Abstract
Key message
Transcriptional activation of genes belonging to the plastidial MEP-derived isoprenoid pathway by elicitation with methyl jasmonate and coronatine enhanced the content of bioactive abietane diterpenes in Salvia sclarea hairy roots.
Abstract
We have shown that aethiopinone, an abietane diterpene synthesized in Salvia sclarea roots is cytotoxic and induces apoptosis in human melanoma cells. To develop a production platform for this compound and other abietane diterpenes, hairy root technology was combined with the elicitation of methyl jasmonate (MeJA) or the phytotoxin coronatine (Cor). Both MeJA and Cor induced a significant accumulation of aethiopinone, but prolonged exposure to MeJA irremediably caused inhibition of hairy root growth, which was unaffected by Cor treatment. Considering together the fold increase in aethiopinone content and the final hairy root biomass, the best combination was a Cor treatment for 28 days, which allowed to obtain up to 105.34 ± 2.30 mg L−1 of this compound to be obtained, corresponding to a 24-fold increase above the basal content in untreated hairy roots. MeJA or Cor elicitation also enhanced the synthesis of other bioactive abietane–quinone diterpenes. The elicitor-dependent steering effect was due to a coordinated transcriptional activation of several biosynthetic genes belonging to the plastidial MEP-derived isoprenoid pathway. High correlations between aethiopinone content and MeJA or Cor-elicited level of gene transcripts were found for DXS2 (r 2 = 0.99), DXR (r 2 = 0.99), and GGPPS (r 2 = 0.98), encoding enzymes acting upstream of GGPP, the common precursor of diterpenes and other plastidial-derived terpenes, as well as CPPS (r 2 = 0.99), encoding the enzyme involved in the first cyclization steps leading to copalyl-diphosphate, the precursor of abietane-like diterpenes. These results point to these genes as possible targets of metabolic engineering approaches to establish a more efficient production platform for such promising anti-proliferative plant-derived compounds.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Change history
10 August 2018
Unfortunately, the second author name was wrongly published in the original publication. The correct author name should read as follows.
27 December 2016
An erratum to this article has been published.
References
Atanasov AG, Waltenberger B, Pferschy-Wenzig EM, Linder T, Wawrosch C, Uhrin P (2015) Discovery and resupply of pharmacologically active plant-derived natural products: a review. Biotechnol Adv 33:1582–1614. doi:10.1016/j.biotechadv.2015.08.001
Baenas N, García-Viguera C, Moreno DA (2014) Biotic elicitors effectively increase the glucosinolates content in Brassicaceae sprouts. J Agr Food Chem 62:1881–1889. doi:10.1021/jf404876z
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 30:1255–1264. doi:10.1016/j.biotechadv.2011.11.004
Bonito MC, Cicala C, Marcotullio MC, Maione F, Mascolo N (2011) Biological activity of bicyclic and tricyclic diterpenoids from Salvia species of immediate pharmacological and pharmaceutical interest. Nat Prod Commun 6:1205–1215
Božić D, Papaefthimiou D, Brückner K, de Vos RC, Tsoleridis CA, Katsarou D, Papanikolaou A, Pateraki I, Chatzopoulou FM, Dimitriadou E, Kostas S, Manzano D, Scheler U, Ferrer A, Tissier A, Makris AM, Kampranis SC, Kanellis AK (2015) Towards elucidating carnosic acid biosynthesis in Lamiaceae: functional characterization of the three first steps of the pathway in Salvia fruticosa and Rosmarinus officinalis. PLoS One 10:e0124106. doi:10.1371/journal.pone.0124106
Chatzopoulou FM, Makris AM, Argiriou A, Degenhardt J, Kanellis AK (2010) EST analysis and annotation of transcripts derived from a trichome-specific cDNA library from Salvia fruticosa. Plant Cell Rep 29:523–534. doi:10.1007/s00299-010-0841-9
Cheng Q, He Y, Li G, Liu Y, Gao W, Huang L (2013) Effects of combined elicitors on tanshinone metabolic profiling and SmCPS expression in Salvia miltiorrhiza hairy root cultures. Molecules 18:7473–7485. doi:10.3390/molecules18077473
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. doi:10.1093/jxb/erp190
De Geyter N, Gholami A, Goormachtig S, Goossens A (2012) Transcriptional machineries in jasmonate-elicited plant secondary metabolism. Trends Plant Sci 17:349–359. doi:10.1016/j.tplants.2012.03.001
Dudareva N, Della Penna D (2013) Plant metabolic engineering: future prospects and challenges. Curr Opin Biotechnol 24:226–228. doi:10.1016/j.copbio.2013.02.002
Feng S, Ma L, Wang X, Xie D, Dinesh-Kumar SP, Wei N, Deng XW (2003) The COP9 signalosome interacts physically with SCF COI1 and modulates jasmonate responses. Plant Cell 15:1083–1094. doi:10.1105/tpc.010207
Fliegmann J, Schüler G, Boland W, Ebel J, Mithöfer A (2003) The role of octadecanoids and functional mimics in soybean defense responses. Biol Chem 384:437–446. doi:10.1515/BC.2003.049
Fonseca S, Chini A, Hamberg M, Adie B, Porzel A, Kramell R, Miersch O, Wasternack C, Solano R (2009) (+)-7-iso-Jasmonoyl-l-isoleucine is the endogenous bioactive jasmonate. Nat Chem Biol 5:344–350. doi:10.1038/nchembio.161
Gallego A, Imseng N, Bonfill M, Cusido RM, Palazon J, Eibl R, Moyano E (2015) Development of a hazel cell culture-based paclitaxel and baccatin III production process on a benchtop scale. J Biotechnol 195:93–102. doi:10.1016/j.jbiotec.2014
Gao W, Sun HX, Xiao H, Cui G, Hillwig ML, Jackson A, Wang X, Shen Y, Zhao N, Zhang L, Wang XJ, Peters RJ, Huang L (2014) Combining metabolomics and transcriptomics to characterize tanshinone biosynthesis in Salvia miltiorrhiza. BMC Genom 28:15–73. doi:10.1186/1471-2164-15-73
Geng X, Jin L, Shimada M, Kim MG, Mackey D (2014) The phytotoxin coronatine is a multifunctional component of the virulence armament of Pseudomonas syringae. Planta 240:1149–1165. doi:10.1007/s00425-014-2151-x
González MA (2015) Aromatic abietane diterpenoids: their biological activity and synthesis. Nat Prod Rep 32:684–704. doi:10.1039/c4np00110a
Guo J, Zhou YJ, Hillwig ML, Shen Y, Yang L, Wang Y, Zhang X, Liu W, Peters RJ, Chen X, Zhao ZK, Huang L (2013) CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts. Proc Natl Acad Sci USA 110:12108–12113. doi:10.1073/pnas.1218061110
Haider G, von Schrader T, Füsslein M, Blechert S, Kutchan TM (2005) Structure-Activity relationships of synthetic analogs of jasmonic acid and coronatine on induction of benzophenanthridine alkaloid accumulation in Eschscholzia californica cell cultures. Biol Chem 381:741–748. doi:10.1515/BC.2000.094
Hao G, Shi R, Tao R, Fang Q, Jiang X, Ji H, Feng L, Huang L (2013) Cloning, molecular characterization and functional analysis of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) gene for diterpenoid tanshinone biosynthesis in Salvia miltiorrhiza Bge. f. alba. Plant Physiol Biochem 70:21–32. doi:10.1016/j.plaphy.2013.05.010
Hao X, Shi M, Cui L, Xu C, Zhang Y, Kai G (2015a) Effects of methyl jasmonate and salicylic acid on tanshinone production and biosynthetic gene expression in transgenic Salvia miltiorrhiza hairy roots. Biotechnol Appl Bioch. 62:24–31. doi:10.1002/bab.1236
Hao C, Chen SL, Osbourn A, Kontogianni VG, Liu LW, Jordán MJ (2015b) Temporal transcriptome changes induced by methyl jasmonate in Salvia sclarea. Gene 558:41–53. doi:10.1016/j.gene.2014.12.043
Holohan C, Van Schaeybroeck S, Longle DB, Johnston PG (2013) Cancer drug resistance: an evolving paradigm. Nat Rev Cancer 13:714–726. doi:10.1038/nrc3599
Hu F, Huang J, Xu Y, Qian X, Zhong JJ (2006) Responses of defense signals, biosynthetic gene transcription and taxoid biosynthesis to elicitation by a novel synthetic jasmonate in cell cultures of Taxus chinensis. Biotechnol Bioeng 94:1064–1071. doi:10.1002/bit.20921
Hussain MS, Fareed S, Ansari S, Rahman MA, Ahmad IZ, Saeed M (2012) Current approaches toward production of secondary plant metabolites. J Pharm Bioallied Sci 4:10–20. doi:10.4103/0975-7406.92725
Kabouche A, Kabouche Z (2008) Bioactive diterpenoids of Salvia species. In: Atta-u-Rahman (ed) Studies in natural products chemistry, vol 35. Elsevier, Amsterdam, pp 753–833 doi:10.1016/S1572-5995(08)80017-8
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 Eng 13:319–327. doi:10.1016/j.ymben.2011.02.003
Kuzma L, Skrypek Z, Wysokinska H (2006) Diterpenoid and triterpenoids in hairy root of Salvia sclarea. Plant Cell Tissue Org Cult 84:171–179. doi:10.1007/s11240-005-9018-6
Kuzma L, Bruchajzer E, Wysokinska H (2009) Methyl jasmonate effect on diterpenoid accumulation in Salvia sclarea hairy root culture in shake flasks and sprinkle bioreactor. Enz Microb Tech 44:406–410. doi:10.1016/j.enzmictec.2009.01.005
Legrand S, Valot N, Nicolé F, Moja S, Baudino S, Jullien F, Magnard JL, Caissard JC, Legendre L (2010) One-step identification of conserved miRNAs, their targets, potential transcription factors and effector genes of complete secondary metabolism pathways after 454 pyrosequencing of calyx cDNAs from the Labiate Salvia sclarea L. Gene 450:55–62. doi:10.1016/j.gene.2009.10.004
Leone A, Grillo S, Monti L, Cardi T (2007) Molecular tailoring and boosting of bioactive secondary metabolites in medicinal plant. In: Ranalli P (ed) Improvement of crop plants for industrial end uses. IX: 471–507. Springer, Netherlands. ISBN: 978-1-4020-5486-0
Ma Y, Yuan L, Wu B, Li X, Chen S, Lu S (2012) Genome-wide identification and characterization of novel genes involved in terpenoid biosynthesis in Salvia miltiorrhiza. J Exp Bot 63:2809–2823. doi:10.1093/jxb/err466
Marchev A, Haas C, Schulz S, Georgiev V, Steingroewer J, Bley T, Pavlov A (2014) Sage in vitro cultures: a promising tool for the production of bioactive terpenes and phenolic substances. Biotechnol Lett 36:211–221
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol 15:473–497. doi:10.1111/j.1399-3054.1962.tb08052
O’Connor SE (2015) Engineering of secondary metabolism. Ann Rev Genet 49:71–94. doi:10.1146/annurev-genet-120213-092053
Onrubia M, Moyano E, Bonfill M, Cusidó RM, Goossens A, Palazón J (2013) Coronatine, a more powerful elicitor for inducing taxane biosynthesis in Taxus media cell cultures than methyl jasmonate. J Plant Physiol 170:211–219. doi:10.1016/j.jplph.2012.09.004
Patil RA, Lenka SK, Normanly J, Walker EL, Roberts SC (2014) Methyl jasmonate represses growth and affects cell cycle progression in cultured Taxus cells. Plant Cell Rep 33:1479–1492. doi:10.1007/s00299-014-1632-5
Pauwels L, Inzé D, Goossens A (2009) Jasmonate-inducible gene: what does it mean? Trends Plant Sci 14:87–91. doi:10.1016/j.tplants.2008.11.005
Pauwels L, Morreel K, De Witte E, Lammertyn F, Van Montagu M, Boerjan W, Inzé 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
Qian ZG, Zhao ZJ, Xu Y, Qian X, Zhong JJ (2005) Highly efficient strategy for enhancing taxoid production by repeated elicitation with a newly synthesized jasmonate in fed-batch cultivation of Taxus chinensis cells. Biotechnol Bioeng 90:516–521. doi:10.1002/bit.20460
Ramirez-Estrada K, Vidal-Limon H, Hidalgo D, Moyano E, Golenioswki M, Cusidó RM, Palazon J (2016) Elicitation, an effective strategy for the biotechnological production of bioactive high-added value compounds in plant cell factories. Molecules 21:2–24. doi:10.3390/molecules21020182
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 C 61:483–488. doi:10.1515/znc-2006-7-804
Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108. doi:10.1038/nprot.2008.73
Sheard LB, Tan X, Mao H, Withers J, Ben-Nissan G, Hinds TR, Kobayashi Y, Hsu FF, Sharon M, Browse J, He SY, Rizo J, Howe GA, Zheng N (2010) Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor. Nature 468:400–405. doi:10.1038/nature09430
Shi M, Zhou W, Zhang J, Huang S, Wang H, Kai G (2016) Methyl jasmonate induction of tanshinone biosynthesis in Salvia miltiorrhiza hairy roots is mediated by JASMONATE ZIM-DOMAIN repressor proteins. Sci Rep 6:20919. doi:10.1038/srep20919
Song X, Lopez-Campistrous A, Sun L, Dower NA, Kedei N, Yang J et al (2013) RasGRPs are targets of the anti-Cancer agent Ingenol-3-Angelate. PLoS One 8:e72331. doi:10.1371/journal.pone.0072331
Tamogami S, Kodama O (2000) Coronatine elicits phytoalexin production in rice leaves (Oryza sativa L.) in the same manner as jasmonic acid. Phytochemistry 54:689–694. doi:10.1016/S0031-9422(00)00190-4
Taurino M, Ingrosso I, D’amico L, De Domenico S, Nicoletti I, Corradini D, Santino A, Giovinazzo G (2015) Jasmonates elicit different sets of stilbenes in Vitis vinifera cv. Negramaro cell cultures. SpringerPlus 4:49. doi:10.1186/s40064-015-0831-z
Vaccaro MC, Malafronte N, Alfieri M, De Tommasi N, Leone A (2014) Enhanced biosynthesis of bioactive abietane diterpenes by overexpressing AtDXS or AtDXR genes in Salvia sclarea hairy roots. Plant Cell Tissue Org Cult 119:65–77. doi:10.1007/s11240-014-0514-4
Vranovà E, Coman D, Gruissem W (2013) Network analysis of the MVA and MEP pathways for isoprenoid synthesis. Annu Rev Plant Biol 64:665–700. doi:10.1146/annurev-arplant-050312-120116
Wasternack C, Hause B (2013) Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in Annals of Botany. Ann Bot 111:1021–1058. doi:10.1093/aob/mct067
Weaver BA (2014) How Taxol/paclitaxel kills cancer cells. Mol Biol Cell 25:2677–2681. doi:10.1091/mbcE14-04-0916
Wenping H, Yuan Z, Jie S, Lijun Z, Zhezhi W (2011) De novo transcriptome sequencing in Salvia miltiorrhiza to identify genes involved in the biosynthesis of active ingredients. Genomics 98:272–279. doi:10.1016/j.ygeno.2011.03.012
Yan J, Li H, Li S, Yao R, Deng H, Xie Q, Xie D (2013) The Arabidopsis F-box protein CORONATINE INSENSITIVE1 is stabilized by SCFCOI1 and degraded via the 26S proteasome pathway. Plant Cell 25:486–498. doi:10.1105/tpc.112.105486
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. doi:10.1371/journal.pone.0080464
Zerbe P, Bohlmann J (2015) Plant diterpene synthases: exploring modularity and metabolic diversity for bioengineering. Trends Biotechnol 33:419–428. doi:10.1016/j.tibtech.2015.04.006
Zhang L, Yao J, Withers J, Xin XF, Banerjee R, Fariduddin Q, Nakamura Y, Nomura K, Howe GA, Bolandf Wilhelm, Yang H, He SY (2015) Host target modification as a strategy to counter pathogen hijacking of the jasmonate hormone receptor. Proc Natl Acad Sci USA 112:14354–14359. doi:10.1073/pnas.1510745112
Zhao J, Davis LC, Verpoorte R (2005) Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol Adv 23:283–333. doi:10.1016/j.biotechadv.2005.01.003
Zhao L, Chang WC, Xiao Y, Liu HW, Liu P (2013) Methylerythritol phosphate pathway of isoprenoid biosynthesis. Ann Rev Biochem 82:497–530. doi:10.1146/annurev-biochem-052010-100934
Zhou H, Li Y, Zhang Q, Ren S, Shen Y, Qin L, Xing Y (2016) Genome-wide analysis of the expression of WRKY family genes in different developmental stages of wild strawberry (Fragaria vesca) fruit. PLoS One 11(5):e0154312. doi:10.1371/journal.pone.0154312
Acknowledgements
This work was supported by FARB 2014 funds of the University of Salerno to AL. MV is also grateful for the short-term mobility funds of the COST Action FA 1006 “Plant Metabolic Engineering for High Value Products”.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Communicated by E. Benvenuto.
The original version of this article was revised: The second author "M. E. Alfieri" was incorrect. This error has been corrected.
Electronic supplementary material
Below is the link to the electronic supplementary material.
299_2016_2076_MOESM1_ESM.tif
Hairy root biomass, expressed as dry weight, of S. sclarea hairy root lines treated with MeJA (100 µM) or Cor (0.1 µM) for 7 or 28 days, compared to control hairy roots, during one month of culture. Data represent mean values ± SD of three experiments (TIFF 77 kb)
Rights and permissions
About this article
Cite this article
Vaccaro, M.C., Mariaevelina, A., Malafronte, N. et al. Increasing the synthesis of bioactive abietane diterpenes in Salvia sclarea hairy roots by elicited transcriptional reprogramming. Plant Cell Rep 36, 375–386 (2017). https://doi.org/10.1007/s00299-016-2076-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-016-2076-x