Plant Molecular Biology

, Volume 95, Issue 1–2, pp 51–62 | Cite as

A MDR transporter contributes to the different extracellular production of sesquiterpene pyridine alkaloids between adventitious root and hairy root liquid cultures of Tripterygium wilfordii Hook.f.



Key message

TwMDR1 transports sesquiterpene pyridine alkaloids, wilforine and wilforgine, into the hairy roots of T. wilfordii Hook.f. resulting in low secretion ratio of alkaloids.


Hairy roots (HRs) exhibit high growth rate and biochemical and genetic stability. However, varying secondary metabolites in HR liquid cultures mainly remain in root tissues, and this condition may affect cell growth and cause inconvenience in downstream extraction. Studies pay less attention to adventitious root (AR) liquid cultures though release ratio of some metabolites in AR liquid cultures is significantly higher than that of HR. In Tripterygium wilfordii Hook.f., release ratio of wilforine in AR liquid cultures reached 92.75 and 13.32% in HR on day 15 of culture. To explore potential roles of transporters in this phenomenon, we cloned and functionally identified a multidrug resistance (MDR) transporter, TwMDR1, which shows high expression levels in HRs and is correlated to transmembrane transportation of alkaloids. Nicotiana tabacum cells with overexpressed TwMDR1 efficiently transported wilforine and wilforgine in an inward direction. To further prove the feasibility of genetically engineered TwMDR1 and improve alkaloid production, we performed a transient RNAi experiment on TwMDR1 in T. wilfordii Hook.f. suspension cells. Results indicated that release ratios of wilforine and wilforgine increased by 1.94- and 1.64-folds compared with that of the control group, respectively. This study provides bases for future studies that aim at increasing secretion ratios of alkaloids in root liquid cultures in vitro.


ABC transporter Hairy root Adventitious root Sesquiterpene pyridine alkaloids 



This study was supported by Educational Commission of Anhui Province of China (KJ2016A668), the National Natural Science Foundation of Anhui Province (1708085QC52), the National Natural Science Foundation of China (Grant No. 31272110) and the Key Project of the Outstanding Young Talent Support Program of the University of Anhui Province (gxyqZD2016264).

Author Contributions

XZ and CZ: conceived and designed the experiments. GM and JH: performed the experiments. JZ: analyzed the data. XZ and CZ: wrote the paper.

Compliance with ethical standards

Conflict of interest

The authors declare no competing financial interest.

Supplementary material

11103_2017_634_MOESM1_ESM.xlsx (32 kb)
Online Resource 1—Protein sequence alignment of TwMDR1 with CjMDR1 (BAB62040.1) and CjABCB2 (BAM11098.1). Two TMDs and two NBDs, arranged in TMD1-NBD1-TMD2-NBD2 direction, are indicated in black frame. Within NBDs, three characteristic motifs common to all ABC transporters, Walker A, Walker B, and ABC-signature motif close to the Walker B domain, are shown in bold. Identical residues in sequences are shadowed. Gaps are represented by dashes and introduced for optimal alignment (XLSX 31 KB)
11103_2017_634_MOESM2_ESM.tif (710 kb)
Online Resource 2—Accumulation of wilforine and reduction percentages in different kinds of media. On day 15 of HR liquid cultures, medium was separated from HR. After adding 100 µM MeJA to the above cultures for 9 h, medium (+MeJA) was filtrated and immediately supplemented with 40 µg/100 mL wilforine. Both media were maintained at 25 °C with 120 rpm rotation and monitored for wilforine contents at 0, 3, 6, 9, 12, and 24 h. Medium with HR (+MeJA) was directly collected from HR liquid cultures treated by 100 µM MeJA at corresponding time points (TIF 710 KB)


  1. Baque MA, Moh SH, Lee EJ, Zhong JJ, Paek KY (2012) Production of biomass and useful compounds from adventitious roots of high-value added medicinal plants using bioreactor. Biotechnol Adv 30:1255–1267. doi: 10.1016/j.biotechadv.2011.11.004 CrossRefPubMedGoogle Scholar
  2. Brinker AM, Ma J, Lipsky PE, Raskin I (2007) Medicinal chemistry and pharmacology of genus Tripterygium (Celastraceae). Phytochemistry 68:732–766. doi: 10.1016/j.phytochem.2006.11.029 CrossRefPubMedGoogle Scholar
  3. Cai Z, Kastell A, Knorr D, Smetanska I (2012) Exudation: an expanding technique for continuous production and release of secondary metabolites from plant cell suspension and hairy root cultures. Plant Cell Rep 31:461–477. doi: 10.1007/s00299-011-1165-0 CrossRefPubMedGoogle Scholar
  4. Crouzet J, Roland J, Peeters E, Trombik T, Ducos E, Nader J, Boutry M (2013) NtPDR1, a plasma membrane ABC transporter from Nicotiana tabacum, is involved in diterpene transport. Plant Molecul Biol 82:181–192. doi: 10.1007/s11103-013-0053-0 CrossRefGoogle Scholar
  5. Cutanda-Perez MC et al. (2009) Ectopic expression of VlmybA1 in grapevine activates a narrow set of genes involved in anthocyanin synthesis and transport. Plant Molecul Biol 69(6):633–648CrossRefGoogle Scholar
  6. Duan H, Takaishi Y, Momota H, Ohmoto Y, Taki T, Jia Y, Li D (2001) Immunosuppressive sesquiterpene alkaloids from Tripterygium wilfordii. J Nat Prod 64:582–587CrossRefPubMedGoogle Scholar
  7. Goossens A, Häkkinen ST, Laakso I, Oksman-Caldentey K-M, Inzé D (2003) Secretion of secondary metabolites by ATP-binding cassette transporters in plant cell suspension cultures. Plant Physiol 131:1161–1164CrossRefPubMedPubMedCentralGoogle Scholar
  8. Horiuch M et al (2006) Tripfordines A-C, sesquiterpene pyridine alkaloids from Tripterygium wilfordii, and structure anti-HIV activity relationships of Tripterygium alkaloids. J Nat Prod 69:1271–1274. doi: 10.1021/np060124a CrossRefPubMedGoogle Scholar
  9. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231CrossRefGoogle Scholar
  10. Karimi M, Inze D, Depicker A (2002) GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195CrossRefPubMedGoogle Scholar
  11. Kell DB, Swainston N, Pir P, Oliver SG (2015) Membrane transporter engineering in industrial biotechnology and whole cell biocatalysis. Trends Biotechnol 33:237–246. doi: 10.1016/j.tibtech.2015.02.001 CrossRefPubMedGoogle Scholar
  12. Kuo YH, Chen CH, Kuo LM, King ML, Wu TS, Haruna M, Lee KH (1990) Antitumor agents, 112. Emarginatine B, a novel potent cytotoxic sesquiterpene pyridine alkaloid from Maytenus emarginata. J Nat Prod 53:422–428CrossRefPubMedGoogle Scholar
  13. Lhinhatrakool T, Prabpai S, Kongsaeree P, Sutthivaiyakit S (2011) Antiplasmodial sesquiterpene alkaloids from the roots of Maytenus mekongensis. J Nat Prod 74:1386–1391. doi: 10.1021/np200014k CrossRefPubMedGoogle Scholar
  14. Li L, He Z, Pandey GK, Tsuchiya T, Luan S (2002) Functional cloning and characterization of a plant efflux carrier for multidrug and heavy metal detoxification. J Biol Chem 277:5360–5368. doi: 10.1074/jbc.M108777200 CrossRefPubMedGoogle Scholar
  15. Liao LM (2003) Sesquiterpene pyridine alkaloids. Alkaloids Chem Biol 60:287–343CrossRefPubMedGoogle Scholar
  16. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif) 25:402–408. doi: 10.1006/meth.2001.1262 CrossRefPubMedGoogle Scholar
  17. Mano Y, Ohkawa H, Yamada Y (1989) Production of tropane alkaloids by hairy root cultures of Duboisia leichhardtii transformed by Agrobacterium rhizogenes. Plant Sci 59:191–201. doi: 10.1016/0168-9452(89)90137-4 CrossRefGoogle Scholar
  18. Miao GP, Zhu CS, Yang YQ, Feng MX, Ma ZQ, Feng JT, Zhang X (2014) Elicitation and in situ adsorption enhanced secondary metabolites production of Tripterygium wilfordii Hook. f. adventitious root fragment liquid cultures in shake flask and a modified bubble column bioreactor. Bioprocess Biosyst Eng 37:641–650. doi: 10.1007/s00449-013-1033-0 CrossRefPubMedGoogle Scholar
  19. Miao GP et al (2015) Identification of genes involved in the biosynthesis of Tripterygium wilfordii Hook. f. secondary metabolites by suppression subtractive hybridization. Plant Mol Biol Rep 33:756–769CrossRefGoogle Scholar
  20. Morita M et al (2009) Vacuolar transport of nicotine is mediated by a multidrug and toxic compound extrusion (MATE) transporter in Nicotiana tabacum. Proc Nat Acad Sci 106:2447–2452. doi: 10.1073/pnas.0812512106 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Murthy HN, Lee EJ, Paek KY (2014) Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation. Plant Cell Tiss Org 118:1–16. doi: 10.1007/s11240-014-0467-7 CrossRefGoogle Scholar
  22. Nour-Eldin HH, Halkier BA (2013) The emerging field of transport engineering of plant specialized metabolites. Curr Opin Biotechnol 24:263–270. doi: 10.1016/j.copbio.2012.09.006 CrossRefPubMedGoogle Scholar
  23. Plasencia A et al. (2016) Eucalyptus hairy roots, a fast, efficient and versatile tool to explore function and expression of genes involved in wood formation. Plant Biotechnol J 14:1381–1393CrossRefPubMedGoogle Scholar
  24. Pomahačová B, Dušek J, Dušková J, Yazaki K, Roytrakul S, Verpoorte R (2009) Improved accumulation of ajmalicine and tetrahydroalstonine in Catharanthus cells expressing an ABC transporter. J Plant Physiol 166:1405–1412. doi: 10.1016/j.jplph.2009.02.015 CrossRefPubMedGoogle Scholar
  25. Ron M et al. (2014) Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model. Plant Physiol 166:455–469CrossRefPubMedPubMedCentralGoogle Scholar
  26. Ruiz-May E, Galaz-Ávalos RM, Loyola-Vargas VM (2008) Differential secretion and accumulation of terpene indole alkaloids in hairy roots of Catharanthus roseus treated with methyl jasmonate. Mol Biotechnol 41:278–285. doi: 10.1007/s12033-008-9111-2 CrossRefPubMedGoogle Scholar
  27. Sakai K, Shitan N, Sato F, Ueda K, Yazaki K (2002) Characterization of berberine transport into Coptis japonica cells and the involvement of ABC protein. J Experiment Bot 53:1879–1886CrossRefGoogle Scholar
  28. Sang Jun S, Ho Nam C, Jang Ryol L, Kyung Hee J (1994) Production and secretion of indole alkaloids in hairy root cultures of Catharanthus roseus: Effects of in situ adsorption, fungal elicitation and permeabilization. J Ferment Bioeng 78:229–234. doi: 10.1016/0922-338X(94)90295-X CrossRefGoogle Scholar
  29. Santos VA et al (2012) Antiprotozoal sesquiterpene pyridine alkaloids from Maytenus ilicifolia. J Nat Prod 75:991–995. doi: 10.1021/np300077r CrossRefPubMedGoogle Scholar
  30. Sharma P, Padh H, Shrivastava N (2013) Hairy root cultures: A suitable biological system for studying secondary metabolic pathways in plants. Eng Life Sci 13:62–75CrossRefGoogle Scholar
  31. Shi BJ, Ji ZQ, Zhang JW, Wu WJ (2007) Insecticidal activities and active ingredients of Tripterygium hypoglaucum (Levl.). Hutch Acta Entomologica Sinica 50:795–800Google Scholar
  32. Shitan N et al (2003) Involvement of CjMDR1, a plant multidrug-resistance-type ATP-binding cassette protein, in alkaloid transport in Coptis japonica. Proc Nat Acad Sci 100:751–756. doi: 10.1073/pnas.0134257100 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Shoji T (2014) Chapter Six—ATP-binding cassette and multidrug and toxic compound extrusion transporters in plants: a common theme among diverse detoxification mechanisms. In: Kwang WJ (ed) International review of cell and molecular biology, vol 309. Academic Press, New York, pp 303–346. doi: 10.1016/B978-0-12-800255-1.00006-5 Google Scholar
  34. Szewczak A, Ziomkiewicz I, Jasinski M (2011) Hiring cell gatekeepers–ABC transporters in plant biotechnology. BioTechnologia J Biotechnol Comput Biol Bionanotechnol 92:132–139Google Scholar
  35. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599. doi: 10.1093/molbev/msm092 CrossRefPubMedGoogle Scholar
  36. Taya M, Mine K, Kino-Oka M, Tone S, Ichi T (1992) Production and release of pigments by culture of transformed hairy root of red beet. J Ferment Bioengin 73:31–36CrossRefGoogle Scholar
  37. Zhi BH, Alfermann A (1993) Diterpenoid production in hairy root cultures of Salvia miltiorrhiza. Phytochemistry 32:699–703CrossRefGoogle Scholar
  38. Zhu JB, Wang MG, Wu WJ, Ji ZQ, Hu ZN (2002) Insecticidal sesquiterpene pyridine alkaloids from Euonymus species. Phytochemistry 61:699–704. doi: 10.1016/S0031-9422(02)00335-7 CrossRefGoogle Scholar
  39. Zhu C, Miao G, Guo J, Huo Y, Zhang X, Xie J, Feng J (2014) Establishment of Tripterygium wilfordii Hook.f. hairy root culture and optimization of its culture conditions for the production of triptolide and wilforine. J Microbiol Biotechnol. doi: 10.4014/jmb.1402.02045 Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  1. 1.Department of BioengineeringHuainan Normal UniversityHuainanChina
  2. 2.Research & Development Center of Biorational PesticidesNorthwest A&F UniversityYanglingChina

Personalised recommendations