Enhanced biosynthesis of bioactive abietane diterpenes by overexpressing AtDXS or AtDXR genes in Salvia sclarea hairy roots

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Diterpenoids are important compounds for plant survival and have beneficial properties for humans. Bioactive abietanic diterpenes are synthesized in roots of Salvia sclarea (e.g. aethiopinone, 1-oxoaethiopinone, salvipisone, and ferruginol), but at a very low level (about 1 % of root dry weight). To enhance the biosynthesis of this interesting class of compounds, heterologous AtDXS (d-xylulose 5-phosphate synthase) or AtDXR (1-deoxy-d-xylulose 5 phosphate reductoisomerase) genes, encoding the up-stream enzymes of the plastidial 2-C-methyl-D-erythritol 4-phosphate (MEP)-dependent terpenoid pathway, were ectopically expressed in S. sclarea hairy roots. Quantitative targeted metabolic analysis (HPLC–DAD) revealed that three independent root lines, expressing different levels of DXS or DXR transcripts and proteins, synthesized a significant higher content of abietanic diterpenes, compared to the control hairy root line transformed with the empty vector. The increase was gene-dependent, since the overexpression of the AtDXR triggered a 4.4-fold increase in aethiopinone, an abietane quinone-type tricyclic diterpene. In addition, aethiopinone was proved to be cytotoxic to different solid tumor cell lines, with the highest effect on human melanoma A375 cell line (IC50 11.4 µM). Overall these results show that it is possible to boost the metabolic flow towards the synthesis of abietanic diterpenes in S. sclarea hairy roots by overexpressing genes involved in the first steps of the MEP-pathway and provide new insights for the large-scale production of this class of compounds, with potential application in cancer treatment.

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Arabidopsis thaliana


d-xylulose 5-phosphate synthase


1-Deoxy-d-xylulose 5 phosphate reductoisomerase


2-C-methyl-D-erythritol 4-phosphate


Cauliflower mosaic virus


  1. Alasbahi RH, Melzig MF (2010) Plectranthus barbatus: a review of phytochemistry, ethnobotanical uses and pharmacology—Part 2. Planta Med 76:753–765

  2. Bede JC, Musser RO, Felton GW, Korth KL (2006) Caterpillar herbivory and salivary enzymes decrease transcript levels of Medicago truncatula genes encoding early enzymes in terpenoid biosynthesis. Plant Mol Biol 60:519–531

  3. Caniard A, Zerbe P, Legrand S, Cohade A, Valot N, Magnard JL, Bohlmann J, Legendre L (2012) Discovery and functional characterization of two diterpene synthase for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture. BMC Plant Biol 12:119–132

  4. Carretero-Paulet L, Ahumada I, Cunillera N, Rodríguez-Concepción M, Ferrer A, Boronat A, Campos N (2002) Expression and molecular analysis of the Arabidopsis DXR gene enconding 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the first committed enzyme of the 2-C-methyl-D-erythritol 4-phosphate pathway. Plant Physiol 129:1581–1591

  5. Carretero-Paulet L, Cairó A, Botella-Pavía P, Besumbes O, Campos N, Boronat A, Rodríguez-Concepción M (2006) Enhanced flux through the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxylulose 5-phosphate reductoisomerase. Plant Mol Biol 62:683–695

  6. Chappell J (1995) Biochemistry and molecular biology of the isoprenoid pathway in plants. Annu Rev Plant Mol Biol 46:521–547

  7. Cordoba E, Salmi M, León P (2009) Unravelling the regulatory mechanisms that modulate the MEP pathway in higher plants. J Exp Bot 60:2933–2943

  8. Dat NT, Jin X, Lee JH, Lee D, Hong YS, Lee K, Kim YH, Lee JJ (2007) Abietane diterpenes from Salvia miltiorrhiza inhibit the activation of hypoxia-inducible factor-1. J Nat Prod 70:1093–1097

  9. Doyle JJ, Doyle JL (1990) A rapid total DNA preparation procedure for fresh plant tissue. Focus 12:13–15

  10. Dudareva N, Andersson S, Orlova I, Gatto N, Reichelt M, Rhodes D, Boland W, Gerchenzon J (2005) The nonmevalonate pathway supportos both monoterpene and sesquiterpenes formation in snapdragon flowers. Proc Natl Acad Sci USA 102:933–938

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

  12. Enfissi EM, Fraser PD, Lois LM, Boronat A, Schuch W, Bramley PM (2005) Metabolic engineering of the mevalonate and non mevalonate isopentenyl diphosphate-forming pathways for the production of health-promoting isoprenoids in tomato. Plant Biotech J 3:17–27

  13. Estévez JM, Cantero A, Reindl A, Reichler S, León P (2001) 1-Deoxy-D-xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants. J Biol Chem 276:22901–22909

  14. Fronza M, Lamy E, Günther S, Heinzmann B, Laufer S, Merfort I (2012) Abietane diterpenes induce cytotoxic effects in human pancreatic cancer cell line MIA PaCa-2 through different modes of action. Phytochemistry 78:107–119

  15. Georgiev M, Agostini E, Ludwig-Muller J, Xu J (2012) Genetically transformed roots: from plant disease to biotechnological resource. Trends Biotech 30:528–537

  16. Haas JH, Moore LW, Ream W, Manulis S (1995) Universal PCR primers for detection of phytopathogenic Agrobacterium strains. Appl Environ Microbiol 61:2879–2884

  17. Hans J, Hause B, Strack D, Walter MH (2004) Cloning, characterization, and immunolocalization of a mycorrhiza-inducible 1-deoxy-D-xylulose 5-phosphate reductoisomerase in arbuscule-containing cells of maize. Plant Physiol 134:614–624

  18. Hasunuma T, Takeno S, Hayashi S, Sendai M, Bamba T, Yoshimura S, Tomizawa K, Fukusaki E, Miyake C (2008) Overexpression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene in chloroplast contributes to increment of isoprenoid production. J Biosci Bioeng 105:518–526

  19. Hasunuma T, Kondo A, Miyake C (2010) Metabolic engineering by plastid transformation as a strategy to modulate isoprenoid yield in plants. Methods Mol Biol 643:213–227

  20. Hsieh MH, Goodman HM (2005) The Arabidopsis IspH homolog is involved in the plastid nonmevalonate pathway of isoprenoid biosynthesis. Plant Physiol 138:641–653

  21. Jennewein S, Croteau R (2001) Taxol: biosynthesis, molecular genetics, and biotechnological applications. Appl Microbiol Biotechnol 57:13–19

  22. Kai G, Xu H, Zhou C, Liao P, Xiao J, Luo X, You L, Zhang L (2011) Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures. Metab Engin 13:319–327

  23. Kuźma Ł, Skrzypek Z, Wysokińska H (2006) Diterpenoids and triterpenoids in hairy roots of Salvia sclarea. Plant Cell Tissue Org Cult 84:171–179

  24. Kuźma L, Rozalsk M, Walencka E, Rozalska B, Wysokinska H (2007) Antimicrobial activity of diterpenoids from hairy roots of Salvia sclarea L: salvipisone as a potential antibiofilm agent active against antibiotic resistant staphylococci. Phytomedicine 14:31–35

  25. Lange BM, Wildung MR, McCaskill D, Croteau R (1998) A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway. Proc Nat Acad Sci USA 95:2100–2104

  26. Lange BM, Rujan T, Martin W, Croteau R (2000) Isoprenoid biosynthesis: the evolution of two ancient and distinct pathways across genomes. Proc Natl Acad Sci USA 97:13172–13177

  27. Li L, Shukla S, Lee A, Garfield SH, Maloney DJ, Ambudkar SV, Yuspa SH (2010) The skin cancer chemotherapeutic agent ingenol-3-angelate (PEP005) is a substrate for the epidermal multidrug transporter (ABCB1)and targets tumor vasculature. Cancer Res 70:4509–4519

  28. Lois LM, Campos N, Putra SR, Danielsen K, Rohmer M, Boronat A (1998) Cloning and characterization of a gene from Escherichia coli encoding a tranketolase-like enzyme that catalyzes the synthesis of D-1-deoxyxylulose 5 –phosphate, a common precursor for isoprenoid, thiamin, and pyridoxol biosynthesis. Proc Natl Acad Sci USA 95:2105–2110

  29. Lu Q, Zhang P, Zhang X, Chen J (2009) Experimental study of the anti-cancer mechanism of tanshinone IIA against human breast cancer. J Int Mol Med 24:773–780

  30. Mahaira LG, Tsimplouli C, Sakellaridis N, Alevizopoulos K, Demetzos C, Han Z, Pantazis P, Dimas K (2011) The labdane diterpene sclareol (labd-14-ene-8, 13-diol) induces apoptosis in human tumor cell lines and suppression of tumor growth in vivo via a p53-independent mechanism of action. Eur J Pharmacol 666:173–182

  31. Mahmoud SS, Croteau RB (2001) Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxy-xylulose phosphate reductoisomerase and menthofuran synthase. Proc Nat Acad Sci USA 98:8915–8920

  32. Mayrhofer S, Teuber M, Zimmer I, Louis S, Fischbach RJ, Schnitzler JP (2005) Diurnal and seasonal variation of isoprene biosynthesis-related genes in grey poplar leaves. Plant Physiol 139:474–484

  33. Miñoz-Bertomeu J, Arrillaga I, Ros R, Segura J (2006) Up-regulation of 1-deoxy-D-xylulose-5-phosphate synthase enhances production of essential oils in transgenic spike lavender. Plant Physiol 142:890–900

  34. Mnonopi N, Levendal RA, Mzilikazi N, Frost CL (2012) Marrubiin, a constituent of Leonotis leonurus, alleviates diabetic symptoms. Phytomedicine 19:488–493

  35. Morris WL, Ducreux LJ, Hedden P, Millam S, Taylor MA (2006) Overexpression of a bacterial 1-deoxy-D-xylulose 5-phosphate synthase gene in potato tubers perturbs the isoprenoid metabolic network: implications for the control of the tuber life cycle. J Exp Bot 57:3007–3018

  36. Moses T, Pollier J, Thevelein JM, Goossens A (2013) Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Phytol 200:27–43

  37. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63

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

  39. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocite apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–279

  40. Noori S, Hassan ZM, Mohammad M, Habibi Z, Sohrabi N, Bavanolhagh S (2010) Sclareol modulates the Treg intra-tumoral infiltrated cell and inhibits tumor growth in vivo. Cell Immunol 263:148–153

  41. Opipari AWJr, Hu HM, Yabkowitz R, Dixit VM (1992) The A20 zinc-finger protein protects cells from turmor necrosis factor cytotoxicity. J Biol Chem 297:12424–12427

  42. Pan TL, Hung YC, Wang PW, Chen ST, Hsu TK, Sintupisut N, Cheng CS, Lyu PC (2010) Functional proteomic and structural insights into molecular targets related to the growth inhibitory effect of tanshinone IIA on HeLa cells. Proteomics 10:914–929

  43. Peebles AM, Sander G, Hughes E, Peacok R, Shanks J, San K (2011) The expression of 1-deoxy-D-xylulose synthase and geraniol-10-hydroxylase or anthranilate synthase increases terpenoid indole alkaloid accumulation in Catharanthus roseus hairy roots. Metab Eng 13:234–240

  44. Phillips M, León P, Boronat A, Rodríguez-Concepción M (2008) The plastidial MEP pathway unified nomenclature and resources. Trend Plant Sci 13:619–623

  45. Pulido P, Toledo-Ortiz G, Phillips MA, Wright LP, Rodríguez-Concepción M (2013) Arabidopsis j-protein j20 delivers the first enzyme of the plastidial isoprenoid pathway to protein quality control. Plant Cell 25:4183–4194

  46. Rigel DS, Carucci JA (2000) Malignant melanoma: prevention, early detection, and treatment in the 21st century. J CA Cancer Clin 50:215–236

  47. Rodríguez-Concepción M, Boronat A (2002) Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics. Plant Physiol 130:1079–1089

  48. Rodríguez-Concepción M, Ahumada I, Diez-Juez E, Sauret-Gueto S, Lois LM, Gallego F, Carretero Paulet L, Campos N, Boronat A (2001) 1-Deoxy-D-xylulose 5-phosphate reductoisomerase and plastid isoprenoids biosynthesis during tomato fruit ripening. Plant J 27:213–222

  49. Rohmer M, Seeman M, Horbach S, Bringer-Meyer S, Sahm H (1996) Glyceraldehyde 3-phosphate and pyruvate as precursors of isoprenic units in an alternative non-mevalonate pathway for terpenoid biosynthesis. J Am Chem Soc 118:2564–2566

  50. Rózalski M, Kuźma L, Krajewska U, Wysokinska H (2006) Cytotoxic and proapoptotic activity of diterpenoids from in vitro cultivated Salvia sclarea roots. Studies on the leukemia cell lines. Z Naturforsch 61:483–488

  51. Schardl CL, Byrd AD, Bezion G, Altschuler MA, Hildebrand DF, Hunt AG (1987) Design and construction of a versatile system for the expression of foreign genes in plants. Gene 61:1–11

  52. Talano M, Oller A, González P, Agostini E (2012) Hairy roots their multiple applications and recents patents. Recent Plant Biotech 6:115–133

  53. Topcu G, Goren AC (2007) Biological activity of diterpenoids isolated from Anatolian laminaceae plants. Rec Nat Prod 1:1–16

  54. Ulubelen A, Eriş C, Sönmez U, Kartal M, Kurucu S, Bozok-Johansson C (1994) Terpenoids from Salvia sclarea. Phytochemistry 36:971–974

  55. Ulubelen A, Sonmez U, Topcu G (1997) Diterpenoids from the roots of Salvia sclarea. Phytochemistry 344:1297–1299

  56. Veau B, Courtois A, Oudin J, Chenieux C, Rideau M, Clastre M (2000) Cloning and expression of cDNAs enconding two enzymes of the MEP pathway in Catharanthus roseus. Biochem Biophys Acta Gene Struct Exp 1517:159–163

  57. Walter M, Fester T, Strack D (2000) Arbuscular mycorrhizal fungi induce the non-mevalonate methylerythritol phosphate pathway of isoprenoid biosynthesis correlated with the accumulation of the yellow pigment and other apocarotenoids. Plant J 21:571–578

  58. Weigel D, Glazebrook J (2006) Transformation of Agrobacterium using the freeze-thaw method. CSH Protoc 7:110–115

  59. Wu S, Shalk M, Clark A, Miles RB, Coates R, Chappell J (2006) Redirection of cytosolic or plastidic isoprenoid precursors elevates terpene production in plants. Nat Biotech 24:1141–1147

  60. Xiang L, Zeng L, Yuan Y, Chen M, Wang F, Liu X, Zeng L, Lan X, Liao Z (2012) Enanchement of artemisinin biosynthesis by overexpressing dxr, cyp71av1 and cpr in the plants of Artemisia annua L. Plant Omics J 5:503–507

  61. Yang L, Ding G, Lin H, Cheng H, Kong Y, Wei Y, Fang X, Liu R, Wang L, Chen X, Yang C (2013) Transcriptome analysis of medicinal plant Salvia miltiorrhiza and identification of genes related to tanshinone biosynthesis. PLoS ONE 8:e80464

  62. Yoon V, Nodwell JR (2014) Activating secondary metabolism with stress and chemicals. J Ind Microbiol Biotechnol 41:415–424

  63. Zerbe P, Hamberger B, Yuen MM, Chiang A, Sandhu HK, Madilao LL, Nguyen A, Hamberger B, Bach SS, Bohlmann J (2013) Gene discovery of modular diterpene metabolism in non model systems. Plant Physiol 162:1073–1091

  64. Zhou L, Chan WK, Xu N, Xiao K, Luo H, Luo KQ, Chang DC (2008) Tanshinone IIA, an isolated compound from Salvia miltiorrhiza Bunge, induces apoptosis in HeLa cells through mitotic arrest. Life Sci 83:394–403

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This work was supported by funds provided to AL by the Research Project of National Interest (PRIN 2005), the Italian Ministry of University of Education (MIUR) and by a Short-Term Scientific Mission to MCV by COST Action FA1006 “Plant Metabolic Engineering for High Value Products”. We would like to acknowledge Prof. M.H. Walter, Plant Biochemical Institute, Halle, Germany, for the gift of ZmDXR antibody.

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Correspondence to Mariacarmela Vaccaro.

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Vaccaro, M., Malafronte, N., Alfieri, M. et al. Enhanced biosynthesis of bioactive abietane diterpenes by overexpressing AtDXS or AtDXR genes in Salvia sclarea hairy roots. Plant Cell Tiss Organ Cult 119, 65–77 (2014) doi:10.1007/s11240-014-0514-4

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  • Bioactive diterpenes
  • Salvia sclarea
  • Hairy roots
  • Metabolic engineering