Skip to main content

Advertisement

SpringerLink
Log in

Heterologous production of a ginsenoside saponin (compound K) and its precursors in transgenic tobacco impairs the vegetative and reproductive growth

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

Main conclusion

Production of compound K (a ginsenoside saponin) and its precursors in transgenic tobacco resulted in stunted growth and seed set failure, which may be caused by strong autotoxicity of heterologously produced phytochemicals against the tobacco itself.

Panax ginseng roots contain various saponins (ginsenosides), which are major bioactive compounds. A monoglucosylated saponin, compound K (20-O-(β-d-glucopyranosyl)-20(S)-protopanaxadiol), has high medicinal and cosmetic values but is present in undetectable amounts in naturally grown ginseng roots. The production of compound K (CK) requires complicated deglycosylation of ginsenosides using physicochemical and/or enzymatic degradation. In this work, we report the production of CK in transgenic tobacco by co-overexpressing three genes (PgDDS, CYP716A47 and UGT71A28) isolated from P. ginseng. Introduction and expression of the transgenes in tobacco lines were confirmed by genomic PCR and RT-PCR. All the lines of transgenic tobacco produced CK including its precursors, protopanaxadiol and dammarenediol-II (DD). The concentrations of CK in the leaves ranged from 1.55 to 2.64 µg/g dry weight, depending on the transgenic line. Interestingly, production of CK in tobacco brought stunted plant growth and gave rise to seed set failure. This seed set failure was caused by both long-styled flowers and abnormal pollen development in transgenic tobacco. Both CK and DD treatments highly suppressed in vitro germination and tube growth in wild-type pollens. Based on these results, metabolic engineering for CK production in transgenic tobacco was successfully achieved, but the production of CK and its precursors in tobacco severely affects vegetative and reproductive growth due to the cytotoxicity of phytochemicals that are heterologously produced in transgenic tobacco.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12

Similar content being viewed by others

Abbreviations

PgDDS :

Panax ginseng dammarenediol-II synthase

CYP716A47 :

Protopanaxadiol synthase

UGT71A28 :

Protopanaxadiol 20-glycosyltransferase

qPCR:

Real-time polymerase chain reaction

CK:

Compound K

DD:

Dammarenediol-II

PPD:

Protopanaxadiol

References

  • Aoyagi K, Beyou A, Moon K, Fang L, Ulrich T (1993) Isolation and characterization of cDNAs encoding wheat 3-hydroxy-3-methylglutaryl coenzyme A reductase. Plant Physiol 102:623–628

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bae EA, Han MJ, Kim EJ, Kim DH (2004) Transformation of ginseng saponins to ginsenoside Rh2 by acids and human intestinal bacteria and biological activities of their transformants. Arch Pharmacal Res 27:61–67

    Article  CAS  Google Scholar 

  • Baena-Gonzalez E, Rolland F, Thevelein JM, Sheen J (2007) A central integrator of transcription networks in plant stress and energy signalling. Nature 448:938–942

    Article  CAS  PubMed  Google Scholar 

  • Bertoldo M, Faure M, Dupont J, Froment P (2015) AMPK: a master energy regulator for gonadal function. Front Neurosci 9:235

    Article  PubMed  PubMed Central  Google Scholar 

  • Chappell J, Wolf F, Proulx J, Cuellar R, Saunders C (1995) Is the reaction catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A reductase a rate-limiting step for isoprenoid biosynthesis in plants? Plant Physiol 109:1337–1343

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen Y, Nose M, Ogihara Y (1987) Alkaline cleavage of ginsenosides. Chem Pharm Bull (Tokyo) 35:1653–1655

    Article  CAS  Google Scholar 

  • Cheng F, Cheng Z (2015) Research progress on the use of plant allelopathy in agriculture and the physiological and ecological mechanisms of allelopathy. Front Plant Sci 6:1020

    PubMed  PubMed Central  Google Scholar 

  • Christensen LP (2008) Ginsenosides: chemistry, biosynthesis, analysis, and potential health effects. Adv Food Nutr Res 55:1–99

    Article  Google Scholar 

  • Chun JH, Adhikari PB, Park SB, Han JY, Choi YE (2015) Production of the dammarene sapogenin (protopanaxadiol) in transgenic tobacco plants and cultured cells by heterologous expression of PgDDS and CYP716A47. Plant Cell Rep 34:1551–1560

    Article  CAS  PubMed  Google Scholar 

  • Du GJ, Dai Q, Williams S, Wang CZ, Yuan CS (2011) Synthesis of protopanaxadiol derivatives and evaluation of their anticancer activities. Anticancer Drugs 22:35–45

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dudareva N, Klempien A, Muhlemann JK, Kaplan I (2013) Biosynthesis, function and metabolic engineering of plant volatile organic compounds. New Phytol 198:16–32

    Article  CAS  PubMed  Google Scholar 

  • Han BH, Park MH, Han YN, Woo LK, Sankawa U, Yahara S, Tanaka O (1982) Degradation of ginseng saponins under mild acidic conditions. Planta Med 44:146–149

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Han JY, Kim HJ, Kwon YS, Choi YE (2011) The Cyt P450 enzyme CYP716A47 catalyzes the formation of protopanaxadiol from dammarenediol-II during ginsenoside biosynthesis in Panax ginseng. Plant Cell Physiol 52:2062–2073

    Article  CAS  PubMed  Google Scholar 

  • Han JY, Wang HY, Choi YE (2014) Production of dammarenediol-II triterpene in a cell suspension culture of transgenic tobacco. Plant Cell Rep 33:225–233

    Article  CAS  PubMed  Google Scholar 

  • Hardie DG (2011) AMP-activated protein kinase—an energy sensor that regulates all aspects of cell function. Genes Dev 25:1895–1908

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hardie DG (2015) AMPK: positive and negative regulation, and its role in whole-body energy homeostasis. Curr Opin Cell Biol 33:1–7

    Article  CAS  PubMed  Google Scholar 

  • Hasegawa H, Sung JH, Matsumiya S, Uchiyama M (1996) Main ginseng saponin metabolites formed by intestinal bacteria. Planta Med 62:453–457

    Article  CAS  PubMed  Google Scholar 

  • Hasegawa H, Sung JH, Benno Y (1997) Role of human intestinal prevotella oris in hydrolyzing ginseng saponins. Planta Med 63:436–440

    Article  CAS  PubMed  Google Scholar 

  • He CN, Gao WW, Yang JX, Bi W, Zhang XS, Zhao YJ (2009) Identification of autotoxic compounds from fibrous roots of Panax quinquefolium L. Plant Soil 318:63–72

    Article  CAS  Google Scholar 

  • Huang LF, Song LX, Xia XJ, Mao WH, Shi K, Zhou YH, Yu JQ (2013) Plant-soil feedbacks and soil sickness: from mechanisms to application in agriculture. J Chem Ecol 39:232–242

    Article  CAS  PubMed  Google Scholar 

  • Jensen K, Møller BL (2010) Plant NADPH-cytochrome P450 oxidoreductases. Phytochemistry 71:132–141

    Article  CAS  PubMed  Google Scholar 

  • Jeong YJ, You HJ, Ji GE (2012) Ginsenoside compound K induces cell cycle arrest and apoptosis in human colon cancer cells. Int J Biomed Pharm Sci 6:113–118

    Google Scholar 

  • Jia W, Yan H, Bu X, Liu G, Zhao Y (2004) Aglycone protopanaxadiol, a ginseng saponin inhibits P-glycoprotein and sensitizes chemotherapy drugs on multidrug resistant cancer cells. J Clin Oncol 22:9663

    Article  Google Scholar 

  • Kang KA, Kim YW, Kim SU, Chae S, Koh YS, Kim HS, Choo MK, Kim DH, Hyun JW (2005) G1 phase arrest of the cell cycle by a ginseng metabolite, compound K, in U937 human monocytic leukaemia cells. Arch Pharmacal Res 28:685–690

    Article  CAS  Google Scholar 

  • Kang KA, Lee KH, Chae S, Kim JK, Seo JY, Ham YH, Lee KH, Kim BJ, Kim HS, Kim DH, Hyun JW (2006) Inhibition of telomerase activity in U937 human monocytic leukemia cells by Compound K, a ginseng saponin metabolite. Biotechnol Bioprocess Eng 11:7

    Article  CAS  Google Scholar 

  • Karikura M, Miyase T, Tanizawa H, Taniyama T, Takino Y (1991) Studies on absorption, distribution, excretion and metabolism of ginseng saponins. VII. Comparison of the decomposition modes of ginsenoside-Rb1 and -Rb2 in the digestive tract of rats. Chem Pharm Bull 39:2357–2361

    Article  CAS  PubMed  Google Scholar 

  • Kim WY, Kim JM, Han SB, Lee SK, Kim ND, Park MK, Kim CK, Park JH (2000) Steaming of ginseng at high temperature enhances biological activity. J Nat Prod 63:1702–1704

    Article  CAS  PubMed  Google Scholar 

  • Kim DY, Park MW, Yuan HD, Lee HJ, Kim SH, Chung SH (2009) Compound K induces apoptosis via CAMK-IV/AMPK pathways in HT-29 colon cancer cells. J Agric Food Chem 57:10573–10578

    Article  CAS  PubMed  Google Scholar 

  • Kim AD, Kang KA, Zhang R, Lim CM, Kim HS, Kim DH, Jeon YJ, Lee CH, Park J, Chang WY, Hyun JW (2010) Ginseng saponin metabolite induces apoptosis in MCF-7 breast cancer cells through the modulation of AMP-activated protein kinase. Environ Toxicol Pharmacol 30:134–140

    Article  CAS  PubMed  Google Scholar 

  • Kim YJ, Jeon JN, Jang MG, Oh JY, Kwon WS, Jung SK, Yang DC (2014) Ginsenoside profiles and related gene expression during foliation in Panax ginseng Meyer. J Ginseng Res 38:66–72

    Article  CAS  PubMed  Google Scholar 

  • Law CKM, Kwok HH, Poon PY, Lau CC, Jiang ZH, Tai WCS, Hsiao WWL, Mak NK, Yue PYK, Wong RNS (2014) Ginsenoside compound K induces apoptosis in nasopharyngeal carcinoma cells via activation of apoptosis-inducing factor. Chin Med 9:11

    Article  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Lee IK, Kang KA, Lim CM, Kim KC, Kim HS, Kim DH, Kim BJ, Chang WY, Choi JH, Hyun JW (2010) Compound K, a metabolite of ginseng saponin, induces mitochondria-dependent and caspase-dependent apoptosis via the generation of reactive oxygen species in human colon cancer cells. Int J Mol Sci 11:4916–4931

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lee HS, Kim MR, Park Y, Park HJ, Chang UJ, Kim SY, Suh HJ (2012) Fermenting red ginseng enhances its safety and efficacy as a novel skin care anti-aging ingredient: in vitro and animal study. J Med Food 15:1015–1023

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lei FJ, Zhang AH, Xu YH, Zhang LX (2010) Allelopathic effects of ginsenosides on in vitro growth and antioxidant enzymes activity of ginseng callus. Allelopathy J 26:13–22

    Google Scholar 

  • Leung KW, Wong AST (2010) Pharmacology of ginsenosides: a literature review. Chin Med 5:20

    Article  PubMed  PubMed Central  Google Scholar 

  • Li Y, Huang X, Ding W, Zhang R (2008) Allelopathic effects of soil extracts on the growth of ginseng seeds and its chemical composition. Ecol Environ Sci 17:1173–1178

    Google Scholar 

  • Liu H, Yang J, Du F, Gao X, Ma X, Huang Y, Xu F, Niu W, Wang F, Mao Y, Sun Y, Lu T, Liu C, Zhang B, Li C (2009) Absorption and disposition of ginsenosides after oral administration of Panax notoginseng extract to rats. Drug Metab Dispos 37:2290–2298

    Article  CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Musende AG, Eberding A, Wood CA, Adomat H, Fazli L, Hurtado-Coll A, Jia W, Bally MB, Tomlinson Guns ES (2012) A novel oral dosage formulation of the ginsenoside aglycone protopanaxadiol exhibits therapeutic activity against a hormone-insensitive model of prostate cancer. Anticancer Drugs 23:543–552

    Article  CAS  PubMed  Google Scholar 

  • Nicol RW, Yousef L, Traquair JA, Bernards MA (2003) Ginsenosides stimulate the growth of soilborne pathogens of American ginseng. Phytochemistry 64:257–264

    Article  CAS  PubMed  Google Scholar 

  • Nukarinen E, Nägele T, Pedrotti L, Wurzinger B, Mair A, Landgraf R, Börnke F, Hanson J, Teige M, Baena-Gonzalez E, Dröge-Laser W, Weckwerth W (2016) Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation. Sci Rep 6:31697

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Popovich DG, Kitts DD (2004) Ginsenosides 20(S)-protopanaxadiol and Rh2 reduce cell proliferation and increase sub-G1 cells in two cultured intestinal cell lines, Int-407 and Caco-2. Can J Physiol Pharmacol 82:183–190

    Article  CAS  PubMed  Google Scholar 

  • Rice EL (1995) Biological control of weeds and plant diseases: advances in applied allelopathy. University of Oklahoma Press, Oklahoma

    Google Scholar 

  • Ro DK, Ehlting J, Douglas CJ (2002) Cloning, functional expression, and subcellular localization of multiple NADPH-cytochrome P450 reductases from hybrid poplar. Plant Physiol 130:1837–1851

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ryder NS (1991) Squalene epoxidase as a target for the allylamines. Biochem Soc Trans 19:774–777

    Article  CAS  PubMed  Google Scholar 

  • Saklani A, Kutty SK (2008) Plant-derived compounds in clinical trials. Drug Discov Today 13:161–171

    Article  CAS  PubMed  Google Scholar 

  • Shibata S (2001) Chemistry and cancer preventing activities of ginseng saponins and some related triterpenoid compounds. J Korean Med Sci 16:S28–S37

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Singh HP, Batish DR, Kohli RK (1999) Autotoxicity: concept, organisms, and ecological significance. Crit Rev Plant Sci 18:757–772

    Article  CAS  Google Scholar 

  • Tawab MA, Bahr U, Karas M, Wurglics M, Schubert-Zsilavecz M (2003) Degradation of ginsenosides in humans after oral administration. Drug Metab Dispos 31:1065–1071

    Article  PubMed  Google Scholar 

  • Verpoorte R, Memelink J (2002) Engineering secondary metabolite production in plants. Curr Opin Biotechnol 13:181–187

    Article  CAS  PubMed  Google Scholar 

  • Wang H, Jiang D, Liu J, Ye S, Xiao S, Wang W, Sun Z, Xie Y, Wang J (2013) Compound K induces apoptosis of bladder cancer T24 cells via reactive oxygen species-mediated p38 MAPK pathway. Cancer Biother Radiopharm 28:607–614

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wu S, Chappell J (2008) Metabolic engineering of natural products in plants; tools of the trade and challenges for the future. Curr Opin Biotechnol 19:145–152

    Article  CAS  PubMed  Google Scholar 

  • Yan X, Fan Y, Wei W, Wang P, Liu Q, Wei Y, Zhang L, Zhao G, Yue J, Zhou Z (2014) Production of bioactive ginsenoside compound K in metabolically engineered yeast. Cell Res 24:770–773

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yang M, Zhang X, Xu Y, Mei X, Jiang B, Liao J, Yin Z, Zheng J, Zhao Z, Fan L, He X, Zhu Y, Zhu S (2015a) Autotoxic ginsenosides in the rhizosphere contribute to the replant failure of Panax notoginseng. PLoS One 10:e0118555

    Article  PubMed  PubMed Central  Google Scholar 

  • Yang XD, Yang YY, Ouyang DS, Yang GP (2015b) A review of biotransformation and pharmacology of ginsenoside compound K. Fitoterapia 100:208–220

    Article  CAS  PubMed  Google Scholar 

  • Yousef LF, Bernards MA (2006) In vitro metabolism of ginsenosides by the ginseng root pathogen Pythium irregulare. Phytochemistry 67:1740–1749

    Article  CAS  PubMed  Google Scholar 

  • Zhang AH, Lei FJ, Guo ZX, Zhang LX (2011) Allelopathic effects of ginseng root exudates on the seeds germination and growth of ginseng and American ginseng. Allelopathy J 28:13–20

    Google Scholar 

  • Zhang AH, Tan SQ, Zhao Y, Lei FJ, Zhang LX (2015) Effects of total ginsenosides on the feeding behavior and two enzymes activities of Mythimna separata (Walker) larvae. Evid Based Complement Alternat Med 2015:451828

    PubMed  PubMed Central  Google Scholar 

  • Zhao Y, Wang Y, Shao D, Yang J, Liu D (2005) Autotoxicity of Panax quinquefolium L. Allelopathy J 15:67–74

    Google Scholar 

Download references

Acknowledgements

This work was supported by the Rural Development Administration, Republic of Korea [Next-Generation BioGreen 21 Program (PJ011285)].

Author information

Authors and Affiliations

  1. Department of Forest Resources, College of Forest and Environmental Sciences, Kangwon National University, Chuncheon, 200-701, Republic of Korea

    Yu Shin Gwak, Jung Yeon Han, Prakash Babu Adhikari, Chang Ho Ahn & Yong Eui Choi

Authors
  1. Yu Shin Gwak
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jung Yeon Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Prakash Babu Adhikari
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chang Ho Ahn
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yong Eui Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yong Eui Choi.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gwak, Y.S., Han, J.Y., Adhikari, P.B. et al. Heterologous production of a ginsenoside saponin (compound K) and its precursors in transgenic tobacco impairs the vegetative and reproductive growth. Planta 245, 1105–1119 (2017). https://doi.org/10.1007/s00425-017-2668-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-017-2668-x

Keywords

Search

Navigation