Regulation of Secondary Metabolism in Tobacco Cell Cultures

  • Suvi T. Häkkinen
  • Kirsi-Marja Oksman-Caldentey
Part of the Biotechnology in Agriculture and Forestry book series (AGRICULTURE, volume 53)


In addition to primary metabolites, which are essential for life and development, plants also synthesize a number of low molecular weight compounds, so-called secondary metabolites. These compounds have important functions for plants in survival and competing in the environment, in protection against UV light as well as in various defence-related reactions. Up to date, about 100,000 plant secondary metabolites have been isolated (Verpoorte 2000). Most plant constituents that are used medicinally are secondary metabolites, and up to 25% of the contemporary drugs contain an active compound originating from plants. In addition, secondary metabolites are of interest for people as flavours, fragrances, pesticides and dyes. An important group of pharmacologically active compounds consists of alkaloids. Up to 15,000 alkaloids have been characterised since the identification of the first alkaloid morphine from the opium poppy in 1806 (Kutchan 1995). One of the most studied plants is tobacco, belonging to the genus Nicotiana, which was named after the French diplomat Jean Nicot who, in the middle of the sixteenth century, started to popularise tobacco in Europe. Tobacco secondary metabolites have been extensively studied and more than 2500 compounds have been identified. However, the biosynthetic pathways and metabolism of these compounds need further elucidation (Nugroho and Verpoorte 2002). A lot of work concerning the biosynthetic studies has been done using plant cell cultures in order to overcome the problems caused by cultivation of the whole plants. The aim of this chapter is to give an overview of the nicotine alkaloid biosynthesis in tobacco callus and cell suspension cultures including tobacco BY-2 cell culture, and offer an insight into the variables affecting the alkaloid production in these systems. In addition, metabolism of other secondary compounds in tobacco cell cultures is discussed. Today, ample possibilities to study secondary metabolism are allowed by novel techniques, such as genome-wide gene identification, which is demonstrated here by using tobacco BY-2 cell culture.


Nicotinic Acid Methyl Jasmonate Fungal Elicitor Tropane Alkaloid Tobacco Callus 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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  1. Alworth WL, Rapoport H (1965) Biosynthesis of the nicotine alkaloids in Nicotiana glutinosa, interrelationships among nicotine, nornicotine, anabasine, and anatabine. Arch Biochem Biophys 112: 45–53PubMedCrossRefGoogle Scholar
  2. Arcavi L, Jacob P III, Hellerstein M, Benowitz NL (1994) Divergent tolerance to metabolic and cardiovascular effects of nicotine in smokers with low and high levels of cigarette consumption. Clin Pharmacol Ther 56: 55–64PubMedCrossRefGoogle Scholar
  3. Barz W, Kettner M, Hüsemann W (1978) On the degradation of nicotine in Nicotiana cell suspension cultures. Planta Med 34: 73–78CrossRefGoogle Scholar
  4. Bate NJ, Orr J, Ni W, Meromi A, Nadler-Hassar T, Doerner PW, Dixon RA, Lamb CJ, Elkind Y (1994) Quantitative relationship between phenylalanine ammoni-lyase levels and phenylpropanoid accumulation in transgenic tobacco identifies a rate-limiting step in natural product synthesis. Proc Natl Acad Sci USA 91: 7608–7612PubMedCrossRefGoogle Scholar
  5. Benowitz NL (1996) Pharmacology of nicotine: addiction and therapeutics. Annu Rev Pharmacol Toxicol 36: 597–613PubMedCrossRefGoogle Scholar
  6. Benowitz NL, Jacob P (1999) Pharmacokinetics and metabolism of nicotine and related alkaloids. In: Arneric SP, Brioni JD (eds) Neuronal nicotinic receptors: pharmacology and therapeutic opportunities. Wiley, New York, pp 213–234Google Scholar
  7. Berlin J (1981) Formation of putrescine and cinnamoyl putrescines in tobacco cell cultures. Phytochemistry 20: 53–55CrossRefGoogle Scholar
  8. Berlin J, Widholm JM (1977) Correlation between phenylalanine ammonia lyase activity and phenolic biosynthesis in p-fluorophenylalanine-sensitive and -resistant tobacco and carrot tissue cultures. Plant Physiol 59: 550–553PubMedCrossRefGoogle Scholar
  9. Berlin J, Mollenschott C, Herminghaus S, Fecker LF (1998) Lysine decarboxylase transgenic tobacco root cultures biosynthesize novel hydroxycinnamoylcadaverines. Phytochemistry 48: 79–84CrossRefGoogle Scholar
  10. Biondi S, Scaramagli S, Capitani F, Altamura MM, Torrigiani P (2001) Methyl jasmonate upregulates biosynthetic gene expression, oxidation and conjugation of polyamines, and inhibits shoot formation in tobacco thin layers. J Exp Bot 52: 231–242PubMedCrossRefGoogle Scholar
  11. Botte M, Mabon F, LeMouillour M, Robins R (1997) Biosynthesis of nornicotine in root cultures of Nicotiana alata does not involve oxidation at C-5 of nicotine. Phytochemistry 46: 117–122CrossRefGoogle Scholar
  12. Breyne P, Zabeau M (2001) Genome-wide expression analysis of plant cell cycle modulated genes. Curr Opin Plant Biol 4: 136–142PubMedCrossRefGoogle Scholar
  13. Cappell J, Von Lanken C, Vögeli U, Bhatt P (1989) Sterol and sesquiterpenoid biosynthesis during a growth cycle of tobacco cell suspension cultures. Plant Cell Rep 8: 48–52CrossRefGoogle Scholar
  14. Chappell J, Nable R (1987) Induction of sesquiterpenoid biosynthesis in tobacco cell suspension cultures by fungal elicitor. Plant Physiol 85: 469–473PubMedCrossRefGoogle Scholar
  15. Chappell J, Nable R, Fleming P, Andersen RA, Burton HR (1987) Accumulation of capsidiol in tobacco cell cultures treated with fungal elicitor. Phytochemistry 26: 2259–2260CrossRefGoogle Scholar
  16. Chelvarajan RL, Fannin FF, Bush LP (1993) Study of nicotine demethylation in Nicotiana otophora. J Agric Food Chem 41: 858–862CrossRefGoogle Scholar
  17. Clark MSG, Rand MJ, Vanov S (1965) Comparison of pharmacological activity of nicotine and related alkaloids occurring in cigarette smoke. Arch Int Pharmacodyn 156: 363–379PubMedGoogle Scholar
  18. Dawson RF (1945) On the biosynthesis of nornicotine and anabasine. J Am Chem Soc 67: 503–504CrossRefGoogle Scholar
  19. Dixon RA, Lamb CJ (1990) Molecular communication in interactions between plants and microbial pathogens. Annu Rev Plant Physiol Plant Mol Biol 41: 339–367CrossRefGoogle Scholar
  20. Flores HE, Protacio CM, Signs MW (1991) Primary and secondary metabolism of polyamines in plants. Recent Adv Phytochem 23: 329–393Google Scholar
  21. Friesen JB, Leete E (1990) Nicotine synthase–an enzyme from Nicotiana species which catalyses the formation of (S)-nicotine from nicotinic acid and 1-methyl-Δ’-pyrrolinium chloride. Tetrahedron Lett 6295–6298Google Scholar
  22. Friesen JB, Burkhouse PC, Biesboer DD, Leete E (1992) Influence of alkaloid precursors on the alkaloid content of Nicotiana alata root cultures. Phytochemistry 31: 3059–3063CrossRefGoogle Scholar
  23. Fujimori T, Tanaka H, Kato K (1983) Stress compounds in tobacco callus infiltrated by Pseudomonas solanacearum. Phytochemistry 22: 1038CrossRefGoogle Scholar
  24. Furuya T, Kojima H, Syono K (1971) Regulation of nicotine biosynthesis by auxins in tobacco callus tissues. Phytochemistry 10: 1529–1532CrossRefGoogle Scholar
  25. Ghosh B (2000) Polyamines and plant alkaloids. Ind J Exp Biol 38: 1086–1091Google Scholar
  26. Goossens A, Häkkinen ST, Laakso I, Seppänen-Laakso T, Biondi S, deSutter V, Lammertyn F, Nuutila AM, Söderlund H, Zabeau M, Inzé D, Oksman-Caldentey K-M (2003) A functional genomics approach toward the understanding of secondary metabolism in plant cells. Proc Natl Acad Sci USA 100: 8595–8600PubMedCrossRefGoogle Scholar
  27. Griffith GD, Griffith T, Byerrum R (1960) Nicotinic acid as a metabolite of nicotine in Nicotina rustica. J Biol Chem 235: 3536–3538PubMedGoogle Scholar
  28. Gross D (1985) Alkaloids derived from nicotinic acid. In: Mothes K, Schütte HR, Luckner M (eds) Biochemistry of alkaloids. VCH, Berlin, pp 163–187Google Scholar
  29. Hahlbrock K, Scheel D (1989) Physiology and molecular biology of phenylpropanoid metabolism. Annu Rev Plant Physiol Plant Mol Biol 40: 347–369CrossRefGoogle Scholar
  30. Hanley KM, Voegeli U, Chappell J (1992) A study of the isoprenoid pathway in elicitor-treated tobacco cell suspension cultures. In: Petroski RJ, McCormick SP (eds) Secondary metabolite biosynthesis and metabolism. Plenum Press, New York, pp 329–336CrossRefGoogle Scholar
  31. Hao D-Y, Yeoman MM (1996) Mechanism of nicotine N -demethylation in tobacco cell suspension cultures. Phytochemistry 41: 477–482CrossRefGoogle Scholar
  32. Hao D-Y, Yeoman MM (1998) Evidence in favour of an oxidative N -demethylation of nicotine to nornicotine in tobacco cell cultures. J Plant Physiol 152: 420–426CrossRefGoogle Scholar
  33. Harborne JB,Williams CA (1982) Flavone and flavonol glycosides. In: Harborne JB, Marby TJ (eds) The flavonoids: advances in research. Chapman and Hall, London, p 261Google Scholar
  34. Hartmann M-A, Wentzinger L, Hemmerlin A, Bach TJ (2000) Metabolism of farnesyl diphosphate in tobacco BY-2 cells treated with squalestatin. Biochem Soc Trans 28: 794–796PubMedCrossRefGoogle Scholar
  35. Hashimoto T, Yamada Y (1986) Hyoscyamine 6p-hydroxylase, a 2-oxoglutarate dependent dioxygenase in alkaloid producing root cultures. Plant Physiol 81: 619PubMedCrossRefGoogle Scholar
  36. Hashimoto T, Yamada Y (1994) Alkaloid biogenesis: molecular aspects. Annu Rev Plant Physiol Plant Mol Biol 45: 257–285CrossRefGoogle Scholar
  37. Hashimoto T, Mitani A, Yamada Y (1990) Diamine oxidase from cultured roots of Hyoscyamus niger. Plant Physiol 93: 216–221PubMedCrossRefGoogle Scholar
  38. Heby O, Persson L (1990) Molecular genetics of polyamine synthesis in eukaryotic cells. TIBS 15: 153–158PubMedGoogle Scholar
  39. Hibi N, Higashiguchi S, Hashimoto T, Yamada Y (1994) Gene expression in tobacco low-nicotine mutants. Plant Cell 6: 723–735PubMedGoogle Scholar
  40. Hino F, Okazaki M, Miura Y (1982) Effect of 2,4-dichlorophenoxyacetic acid on glycosylation of scopoletin to scopolin in tobacco tissue culture. Plant Physiol 69: 810–813PubMedCrossRefGoogle Scholar
  41. Imaishi H, Yamada T, Ohkawa H (1995) Purification and immunochemical characteristics of NADPH-cytochrome P-450 oxidoreductase from tobacco cultured cells. Biochim Biophys Acta 1246: 53–60PubMedCrossRefGoogle Scholar
  42. 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: 1101–1111PubMedCrossRefGoogle Scholar
  43. Ishikawa A, Yoshihara T, Nakamura K (1994) Jasmonate-inducible expression of a potato cathepsin D inhibitor-GUS gene fusion in tobacco cells. Plant Mol Biol 26: 403–414PubMedCrossRefGoogle Scholar
  44. Kartusch R, Mittendorfer B (1990) Ultraviolet radiation increases nicotine production in Nicotiana callus cultures. J Plant Physiol 136: 110–114CrossRefGoogle Scholar
  45. Kisaki T, Tamaki E (1961) Phytochemical studies on the tobacco alkaloids. III. Observations on the interconversion of DL-nicotine and DL-nornicotine in excised tobacco leaves. Arch Biochem Biophys 94: 252–256Google Scholar
  46. Kisaki T, Mizusaki S, Tamaki E (1968) Phytochemical studies on tobacco alkaloids–XI. A new alkaloid in Nicotiana tabacum roots. Phytochemistry 7: 323–327CrossRefGoogle Scholar
  47. Kutchan T (1995) Alkaloid biosynthesis–the basis for metabolic engineering of medicinal plants. The Plant Cell 7: 1059–1070PubMedGoogle Scholar
  48. Leete E (1968) The metabolism of nicotine-2’-14C in Nicotiana glauca. Tetrahedron Lett 42: 4433–4436PubMedCrossRefGoogle Scholar
  49. Leete E (1980) Alkaloids derived from ornithine, lysine and nicotinic acid. In: Bell EA, Charlwood BV (eds) Encyclopedia of plant physiology, new series, secondary plant products, vol 8. Springer, Berlin Heidelberg New York, pp 65–91Google Scholar
  50. Leete E (1983) Biosynthesis and metabolism of the tobacco alkaloids. In: Pelletier SW (ed) Alkaloids: chemical and biological perspectives. Wiley, New York, pp 85–152Google Scholar
  51. Leete E, Chedekel MR (1974) Metabolism of nicotine in Nicotiana glauca. Phytochemistry 13: 1853–1859CrossRefGoogle Scholar
  52. Leete E, Slattery SA (1976) Incorporation of [2–14C]- and [6–14C]nicotinic acid into the tobacco alkaloids. Biosynthesis of anatabine and a-b-dipyridyl. J Am Chem Soc 98: 6326–6330Google Scholar
  53. Lefevre PJ (1989) Pharmacologie des alcaloïdes mineurs du tabac. Sem Hop 65: 2424–2432Google Scholar
  54. Lockwood GB, Essa AK (1984) The effect of varying hormonal and precursor supplementations on levels of nicotine and related alkaloids in cell cultures of Nicotiana tabacum. Plant Cell Rep 3: 109–111CrossRefGoogle Scholar
  55. Lovkova MY, Ill’in GS, Minozhedinova NS (1973) Nicotine transformation to anabasine in Nicotiana glauca shoots. Prikl Biokhim Microbiol 9:595–598; Chem Abstr 79: 133283Google Scholar
  56. Mandujano-Chávez M, Schoenbeck MA, Ralston LF, Lozoya-Gloria E, Chappell J (2000) Differential induction of sesquiterpene metabolism in tobacco cell suspension culture by methyl jasmonate and fungal elicitor. Arch Biochem Biophys 381: 285–294PubMedCrossRefGoogle Scholar
  57. McLauchlan WR, McKee RA, Evans DM (1993) The purification and immunocharacterisation of N -methylputrescine oxidase from transformed root cultures of Nicotiana tabacum l. cv SC58. Planta 191: 440–445CrossRefGoogle Scholar
  58. Mesnard F, Girard S, Fliniaux O, Bhogal RK, Gillet F, Lebreton J, Fliniaux M-F, Robins RJ (2001) Chiral specificity of the degradation of nicotine by Nicotiana plumbaginifolia cell suspension cultures. Plant Sci 161: 1011–1018CrossRefGoogle Scholar
  59. Mizusaki S, Tanabe Y, Noguchi M, Tamaki E (1971) p-Coumaroyl-putrescine, caffeoylputrescine and feruoylputrescine from callus tissue culture of Nicotiana tabacum. Phytochemistry 10: 1347–1350Google Scholar
  60. Mizusaki S, Tanabe Y, Noguchi M, Tamaki E (1972) N-methylputrescine oxidase from tobacco roots. Phytochemistry 11: 2757–2762Google Scholar
  61. Modafar CE, Clerivet A, Fleuriet A, Macheix JJ (1993) Inoculation of Platanus acerifolia with Ceratocystis fimbriata f. sp. Platani induces scopoletin and umbelliferone accumulation. Phytochemistry 34: 1271–1276CrossRefGoogle Scholar
  62. Nagai N, Kojima Y, Shimosaka M, Okazaki M (1988) Effects of kinetin on L-phenylalanine ammonia-lyase activity in tobacco cell culture. Agric Biol Chem 52: 2617–2619CrossRefGoogle Scholar
  63. Nugroho LH, Verpoorte R (2002) Secondary metabolism in tobacco. Plant Cell Tissue Org Cult 68: 105–125CrossRefGoogle Scholar
  64. Ohta S, Yatazawa M (1978) Effect of light on nicotine production in tobacco tissue culture. Agric Biol Chem 42: 873–877CrossRefGoogle Scholar
  65. Ohta S, Matsui O, Yatazawa M (1978) Culture conditions for nicotine production in tobacco tissue culture. Agric Biol Chem 42: 1245–1251CrossRefGoogle Scholar
  66. Okazaki M, Hino F, Konimani K, Miura Y (1982) Effect of hormones on formation of scopoletin and scopolin in tobacco tissue cultures. Agric Biol Chem 46: 609–614CrossRefGoogle Scholar
  67. Robins RJ, Hamill JD, Parr AJ, Smith K, Walton NJ, Rhodes MJ (1987) Potential for use of nicotinic acid as a selective agent for isolation of high-nicotine producing lines of Nicotiana rustica hairy root cultures. Plant Cell Rep 6: 122–126Google Scholar
  68. Robins RJ, Walton NJ, Parr AJ, Aird ELH, Rhodes MJC, Hamill JD (1994) Progress in the genetic engineering of the pyridine and tropane alkaloid biosynthetic pathways of solanaceous plants. In: Ellis BE, Kuroki GW, Stafford HA (eds) Genetic engineering of plant secondary metabolism. Plenum Press, New York, pp 1–33CrossRefGoogle Scholar
  69. Saitoh F, Noma M, Kawashima N (1985) The alkaloid contents of sixty Nicotiana species. Phytochemistry 24: 477–480CrossRefGoogle Scholar
  70. Salminen O, Seppä T, Gäddnäs H, Ahtee L (1999) The effects of acute nicotine on the metabolism of dopamine and the expression of Fos protein in striatal and limbic brain areas of rats during chronic nicotine infusion and its withdrawal. J Neurosci 19: 8145–8151PubMedGoogle Scholar
  71. Schmeltz I, Hoffmann D (1977) Nitrogen containing compounds in tobacco and tobacco smoke. Chem Rev 77: 295–311CrossRefGoogle Scholar
  72. Sharan M, Taguchi G, Gonda K, Jouke T, Shimosaka M, Hayashida N, Okazaki M (1998) Effects of methyl jasmonate and elicitor on the cultivation of phenylalanine ammonia-lyase and the accumulation of scopoletin and scopolin in tobacco cell cultures. Plant Sci 132: 13–19CrossRefGoogle Scholar
  73. Shoji T, Nakajima K, Hashimoto T (2000) Ethylene suppresses jasmonate-inducible gene expression in nicotine biosynthesis. Plant Cell Physiol 41: 1072–1076PubMedCrossRefGoogle Scholar
  74. Shoji T, Winz R, Iwase T, Nakajima K, Yamada Y, Hashimoto T (2002) Expression patterns of two tobacco isoflavone reductase-like genes and their possible roles in secondary metabolism in tobacco. Plant Mol Biol 50: 427–440PubMedCrossRefGoogle Scholar
  75. Snook ME, Chortyk OT, Sisson VA, Costello C (1992) The flower flavanols of Nicotiana species. Phytochemistry 31: 1639–1647CrossRefGoogle Scholar
  76. Sparvoli F, Martin C, Scienza A, Gavazzi G, Tonelli C (1994) Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape (Vitis vinifera L.). Plant Mol Biol 24: 743–755PubMedCrossRefGoogle Scholar
  77. Stolerman IP, Shoaib M (1991) The neurobiology of tobacco addiction. TIPS 12: 467–473PubMedGoogle Scholar
  78. Taguchi G, Fujikawa S, Yazawa T, Kodaira R, Hayashida N, Shimosaka M, Okazaki M (2000) Scopoletin uptake from culture medium and accumulation in the vacuoles after conversion to scopolin in 2,4-D-treated tobacco cells. Plant Sci 151: 153–161PubMedCrossRefGoogle Scholar
  79. Taguchi G, Yoshizawa K, Kodaira R, Hayashida N, Okazaki M (2001) Plant hormone regulation on scopoletin metabolism from culture medium into tobacco cells. Plant Sci 160: 905–911PubMedCrossRefGoogle Scholar
  80. Takahashi M, Yamada Y (1973) Regulation of nicotine production by auxins in tobacco cultured cells in vitro. Agric Biol Chem 37 (7): 1755–1757CrossRefGoogle Scholar
  81. Tanaka H, Fujimori T (1985) Accumulation of phytuberin and phytuberol in tobacco callus inoculated with Pseudomonas solanacearum or Pseudomonas syringae pv. Tabaci. Phytochemistry 24: 1193–1195CrossRefGoogle Scholar
  82. Tiburcio AF, Galston AW (1986) Arginine decarboxylase as the source of putrescine for tobacco alkaloids. Phytochemistry 25: 107–110PubMedCrossRefGoogle Scholar
  83. Tiburcio AF, Kaur-Sawhney R, Ingersoll R, Galston AW (1985) Correlation between polyamines and pyrrolidine alkaloids in developing tobacco callus. Plant Physiol 78: 323–326PubMedCrossRefGoogle Scholar
  84. Verpoorte R (2000) Secondary metabolism. In: Verpoorte R, Alfermann AW (eds) Metabolic engineering of plant secondary metabolism. Kluver Academic Publishers, Dordrecht, The Netherlands, pp 1–29Google Scholar
  85. Wagner R, Wagner KG (1985) The pyridine-nucleotide cycle in tobacco: enzyme activities for the de novo synthesis of NAD. Planta 165: 532–537CrossRefGoogle Scholar
  86. Wahlberg I, Enzell CR (1987) Tobacco isoprenoids. Nat Prod Rep 4: 237–276PubMedCrossRefGoogle Scholar
  87. Waller GR, Heinze TM (1978) Metabolic (catabolic) modifications of alkaloids by plants. In: Waller GR, Nowacki EK (eds) Alkaloid biology and metabolism in plants. Plenum Press, New York, pp 183–274CrossRefGoogle Scholar
  88. Walton NJ, Parr AJ, Robins RJ, Rhodes MJC (1987a) Toxicity of quinoline alkaloids to cultured Cinchona ledgeriana cells. Plant Cell Rep 6: 118–121Google Scholar
  89. Walton NJ, Robins RJ, Rhodes MJC (1987b) Perturbation of alkaloid production by cadaverine in hairy root cultures of Nicotiana rustica. Plant Sci 54: 125–131CrossRefGoogle Scholar
  90. Watanabe R, Wender SH (1965) Flavonoid and certain related compounds in parts of the tobacco flower. Arch Biochem Biophys 112: 111–114PubMedCrossRefGoogle Scholar
  91. Watson DG, Rycroft DS, Freer IM, Brooks CJW (1985) Sesquiterpenoid phytoalexins from suspended callus cultures of Nicotiana tabacum. Phytochemistry 24: 2195–2200CrossRefGoogle Scholar
  92. Whitehead IM, Threlfall DR, Ewing DF (1989) 5-epi-aristolochene is a common precursor of the sesquiterpenoid phytoalexins capsidiol and debneyol. Phytochemistry 28: 775–779Google Scholar
  93. Wonnacott S (1997) Presynaptic nicotinic ACh receptors. Trends Neurosci 20: 92–98PubMedCrossRefGoogle Scholar

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© Springer-Verlag Berlin Heidelberg 2004

Authors and Affiliations

  • Suvi T. Häkkinen
    • 1
  • Kirsi-Marja Oksman-Caldentey
    • 1
  1. 1.VTT BiotechnologyVTTFinland

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