Abstract
Elicitors play an important role in challenging the plant defense system through plant-environment interaction and thus altering the secondary metabolite production. Culture filtrates of four endophytic fungi, namely, Chaetomium globosum, Aspergillus niveoglaucus, Paecilomyces lilacinus, and Trichoderma harzianum were tested on embryogenic cell suspensions of latex-less Papaver somniferum in dose-dependent kinetics. Besides this, abiotic elicitors salicylic acid, hydrogen peroxide, and carbon dioxide were also applied for improved sanguinarine production. Maximum biomass accumulation (growth index (GI) = 293.50 ± 14.82) and sanguinarine production (0.090 ± 0.008 % dry wt.) were registered by addition of 3.3 % v/v T. harzanium culture filtrate. Interestingly, it was further enhanced (GI = 323.40 ± 25.30; 0.105 ± 0.008 % dry wt.) when T. harzanium culture filtrate was employed along with 50 μM shikimate. This was also supported by real-time (RT) (qPCR), where 8–9-fold increase in cheilanthifoline synthase (CFS), stylopine synthase (STS), tetrahydroprotoberberine cis-N-methyltransferase (TNMT), and protopine 6-hydroxylase (P6H) transcripts was observed. Among abiotic elicitors, while hydrogen peroxide and carbon dioxide registered low level of sanguinarine accumulation, maximum sanguinarine content was detected by 250 μM salicylic acid (0.058 ± 0.003 % dry wt.; GI = 172.75 ± 13.40). RT (qPCR) also confirms the downregulation of sanguinarine pathway on CO2 supplementation. Various parameters ranging from agitation speed (70 rpm), impeller type (marine), media volume (2 l), inoculum weight (100 g), and culture duration (9 days) were optimized during upscaling in 5-l stirred tank bioreactor to obtain maximum sanguinarine production (GI = 434.00; 0.119 ± 0.070 % dry wt.). Addition of 3.3 % v/v T. harzanium culture filtrate and 50-μM shikimate was done on the 6th day of bioreactor run.
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Ahmad N, Gupta S, Husain MM, Heiskanen KM, Mukhtar H (2000) Differential antiproliferative and apoptotic response of sanguinarine for cancer cells versus normal cells. Clin Cancer Res 6:1524–1528
Alcantara J, Bird DA, Franceschi VR, Facchini PJ (2005) Sanguinarine biosynthesis is associated with the endoplasmic reticulum in cultured opium poppy cells after elicitor. Plant Physiol 138:173–183
Capasso A, Aquino R, De Tomáis N, Piacente S, Rastrelli L, Pizza C (2002) Neuropharmacology activity of alkaloids from South American medicinal plants. Curr Med Chem Cent Nerv Syst Agents 2:1–15
Chang JH, Shin JH, Chung IS, Lee HJ (1998) Improved menthol production from chitosan-elicited suspension culture of Mentha piperita. Biotechnol Lett 20:1097–1099
Chaturvedi N, Singh SK, Shukla AK, Lal RK, Gupta MM, Dwivedi UN, Shasany AK (2013) Latex-less opium poppy: cause for less latex and reduced peduncle strength. Physiol Plant. doi:10.1111/ppl.12086
Cho H-Y, Lee-Parsons CWT, Yoon S-Y H, Rhee HS, Park JM (2007) Enhanced benzophenanthridine alkaloid production and protein expression with combined elicitor in Eschscholtzia californica suspension cultures. Biotechnol Lett 29:2001–2005
Cho H-Y et al (2008a) Differential induction of protein expression and benzophenanthridine alkaloid accumulation in Eschscholtzia californica suspension cultures by methyl jasmonate and yeast extract. J Microbiol Biotechnol 18:255–262
Cho H-Y et al (2008b) Synergistic effects of sequential treatment with methyl jasmonate, salicylic acid and yeast extract on benzophenanthridine alkaloid accumulation and protein expression in Eschscholtzia californica suspension cultures. J Biotechnol 135:117–122
Dittrich H, Kutchan TM (1991) Molecular cloning, expression, and induction of berberine bridge enzyme, an enzyme essential to the formation of benzophenanthridine alkaloids in the response of plants to pathogenic attack. PNAS (USA) 88:9969–9973
Facchini PJ, De Luca V (2008) Opium poppy and Madagascar periwinkle: model non-model systems to investigate alkaloid biosynthesis in plants. Plant J 54:763–784
Facchini PJ, Park S-U (2003) Developmental and inducible accumulation of gene transcripts involved in alkaloid biosynthesis in opium poppy. Phytochemistry 64:177–186
Farber K, Schumann B, Miersch O, Roos W (2003) Selective desensitization of jasmonate- and pH-dependent signaling in the induction of benzophenanthridine biosynthesis in cells of Eschscholzia californica. Phytochemistry 62:491–500
Fulzele DP, Heble MR (1994) Large-scale cultivation of Catharanthus roseus cells production of ajmalicine in a 20-1-airlift bioreactor. J Biotechnol 35:1–7
Hagel JM, Facchini PJ (2012) Subcellular localization of sanguinarine biosynthetic enzymes in cultured opium poppy cells. In Vitro Cell Dev Biol—Plant 48:233–240
Huang TK, Mc Donald KA (2009) Bioreactor engineering for recombinant protein production in plant cell suspension cultures. Biochem Eng J 45:168–184
Jin H, Shin J, Kim J, Chung S, Lee H (1999) Effect of chitosan elicitation and media components on the production of anthraquinones colorants in Madder (Rubia akane Nakai) cell culture. Biotechnol Bioprocess Eng 4:300–304
Kutchan TM (1995) Alkaloid biosynthesis—the basis for metabolic engineering of medicinal plants. Plant Cell 7:1059–1070
Lee-Parsons CWT, Ertürk S, Tengtrakool J (2004) Enhancement of ajmalicine production in Catharanthus roseus cell cultures with methyl jasmonate is dependent on timing and dosage of elicitation. Biotechnol Lett 26:1595–1599
Mishra S, Triptahi V, Singh S, Phukan UJ, Gupta MM, Shankar K (2013) Wound induced tanscriptional regulation of benzylisoquinoline pathway and characterization of wound inducible PsWRKY transcription factor from Papaver somniferum. PLoS ONE 8:e52784
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Namdeo AG (2007) Plant cell elicitation for production of secondary metabolites: a review. Phcog Rev 1:69–79
Oksman-Caldentey KM, Inze D (2004) Plant cell factories in post genomic era: new ways to produce designer secondary metabolites. Trends Plant Sci 9:433–440
Pan Z-W, Wang H-Q, Zhong J-J (2000) Scale-up study on suspension cultures of Taxus chinensis cells for production of taxane diterpene. Enz Microbiol Technol 27:714–723
Park S-U, Yu M, Facchini PJ (2003) Modulation of berberine bridge enzyme levels in transgenic root cultures of California poppy alters the accumulation of benzophenanthridine alkaloids. Plant Mol Biol 51:153–164
Pitta-Alvarez SI, Spollansky TC, Giulietti AM (2000) The influence of different biotic and abiotic elicitors on the production and profile of tropane alkaloids in hairy root cultures of Brugmansia candida. Enzyme Microbiol Technol 26:252–258
Radman R, Saez T, Bucke C, Keshavarz T (2003) Elicitation of plants and microbial cell systems. Appl Biochem Biotechnol 37:91–102
Sato F, Hashimoto T, Hachiya A, Tamura KI, Choi KB, Murashige T (2001) Metabolic engineering of plant alkaloid biosynthesis. Proc Natl Acad Sci U S A 98:367–372
Savitha BC, Thimmaraju R, Bhagyalakshmi N, Ravishankar GA (2006) Different biotic and abiotic elicitors influence betalain production in hairy root cultures of Beta vulgaris in shake-flask and bioreactor. Process Biochem 41:50–60
Sharma JR, Lal RK, Gupta AP, Misra HO, Pant V, Singh NK, Pandey P (1999) Development of non-narcotic (opiumless and alkaloid-free) opium poppy, Papaver somniferum. Plant Breed 118:449–452
Smetanska I (2008) Production of secondary metabolites using plant cell cultures. In: food biotechnology. Advances in Biochemical Engineering/Biotechnology. Stahl U, Donalies UEB, Nevoigt E (Eds.) Vol 111 pp 187–228
Solarova J, Pospisilova J (1997) Effect of carbon dioxide enrichment during in vitro cultivation and acclimatization to ex vitro conditions. Biol Plant 39:23–30
Sudha G, Ravishankar GA (2002) Involvement and interaction of various signaling compounds on the plant metabolic events during defense response, resistance to stress factors, formation of secondary metabolites and their molecular aspects. Plant Cell Tiss Org Cult 71:181–212
Tanahashi T, Zenk MH (1990) Elicitor induction and characterization of microsomal protopine-6-hydroxylase, the central enzyme in benzophenanthridine alkaloid biosynthesis. Phytochemistry 29:1113–1122
Tripathi U, Sarada R, Ravishankar GA (2001) A culture method for microalgal forms using two-tier vessel providing carbon-dioxide environment: studies on growth and caretenoid production. World J Microbiol Biotechnol 17:325–329
Trujillo-Villanueva K, Rubio-Pine J, Monforte-Gonzalez M, Ramirez-Benitez E, Vazquez-Flota F (2012) The sequential exposure to jasmonate, salicylic acid and yeast extract promotes sanguinarine accumulation in Argemone mexicana cell cultures. Biotechnol Lett 34:379–385
Vasconsuelo A, Boland R (2007) Molecular aspects of the early stages of elicitation of secondary metabolites in plants. Plant Sci 172:861–875
Vasconsuelo AA, Giuletti AM, Picotto G, Rodrıguez-Talou J, Boland R (2003) Involvement of the PLC/PKC pathway in chitosan-induced anthraquinone production by Rubia tinctorum L. cell cultures. Plant Sci 165:429–436
Verma P, Mathur AK, Shankar K (2012) Increased availability of tryptophan in 5-methyltryptophan tolerant shoots of Catharanthus roseus and their post-harvest in vivo elicitation induces enhanced vindoline production. Appl Biochem Biotechnol 168:568–579
Verma P, Mathur AK, Masood N, Luqman S, Shankar K (2013) Tryptophan over producing cell suspensions of Catharanthus roseus (L) G. Don and their up-scaling in stirred tank bioreactor: detection of phenolic compound with antioxidant potential. Protoplasma 250:371–380
Verpoorte R, Alfermann AW (2000) Metabolic engineering of plant secondary metabolism. Kluwer Academic Publishers, Dordrecht
Yamaguchi T, Maehara Y, Kodama O, Okada M, Matsumura M, Shibuya N (2002) Two purified oligosaccharide elicitors, N-acetylchitohepatose and tetraglucosyl glucitol, derived from Magnaporthe grisea cell walls, synergistically activate biosynthesis of phytoalexin in suspension-cultured rice cells. J plant physiol 159:1147–1149
Yamamoto H, Yazaki K, Inoue K (2000) Simultaneous analysis of shikimate-derived secondary metabolites in Lithospermum erythrorhizon cell suspension cultures by high-performance liquid chromatography. J Chromato B Biomed Sci App 738:3–15
Yuan Y-J, Wei Z-J, Miao Z-Q, Wu J-C (2002) Acting paths of elicitors on Taxol biosynthesis pathway and their synergistic effect. Biochem Eng J 10:77–83
Ziegler J, Facchini PJ (2008) Alkaloid biosynthesis: metabolism and trafficking. Annu Rev Plant Biol 59:735–769
Zulak KG, Cornish A, Daskalchuk TE, Deyholos MK, Goodenowe DB, Gordon PMK, Klassen D, Pelcher LE, Sensen CW, Facchini PJ (2007) Gene transcript and metabolite profiling of elicitor-induced opium poppy cell cultures reveals the coordinate regulation of primary and secondary metabolism. Planta 225:1085–1106
Acknowledgments
The work presented here has been supported by DST-FAST TRACK SERC/LS-261/2012. PV is highly thankful to CSIR for awarding CSIR-RA. Help rendered by Dr. Shiv Shanker Pandey, Sonal Mishra, Dr. Rakesh Shukla, and Jyotsana Priya, during experimentation, has been highly acknowledged.
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Verma, P., Khan, S.A., Mathur, A.K. et al. Improved sanguinarine production via biotic and abiotic elicitations and precursor feeding in cell suspensions of latex-less variety of Papaver somniferum with their gene expression studies and upscaling in bioreactor. Protoplasma 251, 1359–1371 (2014). https://doi.org/10.1007/s00709-014-0638-8
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DOI: https://doi.org/10.1007/s00709-014-0638-8