Buccafusco JJ (1995) A rapid microtechnique for the estimation of muscarinic and nicotinic receptor binding parameters using 96-well filtration plates. J Neurosci Methods 63:121–125

    Article  PubMed  CAS  Google Scholar 

  • Gechev TS, Van Breusegem F, Stone JM, Denev I, Laloi C (2006) Reactive oxygen species as signals that modulate plant stress responses and programmed cell death. Bioessays 28:1091–1101

    Article  PubMed  CAS  Google Scholar 

  • George J, Bais HP, Ravishankar GA (2000) Biotechnological production of plant-based Insecticides. Crit Rev Biotechnol 20:49–77

    Article  PubMed  CAS  Google Scholar 

  • Goossens A, Häkkinen ST, Laakso I, Seppanen-Laakso T, Biondi S, De Sutter V, Lammertyn F, Nuutila AM, Saderlund H, Zabeau M, Inze’ D, Oksman-Caldentey K-M (2003) A functional genomics approach toward the understanding of secondary metabolism in plant cells. PNAS 100:8595–8600

    Article  PubMed  CAS  Google Scholar 

  • Guo ZJ, Lamb C, Dixon RA (1998) Potentiation of the oxidative burst and isoflavonoid phytoalexin accumulation by serine protease inhibitors. Plant Physiol 118:1487–1494

    Article  PubMed  CAS  Google Scholar 

  • Häkkinen ST, Tilleman S, Swiatek A, De Sutter V, Rischer H, Vanhoutte I, Van Onckelen H, Hilson P, Inzé D, Oksman-Caldentey K-M, Goossens A (2007) Functional characterisation of genes involved in pyridine alkaloid biosynthesis in tobacco. Phytochemistry 68:2773–2785

    Article  PubMed  Google Scholar 

  • Hashimoto T, Yamada Y (1994) Alkaloid biogenesis: molecular aspects. Ann Rev Plant Physiol Plant Mol Biol 45:257–285

    Article  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  • He Y, Gan S (2001) Identical promoter elements are involved in regulation of the OPR1 gene by senescence and jasmonic acid in Arabidopsis. Plant Mol Biol 47:595–605

    Article  PubMed  CAS  Google Scholar 

  • Henzler T, Steudle E (2000) Transport and metabolic degradation of hydrogen peroxide in Chara corallina: model calculations and measurements with the pressure probe suggest transport of H2O2 across water channels. J Exp Bot 51:2053–2066

    Article  PubMed  CAS  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  CAS  Google Scholar 

  • Hobbs MC, Yeoman MM (1991a) Biotransformation of nicotine to nornicotine by cell suspensions of Nicotiana tabacum l cv wisconsin-38. New Phytol 119:477–482

    Article  CAS  Google Scholar 

  • Hobbs MC, Yeoman MM (1991b) Effect of light on alkaloid accumulation in cell cultures of Nicotiana spp. J Exp Bot 42:1371–1378

    Article  CAS  Google Scholar 

  • Houghtling RA, Davila-Garcia MI, Kellar KJ (1995) Characterization of (±)(-)[3H]epibatidine binding to nicotinic cholinergic receptors in rat and human brain. Mol Pharmacol 48:280–287

    PubMed  CAS  Google Scholar 

  • Imanishi S, Hashizume K, Nakakita M, Kojima H, Matsubayashi Y, Hashimoto T, Sakagami Y, Yamada Y, Nakamura K (1998) Differential induction by methyl jasmonate of genes encoding ornithine decarboxylase and other enzymes involved in nicotine biosynthesis in tobacco cell cultures. Plant Mol Biol 38:1101–1111

    Article  PubMed  CAS  Google Scholar 

  • Iwase A, Aoyagi H, Ohme-Takagi M, Tanaka H (2005) Development of a novel system for producing ajmalicine and serpentine using direct culture of leaves in Catharanthus roseus intact plant. J Biosci Bioeng 99:208–215

    Article  PubMed  CAS  Google Scholar 

  • Jabs T, Tschope M, Colling C, Hahlbrock K, Scheel D (1997) Elicitor-stimulated ion fluxes and O2 − from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley. PNAS 94:4800–4805

    Article  PubMed  CAS  Google Scholar 

  • Karpinski S, Reynolds H, Karpinska B, Wingsle G, Creissen G, Mullineaux P (1999) Systemic signaling and acclimation in response to excess excitation energy in Arabidopsis. Science 284:654–657

    Article  PubMed  CAS  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  PubMed  CAS  Google Scholar 

  • Kroj T, Rudd JJ, Nurnberger T, Gabler Y, Lee J, Scheel D (2003) Mitogen-activated protein kinases play an essential role in oxidative burst-independent expression of pathogenesis-related genes in parsley. J Biol Chem 278:2256–2264

    Article  PubMed  CAS  Google Scholar 

  • Levine A, Tenhaken R, Dixon R, Lamb C (1994) H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79:583–593

    Article  PubMed  CAS  Google Scholar 

  • Littleton J, Rogers T, Falcone D (2005) Novel approaches to plant drug discovery based on high throughput pharmacological screening and genetic manipulation. Life Sci 78:467–475

    Article  PubMed  CAS  Google Scholar 

  • Maliga P, Sz-Breznovits A, Marton L (1973) Streptomycin resistant plants from callus culture of haploid tobacco. Nat New Biol 244:29–30

    PubMed  CAS  Google Scholar 

  • Mehdy MC (1994) Active oxygen species in plant defense against pathogens. Plant Physiol 105:467–472

    PubMed  CAS  Google Scholar 

  • Mizusaki S, Tanebe Y, Noguchi M, Tamaki E (1973) Changes in activities of ornithine decarboxylase, putrescine N-methyltransferase and N-methylputrescine oxidase in tobacco roots in relation to nicotine biosynthesis. Plant Cell Physiol 14:103–110

    CAS  Google Scholar 

  • Moyano E, Fornale S, Palazon J, Cusido RM, Bagni N, Pinol MT (2002) Alkaloid production in Duboisia hybrid hairy root cultures overexpressing the pmt gene. Phytochemistry 59:697–702

    Article  PubMed  CAS  Google Scholar 

  • Neill SJ, Desikan R, Clarke A, Hurst RD, Hancock JT (2002) Hydrogen peroxide and nitric oxide as signalling molecules in plants. J Exp Bot 53:1237–1247

    Article  PubMed  CAS  Google Scholar 

  • O’Kane D, Gill V, Boyd P, Burdon R (1996) Chilling, oxidative stress and antioxidant responses in Arabidopsis thaliana callus. Planta 198:371–377

    Article  PubMed  Google Scholar 

  • Ogino T, Hiraoka N, Tabata M (1978) Selection of high nicotine producing cell lines of tobacco callus by single cell cloning. Phytochemistry 17:1907–1910

    Article  CAS  Google Scholar 

  • Ohnmeiss TE, McCloud ES, Lynds GL, Baldwin IT (1997) Within-plant relationships among wounding, jasmonic acid, and nicotine: Implications for defence in Nicotiana sylvestris. New Phytol 137:441–452

    Article  CAS  Google Scholar 

  • Ohta S, Matsui O, Yatazawa M (1978) Culture conditions for nicotine production in tobacco tissue culture. Agric Biol Chem 42:1245–1252

    CAS  Google Scholar 

  • Pfeiffer W, Hoeftberger M (2001) Oxidative burst in Chenopodium rubrum suspension cells: Induction by auxin and osmotic changes. Physiol Plant 111:144–150

    Article  CAS  Google Scholar 

  • Phillips I (1975) Apical dominance. Annu Rev Plant Physiol 26:341–365

    Article  CAS  Google Scholar 

  • Preston CA, Lewandowski C, Enyedi AJ, Baldwin IT (1999) Tobacco mosaic virus inoculation inhibits wound-induced jasmonic acid-mediated responses within but not between plants. Planta 209:87–95

    Article  PubMed  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  PubMed  CAS  Google Scholar 

  • Rogers DT, Falcone DL, Littleton JM (2003) A functional genomics strategy for plant drug discovery. Pharmagenomics 3:26–34

    CAS  Google Scholar 

  • Rossel JB, Wilson PB, Hussain D, Woo NS, Gordon MJ, Mewett OP, Howell KA, Whelan J, Kazan K, Pogson BJ (2007) Systemic and intracellular responses to photooxidative stress in Arabidopsis. Plant Cell 19:4091–4110

    Article  PubMed  CAS  Google Scholar 

  • Rothe G, Hachiya A, Yamada Y, Hashimoto T, Drager B (2003) Alkaloids in plants and root cultures of Atropa belladonna overexpressing putrescine N-methyltransferase. J Exp Bot 54:2065–2070

    Article  PubMed  CAS  Google Scholar 

  • Sachan N, Falcone DL (2002) Wound-induced gene expression of putrescine N-methyltransferase in leaves of Nicotiana tabacum. Phytochemistry 61:797–805

    Article  PubMed  CAS  Google Scholar 

  • Saedler R, Baldwin IT (2004) Virus-induced gene silencing of jasmonate-induced direct defences, nicotine and trypsin proteinase-inhibitors in Nicotiana attenuata. J Exp Bot 55:151–157

    Article  PubMed  CAS  Google Scholar 

  • Sato F, Hashimoto T, Hachiya A, Tamura K, Choi KB, Morishige T, Fujimoto H, Yamada Y (2001) Metabolic engineering of plant alkaloid biosynthesis. PNAS 98:367–372

    Article  PubMed  CAS  Google Scholar 

  • Saunders JW, Bush LP (1979) Nicotine biosynthetic enzyme activities in Nicotiana tabacum L. genotypes with different alkaloid levels. Plant Physiol 64:236–240

    Article  PubMed  CAS  Google Scholar 

  • Shoji T, Hashimoto T (2008) Why does snatabine, but not nicotine, accumulate in jasmonate-elicited cultured tobacco BY-2 cells? Plant Cell Physiol 49:1209–1216

    Article  PubMed  CAS  Google Scholar 

  • Shoji T, Yamada Y, Hashimoto T (2000) Jasmonate induction of putrescine N-methyltransferase genes in the root of Nicotiana sylvestris. Plant Cell Physiol 41:831–839

    Article  PubMed  CAS  Google Scholar 

  • Steppuhn A, Gase K, Krock B, Halitschke R, Baldwin IT (2004) Nicotine’s defensive function in nature. PLoS Biol 2:E217

    Article  PubMed  Google Scholar 

  • Suzuki K, Yamada Y, Hashimoto T (1999) Expression of Atropa belladonna putrescine N-methyltransferase gene in root pericycle. Plant Cell Physiol 40:289–297

    PubMed  CAS  Google Scholar 

  • Tabata M, Hiraoka N (1976) Variation of alkaloid production in Nicotiana rustica var–Brasilia callus cultures. Physiol Plant 38:19–23

    Article  CAS  Google Scholar 

  • Tabata M, Yamamoto H, Hiraoka N, Marumoto Y, Konoshima M (1971) Regulation of nicotine production in tobacco-d tissue culture by plant growth regulators. Phytochemistry 10:723–729

    Article  CAS  Google Scholar 

  • Takahashi M, Yamada Y (1973) Regulation of nicotine production by auxins in tobacco cultured cells in vitro. Agric Biol Chem 37:1755–1757

    CAS  Google Scholar 

  • Tiburcio AF, Kaur-Sawhney R, Ingersoll RB, Galston AW (1985) Correlation between polyamines and pyrrolidine alkaloids in developing tobacco callus. Plant Physiol 78:323–326

    Article  PubMed  CAS  Google Scholar 

  • Voelckel C, Kruegel T, Gase K, Heidrich N, van Dam NM, Winz R, Baldwin IT (2001) Anti-sense expression of putrescine N-methyltransferase confirms defensive role of nicotine in Nicotiana sylvestris against Manduca sexta. Chemoecology 11:121–126

    Article  CAS  Google Scholar 

  • Wink M (1998) Modes of action of alkaloids. In: Roberts M, Wink M (eds) Alkaloids. Plenum Press, New York, pp 301–326

    Google Scholar 

  • Wise RR, Naylor AW (1987) Chilling-enhanced photooxidation : evidence for the role of singlet oxygen and superoxide in the breakdown of pigments and endogenous antioxidants. Plant Physiol 83:278–282

    Article  PubMed  CAS  Google Scholar 

  • Wu J, Ge X (2004) Oxidative burst, jasmonic acid biosynthesis, and taxol production induced by low-energy ultrasound in Taxus chinensis cell suspension cultures. Biotechnol Bioeng 85:714–721

    Article  PubMed  CAS  Google Scholar 

  • Xu B, Timko M (2004) Methyl jasmonate induced expression of the tobacco putrescine N-methyltransferase genes requires both G-box and GCC-motif elements. Plant Mol Biol 55:743–761

    Article  PubMed  CAS  Google Scholar 

  • Yin S, 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 

  • Zhang Z-P, Krumm T, Baldwin IT (1997) Structural requirements of jasmonates and mimics for nicotine induction in Nicotiana sylvestris. J Chem Ecol 23:2777–2789

    Article  CAS  Google Scholar 

  • Zhao J, Sakai K (2003) Multiple signaling pathways mediate fungal elicitor induced b-thujaplicin accumulation in Cupressus lusitanica cell cultures. J Exp Bot 54:647–656

    Article  PubMed  CAS  Google Scholar 

  • Zhao J, Fujita K, Sakai K (2005) Oxidative stress in plant cell culture: a role in production of beta-thujaplicin by Cupresssus lusitanica suspension culture. Biotechnol Bioeng 90:621–631

    Article  PubMed  CAS  Google Scholar 

  • Zhao J, Matsunaga Y, Fujita K, Sakai K (2006) Signal transduction and metabolic flux of beta-thujaplicin and monoterpene biosynthesis in elicited Cupressus lusitanica cell cultures. Metab Eng 8:14–29

    Article  PubMed  Google Scholar 

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Acknowledgments

We wish to thank Irina Artiushin and May Fu for expert technical assistance with protoplast preparations and alkaloid binding assays, respectively. This work was supported by the Kentucky Tobacco Research Board and the University of Massachusetts Lowell.

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  1. Nita Sachan & Kil-Young Yun

    Present address: University of Texas Southwestern Medical Center, Dallas, TX, USA

Authors and Affiliations

  1. Kentucky Research and Development Center, University of Kentucky, Naprogenix, Inc, Lexington, KY, 40515, USA

    Nita Sachan, Dennis T. Rogers, John M. Littleton & Deane L. Falcone

  2. Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY, 40515, USA

    John M. Littleton

  3. Fungi and Plant Co. Ltd, Chungcheongbuk-do, Republic of Korea

    Kil-Young Yun

  4. Department of Biological Sciences, University of Massachusetts, One University Avenue, Lowell, MA, 01854, USA

    Kil-Young Yun & Deane L. Falcone

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  1. Nita Sachan
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  2. Dennis T. Rogers
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Correspondence to Deane L. Falcone.

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Communicated by D. Somers.

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Sachan, N., Rogers, D.T., Yun, KY. et al. Reactive oxygen species regulate alkaloid metabolism in undifferentiated N. tabacum cells. Plant Cell Rep 29, 437–448 (2010). https://doi.org/10.1007/s00299-010-0833-9

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