Upregulation of ginsenoside and gene expression related to triterpene biosynthesis in ginseng hairy root cultures elicited by methyl jasmonate

  • Ok Tae Kim
  • Kyong Hwan Bang
  • Young Chang Kim
  • Dong Yun Hyun
  • Min Young Kim
  • Seon Woo Cha
Original Paper

Abstract

In this study, methyl jasmonate (MJ)-induced changes of triterpene saponins in ginseng (Panax ginseng C.A. Meyer) hairy roots and expression profiling of relevant responsive genes were analyzed. The transcription of PgSS (squalene synthase), PgSE (squalene epoxidase), and PNA (dammarenediol synthase-II) genes in hairy root cultures elicited by MJ treatment increased as compared with the controls, whereas that of PNX (cycloartenol synthase) decreased slightly. In order to select candidate genes encoding for cytochrome P450-dependent hydroxylase or glucosyltransferase associated with triterpene biosynthesis, RT-PCR analysis was conducted following MJ elicitation. No differences were observed in any expression among the five genes associated with the cytochrome P450 family, when compared to that of control. For candidates of the glucosyltransferase gene,expression of EST IDs PG07020C06, PG07025D04, and PG07029G02 was upregulated. In an effort to assess the effects of MJ elicitation on the biosynthesis of triterpene saponin, protopanaxadiol saponin (Rb group) and protopanaxatriol saponin (Rg group) contents in hairy roots were evaluated by HPLC analysis. With 7 days of MJ elicitation, levels of all ginseonsides of the two-groups increased much higher than that observed in the control. In particular, protopanaxadiol-type saponin contents increased by 5.5–9.7 times that of the control, whereas protopanaxatriol-type saponin contents were increased by 1.85–3.82-fold. In the case of Rg1 ginsenoside after MJ elicitation, the content was affected negatively in ginseng hairy root cultures.

Keywords

Cytochrome P450-depentent hydroxylase Glucosyltransferase Methyl jasmonate Protopanaxadiol Protopanaxatriol 

Abbreviation

MJ

Methyl jasmonate

PgFPS

Farnesyl diphosphate synthase

PgSS

Squalene synthase

PgSE

Squalene epoxidase

PNA

Dammarenediol-II synthase

PNY

β-Amyrin synthase

PNX

Cycloartenol synthase

Notes

Acknowledgments

We thank Min Jeong Lee, College of Agriculture, Life and Environment Sciences for quantitative analysis of ginsenoside contents.

References

  1. Achnine L, Huhman DV, Farag MA, Sumner LW, Blount JW, Dixon RA (2005) Genomic-based selection and functional characterization of triterpene glycosyltransferases from the model legume Medicago truncatula. Plant J 41:875–887. doi: 10.1111/j.1365-313X.2005.02344.x PubMedCrossRefGoogle Scholar
  2. Bae KH, Choi YE, Shin CG, Kim YY, Kim YS (2006) Enhanced ginsenoside productivity by combination of ethephon and methyl jasmonate in ginseng (Panax ginseng C.A. Meyer) adventitious root cultures. Biotechnol Lett 28:1163–1166. doi: 10.1007/s10529-006-9071-1 PubMedCrossRefGoogle Scholar
  3. Bang KH, Kim OT, Jung SJ, Kim YC, Park HW, Cha SW (2009) Molecular characterization of ginseng farnesyl diphosphate synthase gene and its up-regulation by methyl jasmonate. Biol Plant (in press)Google Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. doi: 10.1016/0003-2697(76)90527-3 PubMedCrossRefGoogle Scholar
  5. Briskin DP (2000) Medicinal plants and phytomedicines. Linking plant biochemistry and physiology to human health. Plant Physiol 124:507–514. doi: 10.1104/pp.124.2.507 PubMedCrossRefGoogle Scholar
  6. Choi DW, Jung JD, Ha YI, Park HW, In DS, Chung HJ, Liu JR (2005) Analysis of transcripts in methyl jasmonate-treated ginseng hairy roots to identify genes involved in the biosynthesis of ginsenosides and other secondary metabolites. Plant Cell Rep 23:557–566. doi: 10.1007/s00299-004-0845-4 PubMedCrossRefGoogle Scholar
  7. Creelman RA, Mullet JE (1997) Biosynthesis and action of jasmonates in plants. Annu Rev Plant Physiol Plant Mol Biol 48:355–381. doi: 10.1146/annurev.arplant.48.1.355 PubMedCrossRefGoogle Scholar
  8. Daudonnet S, Karst F, Tourte Y (1997) Expression of the farnesyl diphosphate synthase gene of Saccharomyces cerevisiae in tobacco. Mol Breed 3:137–145. doi: 10.1023/A:1009685032495 CrossRefGoogle Scholar
  9. Gorpenchenko TY, Kiselev KV, Bulgakov VP, Tchernoded GK, Bragina EA, Khodakovskaya MV, Koren OG, Batygina TB, Zhuravlev YN (2006) The Agrobacterium rhizogenes rolC-gene-induced somatic embryogenesis and shoot organogenesis in Panax ginseng transformed calluses. Planta 223:457–467. doi: 10.1007/s00425-005-0102-2 PubMedCrossRefGoogle Scholar
  10. Han JY, Kwon YS, Yang DC, Jung YR, Choi YE (2006) Expression and RNA interference-induced silencing of the dammarenediol synthase gene in Panax ginseng. Plant Cell Physiol 47:1653–1662. doi: 10.1093/pcp/pcl032 PubMedCrossRefGoogle Scholar
  11. Hu FX, Zhong JJ (2007) Role of jasmonic acid in alteration of ginsenoside heterogeneity in elicited cell cultures of Panax notoginseng. J Biosci Bioeng 104:513–516. doi: 10.1263/jbb.104.513 PubMedCrossRefGoogle Scholar
  12. Hu FX, Zhong JJ (2008) Jasmonic acid mediates gene transcription of ginsenoside biosynthesis in cell cultures of Panax notoginseng treated with chemically synthesized 2-hydroxyethyl jasmonate. Process Biochem 43:113–118. doi: 10.1016/j.procbio.2007.10.010 CrossRefGoogle Scholar
  13. Hwang B, Ko KM (1989) Induction and culture of hairy roots from ginseng (Panax ginseng C.A. Meyer) roots discs by Agrobacterium rhizogenes. Korean J Biotechnol Bioeng 4:288–292Google Scholar
  14. Kaminaga Y, Sahin FP, Mizukami H (2004) Molecular cloning and characterization of a glucosyltransferase catalyzing glucosylation of curcumin in cultured Catharanthus roseus cells. FEBS Lett 567:197–202. doi: 10.1016/j.febslet.2004.04.056 PubMedCrossRefGoogle Scholar
  15. Kessler A, Baldwin IT (2002) Plant responses to insect herbivory: the emerging molecular analysis. Annu Rev Plant Biol 53:299–328. doi: 10.1146/annurev.arplant.53.100301.135207 PubMedCrossRefGoogle Scholar
  16. Kim YS, Hahn EJ, Murthy HN, Paek KY (2004) Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate. Biotechnol Lett 26:1619–1622. doi: 10.1007/s10529-004-3183-2 PubMedCrossRefGoogle Scholar
  17. Kushiro T, Shibuya M, Ebizuka Y (1998) β-amyrin synthase-cloning of oxidosqualene cyclase that catalyzes the formation of the most popular triterpene among higher plants. Eur J Biochem 256:238–244. doi: 10.1046/j.1432-1327.1998.2560238.x PubMedCrossRefGoogle Scholar
  18. Lee MH, Jeong JH, Seo JW, Shin CG, Kim YS, In JG, Yang DC, Yi JS, Choi YE (2004) Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene. Plant Cell Physiol 45:976–984. doi: 10.1093/pcp/pch126 PubMedCrossRefGoogle Scholar
  19. Lu MB, Wong HL, Teng WL (2001) Effects of elicitation on the production of saponin in cell culture of Panax ginseng. Plant Cell Rep 20:647–677. doi: 10.1007/s002990100349 CrossRefGoogle Scholar
  20. Ma XQ, Liang XM, Xu Q, Zhang XZ, Xiao HB (2005) Identification of ginsenosides in roots of Panax ginseng by HPLC-APCI/MS. Phytochem Anal 16:181–187. doi: 10.1002/pca.842 PubMedCrossRefGoogle Scholar
  21. Meesapyodsuk D, Balsevich J, Reed DW, Covello PS (2007) Saponin biosynthesis in Saponaria vaccaria. cDNAs encoding β-amyrin synthase and a triterpene carboxylic acid glucosyltransferase. Plant Physiol 143:959–969. doi: 10.1104/pp.106.088484 PubMedCrossRefGoogle Scholar
  22. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue. Physiol Plant 15:473–497. doi: 10.1111/j.1399-3054.1962.tb08052.x CrossRefGoogle Scholar
  23. Palazón J, Cusidó RM, Bonfill M, Mallol A, Moyano E, Morales C, Piñol MT (2003) Elicitation of different Panax ginseng transformed root phenotypes for an improved ginsenoside production. Plant Physiol Biochem 41:1019–1025. doi: 10.1016/j.plaphy.2003.09.002 CrossRefGoogle Scholar
  24. Pauwels L, Morreel K, Witte ED, Lammertyn F, Montagu MV, Boerjan W, Inźe D, Goossens A (2008) Mapping methyl jasmonate-mediated transcriptional reprogramming of metabolism and cell cycle progression in cultured Arabidopsis cells. Proc Natl Acad Sci USA 105:1380–1385. doi: 10.1073/pnas.0711203105 PubMedCrossRefGoogle Scholar
  25. Shibata S (2001) Chemistry and cancer preventing activities of ginseng saponins and some related triterpenoid compounds. J Korean Med Sci 16:S28–S37PubMedGoogle Scholar
  26. Shibuya M, Hoshino M, Katsube Y, Hayashi H, Kushiro T, Ebizuka Y (2006) Identification of β-amyrin and sophoradiol 24-hydroxylase by expressed sequence tag mining and functional expression assay. FEBS J 273:948–959. doi: 10.1111/j.1742-4658.2006.05120.x PubMedCrossRefGoogle Scholar
  27. Sticher O (1998) Getting to the root of ginseng. Chemtech 28:26–32Google Scholar
  28. Suzuki H, Achnine L, Xu R, Matsuda SPT, Dixon RA (2002) A genomic approach to the early stages of triterpene saponin biosynthesis in Medicago truncatula. Plant J 32:1033-1048. doi: 10.1046/j.1365-313X.2002.01497.x PubMedCrossRefGoogle Scholar
  29. Tansakul P, Shibuya M, Kushiro T, Ebizuka Y (2006) Dammarenediol-II synthase, the first dedicated enzyme for ginsenoside biosynthesis, in Panax ginseng. FEBS Lett 580:5143–5149. doi: 10.1016/j.febslet.2006.08.044 PubMedCrossRefGoogle Scholar
  30. Wang W, Zhao ZJ, Xu Y, Qian X, Zhong JJ (2006) Efficient induction of ginsenoside biosynthesis and alteration of ginsenoside heterogeneity in cell cultures of Panax notoginseng by using chemically synthesized 2-hyroxylethyl jasmonate. Appl Microbiol Biotechnol 70:298–307. doi: 10.1007/s00253-005-0089-4 PubMedCrossRefGoogle Scholar
  31. Yu KW, Gao W, Hahn EJ, Paek KY (2002) Jasmonic acid improves ginsenoside accumulation in adventitious root culture of Panax ginseng C.A. Meyer. Biochem Eng J 11:211–215. doi: 10.1016/S1369-703X(02)00029-3 CrossRefGoogle Scholar
  32. Yue CJ, Zhong JJ (2005) Purification and characterization of UDPG:ginsenoside Rd glucosyltransferase from suspend cells of Panax notoginseng. Process Biochem 40:3742–3748. doi: 10.1016/j.procbio.2005.05.001 CrossRefGoogle Scholar
  33. Yue CJ, Zhou X, Zhong JJ (2008) Protopanaxadiol 6-hydroxylase and its role in regulating the ginsenoside heterogeneity in Panax notoginseng cells. Biotechnol Bioeng 100:933–940. doi: 10.1002/bit.21829 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Ok Tae Kim
    • 1
  • Kyong Hwan Bang
    • 1
  • Young Chang Kim
    • 1
  • Dong Yun Hyun
    • 1
  • Min Young Kim
    • 2
  • Seon Woo Cha
    • 1
  1. 1.Department of Herbal Crop ResearchNational Institute of Horticultural and Herbal Science, RDAEumseongSouth Korea
  2. 2.Jeonnam Agricultural Research and Extension ServiceNajuSouth Korea

Personalised recommendations