Enhancing Production of Terpenoids in Metabolically Engineered Transgenic Spearmint (Mentha spicata L.) by Salt and Fungal Elicitors

Choi, Myung Suk;Park, Dong Jin;Song, Hyun Jin;Min, Ji Yun;Kang, Seung Mi;Lee, Chong Kyu;Cho, Kye Man;Karigar, Chandrakant;Kim, Ho Kyoung;Kang, Young Min

  • 투고 : 2013.10.28
  • 심사 : 2014.01.08
  • 발행 : 2014.05.31


Forest tree species usually takes for long periods to be harvested and cultivated but spearmints are a good model system for woody plant because of reducing and shortening cultivation time. Spearmints are good model plants (Mentha species) for research about terpenoids production and industrial essential oil manufacture. Isopentenyl pyrophosphate isomerase (Iso) and limonene synthase (Limo) are the key enzymes of terpenoid biosynthesis pathway. Transgenic and wild spearmints (Mentha spicata, MS) were cultured in vitro and assessed for the essential oil contents. The content of essential oil of transgenic spearmint also was enhanced slightly depending on the target terpenoid genes. In an attempt to increase productivity of terpenoids further, salt and fungal elicitation strategy was adopted on transgenic Mentha spicata. The salt (800 mM NaCl) as abiotic and two fungi (Botrytis cinerea and Glomerella cingulata) as biotic were used for elicitors. In the absence of salt stress four terpenoids were detected from the spearmint extracts, all of them being monoterpenes. On the other hand, the transgenic (MSIso) extracts contained eleven terpenoids (10 monoterpenes and 1 phenylpropene) while transgenic (MSLimo) extracts contained seven monoterpenes. After 3 days of fungal infection, the resistance indices further increased to 4.38, 3.89 and 2.04 for wild type, MSIso and MSLimo, respectively. The salt and fungal elicitators proved beneficial towards modifying both the terpenoids profile and improvement in the composition of essential oil. These results have important applications for the large-scale production of essential oils and forest biotechnology with respect to spearmint.


Essential oils;Elicitors;Forest biotechnology;Metabolically engineered transgenic spearmint;Terpenoids


  1. Abraham WR, Washausen P, Wieslich KK. 1987. Microbial hydroxylation of cedrol and cedrene. Z Naturforsch 42C: 414-419.
  2. Tegelberg R, Julkunen-Tiitto R. 2001. Quantitative changes in secondary metabolites of dark-leaved willow (Salix myrsinifolia) exposed to enhanced ultraviolet-B radiation. Physiologia Plantarum 113: 541-547.
  3. Tegelberg R , Julkunen-Tiitto R, Aphalo PJ. 2001. The effects of long-term elevated UV-B on the growth and phenolics of field-grown silver birch (Betula pendula). Global Change Biology 7: 839-848.
  4. Ultee A, Bennik MH, Moezelaar R. 2002. The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen Bacillus cereus. Appl Environ Microbiol 68: 1561-1568.
  5. Ultee A, Slump RA, Steging G, Smid EJ. 2000. Antimicrobial activity of carvacrol toward Bacillus cereus on rice. J Food Prot 63: 620-624.
  6. Waterman PG, Mole S. 1994. Analysis of phenolic plant metabolites. Blackwell Scientific Publications, Oxford, UK.
  7. Park DJ. 2008. Metabolites profiling and its function through terpenoids biosynthesis genes overexpression of spearmint (MenthaspicataL.). Master thesis. Gyeongsang National University, Jinju, Korea.
  8. Menary RC, Dragar VA, Garland SM. 1999. Tasmannia lanceolata: developing a new commercial flavour product: a report for the Rural Industries Research and Development Corporation, USA.
  9. Mitsuhara I, Ugaki M, Hirochika H, Ohshima M, Murakami T, Gotoh Y, Katayose Y, Nakamura S, Honkura R, Nishimiya S, Ueno K, Mochizuki A, Tanimoto H, Tsugawa H, Otsuki Y, Ohashi Y. 1996. Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol 37: 49-59.
  10. Nickerson GB, Likens ST. 1996. Gas chromatographic evidence for the occurrence of hop oil components in beer. J Chromatogr 21: 1-5.
  11. Sambrook J, Fritch EF, Maniatis T. 1998. Molecular cloning. In: A laboratory manual. Cold Spring Harbor Lab, Cold Spring Harbor, New York.
  12. Seigler DS. 1998. Plant Seconday Metabolism. Kluwer Academic Publishers, Dordrecht, The Netherlands.
  13. Singh HP, Batisha DR, Kohlia RK. 1999. Autotoxicity: concept, organisms, and ecological significance. CR Rev Plant Sci 18: 757-772.
  14. Skold M, Karlberg AT, Matura M, Borje A. 2006. The fragrance chemical beta-caryophyllene-air oxidation and skin sensitization. Food Chem Toxicol 44: 538-545.
  15. Smith BPC, Hayasaka Y, Tyler MJ, Williams BD. 2004. $\beta$-caryophyllene in the skin secretion of the Australian green tree frog, Litoria caerulea: an investigation of dietary sources. Aust J Zool 52: 521-530.
  16. Tambe Y, Tsujiuchi H, Honda G, Ikeshiro Y, Tanaka S. 1996. Gastric cytoprotection of the non-steroidal anti-inflammatory sesquiterpene, beta-caryophyllene. Planta Med 62: 469-470.
  17. Laitinen ML, Julkunen-Tiitto R, Yamaji K, Heinonen J, Rousi M. 2004. Variation in birch bark secondary chemistry between and within clones: implications for herbivory by hares. Oikos 104: 316-326.
  18. Kang YM, Park DJ, Song HJ, Ma HS, Karigar C, Choi M. 2012. Comparative analysis of terpenoids in in vitro culture media of metabolically engineered transgenic and wild type spearmint (Mentha spicata L.). Korean J Medicinal Crop Sci 20: 301-307.
  19. Kieslich K, Abraham WR, Stumpf B, Thede B, Washausen P. 1986. Transformations of terpenoids, Walter de Gruyter and Co, Berlin, Germany.
  20. Laitinen ML, Julkunen-Tiitto R, Rousi M. 2002. Foliar phenolic composition of European white birch during bud unfolding and leaf development. Physiol Plant 114: 450-460.
  21. Lamare V, Fourneron JD, Furstoss R, Ehret C, Corbier B. 1987. Microbial transformations. IX: Biohydroxylation of alpha-cedrene and cedrol. Synthesis of an odoriferous minor component of cedar wood essential oil. Tetrahedron Lett 28: 6269-6272.
  22. Lamare V, Furstoss R. 1990. Bioconversion of sesquiterpenes. Tetrahedron 46: 4109-4132.
  23. Lange BM, Mahmoud SS, Wildung MR, Turner GW, Davis EM, Lange I, Baker RC, Boydston RA, Croteau RB. 2011. Improving peppermint essential oil yield and composition by metabolic engineering. Proc Natl Acad Sci USA 108: 16944-16949.
  24. Lawrence RV. 1989. Processing pine gum into turpentine and rosin. Pulp Chemicals Association, New York, USA.
  25. Lee DW, Seo JB, Kang JS, Koh SH, Lee SH, Koh YH. 2012. Identification and Characterization of Expansins from Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae). Plant Pathology J 28: 409-417.
  26. Li YH, Sun ZH, Zheng P. 2004. Determination of Vanillin, Eugenol and Isoeugenol by RP-HPLC. Chromatographia 60: 709-713.
  27. Erman WF. 1985. Chemistry of the monoterpenes: an encyclopedic handbook. Marcel Dekker, New York.
  28. Corazza M, Levratti A, Virgili A. 2002. Allergic contact cheilitis due to carvone in toothpastes. Contact Dermatitis 46: 366-367.
  29. Davidson PM, Naidu AS. 2000. Phyto-phenols. In: Natural Food Antimicrobial Systems (Naidu AS, ed). CRC Press, Boca Raton, Florida, USA, pp 265-293.
  30. Dorman HJ, Deans SG. 2000. Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J Appl Microbiol 88: 308-316.
  31. Ghelardini C, Galeotti N, Di Cesare Mannelli L, Mazzanti G, Bartolini A. 2001. Local anaesthetic activity of beta- caryophyllene. Farmaco 56: 387-389.
  32. Gouinguene SP, Turlings TC. 2002. The effects of abiotic factors on induced volatile emissions in corn plants. Plant Physiol 129: 1296-1307.
  33. Hall RL, Oser BL. 1965. Recent progress in the consideration of flavoring ingredients under the food additives amendment. III GRAS Substances. Food Technol 19: 151-197.
  34. Himejima M, Hobson KR, Otsuka T, Wood DL, Kubo I. 1992. Antimicrobial terpenes from oleoresin of ponderosa pine tree Pinus ponderosa: A defense mechanism against microbial invasion. J Chem Ecol 18: 1809-1818.
  35. Julkunen-Tiitto R, Bryant JP, Kuropat P, Roininen H. 1995. Slight tissue wounding fails to induce consistent chemical defense in three willow (Salix spp.) clones. Oecologia 101: 467-471.
  36. Jung HY, Kang SM, Kang YM, Kang MJ, Yun DJ, Bahk JD, Yang JK, Choi MS. 2003. Enhanced production of scopolamine by bacterial elicitors in adventitious hairy root cultures of Scopolia parviflora. Enzyme Microb Tech 33: 987-990.
  37. Bryant JP, Julkunen-Tiitto R. 1995. Ontogenic development of chemical defense by seedling resin birch: Energy cost of defense production. J Chem Ecol 21: 883-896.
  38. Aoki T, O'Donnell K, Homma Y, Lattanzi AR. 2003. Suddendeath syndrome of soybean is caused by two morphologically and phylogenetically distinct species within the Fusarium solani species complex--F. virguliforme in North America and F. tucumaniae in South America. Mycologia 95: 660-684.
  39. Aoki T, O'Donnell K, Scandiani MM. 2005. Sudden death syndrome of soybean in South America is caused by four species of Fusarium: Fusarium brasiliense sp. nov., F. cuneirostrum sp. nov., F. tucumaniae, and F. virguliforme. Mycoscience 46: 162-183.
  40. Bienvenu F, Peterson L, Edwards J. 1999. Native and Scotch Spearmint Oil Production in Tasmania and Victoria. A report for the Rural Industries Research and Development Corporation, pp 99-147.
  41. Bryant JP, Provenza FD, Pastor J, Reichardt PB, Clausen TP, du Toit JT. 1991. Interactions between woody plants and browsing mammals mediated by secondary metabolites. Annu Rev Ecol Syst 22: 431-446.
  42. Budavari S. 1996. The Merck index: an encyclopedia of chemicals, drugs, and biologicals. 12th ed. Merck & Co Inc., New Jersey, USA.
  43. Burdock GA. 1995. Fenaroli's Hanbook of Flavor In-gredients: Adapted from the Italian Language Works of Giovanni Fenaroli. 3rd ed. CRC Press, Boca Raton, USA.
  44. Burt SA, Vlielander R, Haagsman HP, Veldhuizen EJ. 2005. Increase in activity of essential oil components carvacrol and thymol against Escherichia coli O157:H7 by addition of food stabilizers. J Food Prot 68: 919-926.
  45. Choi MS, Heu S, Paek NC, Koh HJ, Lee JS, Oh CS. 2012. Expression of hpa1 gene encoding a bacterial harpin protein in Xanthomonas oryzae pv. oryzae enhances disease resistance to both fungal and bacterial pathogens in rice and arabidopsis. Plant Pathology J 28: 364-372.