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Influence of medium and elicitors on the production of cocaine, amino acids and phytohormones by calli

Article in Cell Tissue and Organ Culture · November 2014 DOI: 10.1007/s11240-014-0660-8

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ORIGINAL PAPER

Influence of medium and elicitors on the production of cocaine, amino acids and phytohormones by Erythroxylum coca calli

T. Docimo • A. J. Davis • K. Luck • C. Fellenberg • M. Reichelt • M. Phillips • J. Gershenzon • J. C. D’Auria

Received: 26 August 2014 / Accepted: 17 November 2014 / Published online: 22 November 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Erythroxylum coca () is the indole butyric acid (0.06 mg L-1), and benzylaminopurine source of the tropane alkaloid cocaine. Several lines of evi- (0.5 mg L-1). All accumulated cocaine and cinnamoylco- dence suggest that tropane alkaloid biosynthesis in E. coca caine at levels of 0.05–0.5 nmol per gram dry weight, as differs from that in solanaceous species, but there are many determined by LC–MS, several orders of magnitude below gaps in our understanding of the pathways in both groups. The the concentration found in the intact plant. Anderson’s development of an E. coca cell culture that produces cocaine medium supported the highest level of tro- could provide a reproducible model system for discovering pane alkaloid production, as well as the highest level of the novel biosynthetic genes and study pathway regulation. Calli amino acids arginine, glutamate, proline and phenylalanine, cultures were successfully established from young leaf all thought to be precursors of cocaine, but contained gen- explants on three different media: Anderson’s Rhododen- erally lower levels of hydroxycinnamate-quinate esters, such dron, Gamborg B5, and modified Murashige-Tucker, all as chlorogenic acid. These differences may be ascribed to its supplemented with growth regulators: 2,4-D (0.6 mg L-1), relatively low content of nitrate or salts, or its high content of adenine. Addition of 100 lM salicylic acid or coronalon, an analog of the bioactive jasmonic acid-isoleucine conjugate, Electronic supplementary material The online version of this did not result in any increase in tropane alkaloid production. article (doi:10.1007/s11240-014-0660-8) contains supplementary These E. coca calli could provide valuable material for material, which is available to authorized users. studies on tropane alkaloid biosynthesis and regulation. T. Docimo Á A. J. Davis Á K. Luck Á M. Reichelt Á M. Phillips Á J. Gershenzon Á J. C. D’Auria Keywords Erythroxylaceae Á Callus induction Á Tropane Department of Biochemistry, Max Planck Institute for Chemical alkaloids Á Elicitation Á Coronalon Á Salicylic acid Ecology, Hans-Knoell Strasse 8, 07745 Jena, Germany

Present Address: Abbreviations T. Docimo LC–MS Liquid chromatography-mass spectrometry Istituto di Biologia e Biotecnologia Agraria (IBBA), CNR Via 2,4 D 2,4-D-dichlorophenoxyacetic acid Bassini 8, 20133 Milan, Italy IBA Indole 3-butyric acid C. Fellenberg Á J. C. D’Auria (&) BAP Benzyl amino purine Center for Chemical Biology, Department of Chemistry and RH Relative humidity Biochemistry, Texas Tech University, Memorial Circle & Boston, Lubbock, TX 79409-1061, USA e-mail: [email protected]

Present Address: M. Phillips Introduction Plant Metabolism and Metabolic Engineering Program, Centre for Research in Agricultural Genomics (Consorci CRAG-CSIC- IRTA-UAB-UB), Edifici CRAG, 08193 Bellaterra, Barcelona, Tropane alkaloids (TAs) are an important class of medic- Spain inal alkaloids that have a scattered distribution throughout 123 1062 Plant Cell Tiss Organ Cult (2015) 120:1061–1075 the plant kingdom. They are most commonly found in the in TA biosynthesis in coca is a reduction catalyzed by a Solanaceae, but have been described from six further plant completely different class of enzyme than those catalyzing families: the Erythroxylaceae, Proteaceae, Convolvulaceae, this reduction in members of the Solanaceae (Jirschitzka Brassicaceae, Euphorbiaceae, and Rhizophoraceae (Drager et al. 2012). This distinction further emphasizes the need to 2002; Fodor and Dharanipragada 1994; Griffin and Lin study this pathway in E. coca and other non-solanaceous 2000; Lounasmaa and Tamminen 1993). Some of these species. families are only distantly related suggesting that TA bio- The use of plant in vitro tissue or cell culture is a useful synthesis has evolved independently several times. Inde- tool for the study of the regulation and biosynthesis of pendent evolution might mean that the biosynthetic secondary metabolites permitting the generation of large pathways differ among families or are regulated in dis- quantities of plant material under sterile and controlled tinctive ways. conditions. This minimizes variations that commonly arise Much of the available information on the enzymes and in field–grown . A further advantage is that it allows genes involved in TA biosynthesis comes from studies on large scale experiments to be performed in relatively little species of the Solanaceae (Humphrey and O’Hagan 2001). space to which the power of multivariate statistical analysis Some of the major TAs found in this family, such as sco- can be applied. polamine and atropine, are commercially important phar- Plant cell culture has been particularly helpful in maceuticals (Grynkiewicz and Gadzikowska 2008). In studying alkaloid biosynthesis. Although cultured cells do addition, many genomic tools have been developed for not always exhibit the same secondary metabolite profiles members of the Solanaceae, such as tobacco, tomato, and as their parent plants (Zhou et al. 2009), they can be used to potato, making these plants attractive models for molecular discover biosynthetic genes and elucidate the factors that studies. regulate these genes. For example, benzophenanthridine Much less is known about TA biosynthesis in other biosynthesis has been studied in cultured cells of the Cal- families, such as the Erythroxylaceae. Cocaine is a TA ifornia poppy (Eschscholzia californica) which are able to produced exclusively by members of the Erythroxylaceae. produce sanguinarine but neither noscapine nor morphine Of the roughly 230 species in the genus Erythroxylum,23 (Haider et al. 1997; Hauschild et al. 1998). In addition, wild species and two cultivated species are known to many biosynthetic steps involved in benzylisoquinoline produce cocaine (Ehleringer et al. 2000; Weil 1980). While alkaloid biosynthesis have been characterized from cell the pure compound is a highly addictive illicit drug, the cultures of the opium poppy (Papaver somniferum) (Des- coca plant, E. coca, has a long history of medico-cultural gagne-Penix et al. 2010; Facchini and De Luca 2008; use by the indigenous peoples of South America (Grin- Facchini et al. 1996; Facchini and Park 2003; Farrow et al. spoon and Bakalar 1981; Plowman and Hensold 2004; 2012; Liscombe et al. 2005). Furthermore, indole alkaloid Plowman and Rivier 1983). E. coca has been cultivated for biosynthesis has been widely studied using two cell lines at least 8,000 years (Dillehay et al. 2010; Weil 1980). The from the Madagascar periwinkle (Catharanthus roseus) medicinal effects of cocaine result from the tropane (Champagne et al. 2012; ten Hoopen et al. 2002). Not only nucleus which confers strong activity on the vertebrate have genes encoding enzymes responsible for the biosyn- central nervous system. In particular, cocaine acts by thesis of medicinally important compounds been identified inhibiting dopamine re-uptake (Ruetsch et al. 2001). It in C. roseus, but several aspects of the regulation of the thereby induces general euphoria, which is responsible for pathway have been studied in detail (Guo et al. 2013; Ri- its addictive nature and its abuse as a narcotic. scher et al. 2006; ten Hoopen et al. 2002). The biosynthesis of cocaine and other TAs found in Cell cultures have also been employed in studying TA members of the Erythroxylaceae has only recently begun to biosynthesis. In the Solanaceae, cell differentiation seems be elucidated (Fig. 1). Early studies involving the feeding to be essential for TA formation (Ford et al. 1998; Khanam of radiolabeled precursors to E. coca leaves helped indicate et al. 2000; Medinabolivar and Flores 1995). Undifferen- some of the possible precursors. However, no structural tiated Scopolia parviflora cells, for example, failed to genes or enzymes have been isolated. Cocaine biosynthesis produce scopolamine, while cell cultures derived from in E. coca is regulated differently from the synthesis of roots had the capacity for biosynthesis (Kim et al. 2010). TAs in other plant families (Jirschitzka et al. 2012). In the Hairy root cultures from several species such as Atropa Solanaceae, TA biosynthesis occurs in the root tissue. TAs belladonna, Datura stramonium and Hyoscyamus muticus are first accumulated there and then translocated to the are also able to produce TAs (Oksman-Caldentey 2000). 13 aerial parts. In contrast, CO2 application demonstrated In Solanaceae tissue cultures, as well as in those of other that biosynthesis in E. coca occurs in the young leaf tissue. plant species, the production of commercially important Only trace amounts of cocaine and related TAs occur in the secondary metabolites can be increased by using appro- roots (Docimo et al. 2012). Moreover, the penultimate step priate elicitors that mimic biotic or abiotic stresses which 123 Plant Cell Tiss Organ Cult (2015) 120:1061–1075 1063

Fig. 1 General theoretical tropane alkaloid biosynthetic pathway in Erythroxylum coca starting from L- glutamic acid and leading to cocaine. The abbreviations for the biosynthetic enzymes are as follows P5CS, pyrroline-5- carboxylate synthetase; P5CR, pyrroline-5-carboxylate reductase; ADC, arginine decarbocylase; POX2, phenol oxidase 2; AIH, agmatine imino hydrolase; ODC, ornithine decarboxylase; NCPAH, N-carbamoylputrescine amido hydrolase; PMT, putrescine N- methyltransferase; MPO, methyl putrescine oxidase; MecgoR, methylecgonone reductase; CS, cocaine synthase

123 1064 Plant Cell Tiss Organ Cult (2015) 120:1061–1075 increase the quantity of secondary metabolites in intact the concentrations above together with 10.0 g L-1 sucrose plants (Coste et al. 2011; el Jaber-Vazdekis et al. 2008; and 4 g L-1 Gelrite (Supplementary Table 1). Media were Facchini et al. 1996; Gadzovska et al. 2013; Karuppusamy adjusted to pH 5.8 with 1 N potassium hydroxide and then 2009; Zhao et al. 2005). Therefore the use of elicitors may sterilized at 121 °C for 20 min. After sterilization, the represent a viable strategy for investigating the formation media were cooled to 50 °C in a water bath and the plates of TAs in other families as well. poured. As part of a long-term study on TA biosynthesis in On all media, calli first emerged at the cut leaf edges species of the Erythroxylaceae, we investigated the possi- after 15 days of culture. They were excised from the bilities of using E. coca cell cultures. The only existing explants and cultured further. Calli cultures were routinely studies on tissue culture of this species focused on shoot sub–cultured every 4 weeks on freshly made medium of proliferation (Lydon et al. 1993), and there are no studies the same type. on the metabolism of cultured cells. We therefore tested various culture media for their ability to support callus Effects of medium and elicitor formation and cocaine production, and their influence on other metabolites. In addition, we assessed the effects of The three media described above were each tested with two two elicitors on calli metabolism: the jasmonic acid-iso- elicitor and two non-elicitor controls. The elicitors were SA leucine (JA-Ile) mimic coronalon and salicylic acid (SA). (Fluka, Germany) and coronalon (Schuler et al. 2004), kindly supplied by A. Mitho¨fer, Department of Bio-organic Chemistry, Max Planck Institute of Chemical Ecology. Materials and methods Elicitors were tested at a final concentration of 100 lM after filtering a 100 mM stock solution in analytical grade Plants, calli induction and culturing ethanol (Merck, Darmstadt, Germany) through a 0.22 lm nylon membrane (Schleicher and Schuell, UK). For con- Seeds of Erythroxylum coca var coca were obtained from trols, equivalent amounts of filtered analytical grade etha- the Botanical Garden, Bonn, Germany. The seeds were nol were added to the medium. germinated and the plants grown in sterilized potting soil in E. coca callus was transferred to the 12 possible com- growth chambers set at 24 °C, 60 % RH, long-day photo- binations of medium, elicitor and elicitor control, with five period 16:8 using daylight fluorescent tubes at *790 lmol replicates each to give 60 treatments in all. Treatment photons m-2 s-1. New plants were propagated from cut- combinations were distributed over five 12-well plates so tings. For callus initiation, leaves were taken from a single, that no treatment occupied the same position or shared the well–growing plant with two fully developed primary same neighbor on any plate. Each well contained 2.5 ml of leaves (Docimo et al. 2012) to insure the best possible cell one of the medium 9 elicitor combinations, and one callus proliferation rate and to reduce extraneous variability. was transferred to each. Media type was maintained To create explants for calli production, young leaves between transfers. Transfer order was performed in random were cut and washed with tap water for 30 min. They were order to avoid bias in assigning calli to treatments. A then subjected to surface-sterilization by bleaching for complete experiment consisted of three replicates of the 60 2 min with 0.5 % sodium hypochlorite solution (containing treatments. The plates containing calli were maintained in 0.1 % Tween) and rinsing with sterile distilled water three an incubator (Percival model CU–36L5) at 24 °C, 45 % times over 5 min. We then aseptically removed the mid- RH for varying periods: one, three, or 7 days. Plates for the vein and dissected the leaf blades into 5 9 5 mm explants different time points were subcultured on the same date and which were placed on solid nutrient media and incubated at harvested at the indicated intervals. The entire experiment 24 °C in total darkness. was repeated twice using identical procedures, incubators The media we used for culturing calli were Anderson and incubator shelves, thus providing two series and ten rhododendron medium (ARM, (Anderson 1978)), Gamborg replicates for every treatment combination. B5 (GB5, (Gamborg et al. 1976)) and Murashige–Tucker medium (Murashige and Tucker 1962) modified by Extraction and analysis of tropane alkaloids, amino changing the amount of 2,4-D-dichlorophenoxyacetic acid acids and phytohormones (2,4-D) to 0.6 mg L-1, indole 3-butyric acid (IBA) to 0.06 mg L-1 and benzylaminopurine (BAP) to 0.5 mg L-1. At each time point, 60 calli were harvested and the fresh This modified Murashige–Tucker medium (MMT) was weight of each callus was recorded. Each set of calli was superior in terms of healthy calli produced compared to the transferred to a 96-well plate for chemical analysis. Meth- unmodified medium in preliminary tests. The mastermix for anol (100 lL) was added to each well, vortexed for 3 min all three media thus contained the three growth regulators at after 15 min standing, vortexed for 1 min after 1 h standing, 123 Plant Cell Tiss Organ Cult (2015) 120:1061–1075 1065 and then centrifuged for 2 min at 3009g. The methanol instrument parameters were optimized by infusion experi- contained the following internal standards: 28.9 ng mL-1 ments with pure standards, where available. The ion spray -1 atropine, 40 ng mL 9,10-D2-9,10-dihydrojasmonic acid, voltage was maintained at -4,500 eV. The turbo gas tem- -1 -1 40 ng mL D4-SA(Sigma-Aldrich), 40 ng mL D6- perature was set at 700 °C. Nebulizing gas was set at 60 psi, abscisic acid (Santa Cruz Biotechnology, Santa Cruz, USA), curtain gas at 25 psi, heating gas at 60 psi and collision gas at -1 13 and 8 ng mL jasmonic acid- C6-isoleucine conjugate 7 psi. Multiple reaction monitoring (MRM) was used to aliquots of the supernatant were used for TA, phytohor- follow parent ion ? product ion transitions for each analyte: mone, and amino acid analysis by LC–MS/MS. m/z 136.9 ? 93.0 (CE -22 V; DP -35 V) for salicylic acid;

For analysis of TAs, chlorogenic acid (CGA) and m/z 140.9 ? 97.0 (CE -22 V; DP -35 V) for D4-SA; m/ 4-coumaroyl quinate (CQA), chromatography was per- z 209.1 ? 59.0 (CE -24 V; DP -35 V) for jasmonic acid; formed on an Agilent 1200 HPLC system (Agilent Tech- m/z 213.1 ? 56.0 (CE -24 V; DP -35 V) for 9,10-D2- nologies). Separation was achieved on a Zorbax Eclipse 9,10-dihydrojasmonic acid; m/z 263.0 ? 153.2 (CE -22 V; XDB-C18 column (50 9 4.6 mm, 1.8 lm, Agilent). Formic DP -35 V) for abscisic acid; m/z 269.0 ? 159.2 (CE acid (0.05 %) in water was employed as mobile phase A and -22 V; DP -35 V) for D6-abscisic acid; m/z 322.2 ? 130.1 acetonitrile as mobile phase B. The elution profile was: (CE -30 V; DP -50 V) for jasmonic acid-isoleucine con- 0–0.5 min, 5 % B; 0.5–4 min, 5–60 % B; 4–4.1 min jugate; m/z 328.2 ? 136.1 (CE -30 V; DP -50 V) for 13 60–100 % B; 4.1–5 min 100 % B and 5.1–8 min 5 % B. jasmonic acid- C6-isoleucine conjugate; m/z 163.0 ? 119.0 The mobile phase flow rate was 1.1 mL min-1. The column (CE -20 V; DP -30 V) for p-coumaric acid. Both Q1 and temperature was maintained at 25 °C. An API 3200 tandem Q3 quadrupoles were maintained at unit resolution. Analyst mass spectrometer (Applied Biosystems) equipped with a 1.5 software (Applied Biosystems) was used for data acqui- turbospray ion source was operated in positive ionization sition and processing. Linearity in ionization efficiencies was mode. The ion spray voltage was maintained at 5,500 eV. verified by analyzing dilution series of standard mixtures. The turbo gas temperature was set at 700 °C. Nebulizing gas Phytohormones were quantified relative to the signal of their was set at 60 psi, curtain gas at 20 psi, heating gas at 70 psi corresponding internal standard. For quantification of p- and collision gas at 6 psi. Multiple reaction monitoring coumaric acid, D6-ABA was used as the internal standard (MRM) was used to follow parent ion ? product ion tran- applying an experimentally determined relative weight sitions for each analyte: m/z 304.3 ? 182.3 (collision response factor of 0.722. energy (CE) 26 V; declustering potential (DP) 45 V) for cocaine; m/z 330.3 ? 182.3 (CE 26 V; DP 45 V) for cin- Dry weight determination namoylcocaine; m/z 355.1 ? 163.1 (CE 21 V; DP 41 V) for CGA; m/z 339.1 ? 147.1 (CE 21 V; DP 41 V) for Callus dry weight was determined separately due to the CQA; m/z 290.1 ? 124.1 (CE 31 V; DP 51 V) for atropine destructive nature of the chemical analysis. Chemical con- (internal standard). Both Q1 and Q3 quadrupoles were tent was then expressed per unit dry weight. For dry weight maintained at unit resolution. Analyst 1.5 software (Applied calibration, thirty calli were grown exactly as the calli used Biosystems) was used for data acquisition and processing. for analysis. Ten of these were plated onto each of the three Linearity in ionization efficiencies was verified by analyzing test media and cultured and harvested in the same way as dilution series of standard mixtures. The compounds were test calli. After their fresh weights were determined, they quantified relative to the signal of the internal standard, were dried at 60 °C for 2 days, and their dry weights applying the experimentally determined relative molar recorded. The calculated dry weight to fresh weight ratio response factors: cocaine 0.287; cinnamoylcocaine 0.571; (n = 30) was used to infer dry weights of calli used in CGA and CQA 17.2. extractions. There was no difference between the three For amino acid analysis, 30 lL of the methanol super- media. We applied winsorization and used Kendal-Thiel natant was mixed with 270 lL of water containing (KT) robust correlation to prevent outlier values from 10 lgmL-1 algal amino acids uniformly labeled with 13C unduly influencing the correlation and thus introducing bias and 15N (Isotec, Miamisburg, US). The LC–MS/MS con- into the calibration. Winsorization removes the highest and ditions were those used previously (Docimo et al. 2012). lowest extreme values and KT correlation fits the median of For phytohormone and p-coumaric acid analysis, the all pairwise fitted lines. chromatographic conditions were as described above except the elution profile was: 0–0.5 min, 5 % B; 0.5–4 min, Statistical analysis 5–90 % B; 4–4.02 min 90–100 % B; 4.02–4.5 min 100 % B and 4.51–7 min 5 % B. An API 3200 tandem mass spec- We employed repeated measure multivariate anova trometer (Applied Biosystems) equipped with a turbospray (RMMA). A repeated measure design is required because ion source was operated in negative ionization mode. The measures were taken at intervals over time. A multivariate 123 1066 Plant Cell Tiss Organ Cult (2015) 120:1061–1075 design is required because several measures, the amounts This is accomplished by assigning observations to specific of chemical compounds produced, were made on every locations in a conceptual low-dimensional space such that individual callus. In RMMA, significance of the overall the distances between points in the space match as closely design is required before significance is sought in compo- as possible the similarities calculated between objects. The nents of the design. This requirement guards against result is a least-squares representation of the objects in the wrongly identifying as significant those effects that occur low–dimensional space. The dimension axes can be con- within the design only as a consequence of the many dif- sidered as axes of informational content. Objects that are ferences tested. The between–subjects factors in the ana- close together in the representation plot are those that lysis were medium, elicitor and elicitor level. So that we behave in a similar way in response to experimental con- could check for any differences between series, this was ditions. The objects in our MDS analysis were the different also included as a between–subjects factor. The within- compounds produced by the calli. This analysis was also subjects factor was time, the three collection days. The carried out in SPSS 21. We calculated the proximity three groups of chemical compounds, TAs, amino acids between pairs of metabolites by squared euclidian distance and phytohormones, were tested separately. This design and applied the simplex optimization method on interval allowed us to test simultaneously the effect of medium, transformed data. The convergence criterion for iterative elicitor and elicitor level, as well as the time course of optimization was further reduced to\0.0001 in normalized chemical production. It also allowed testing all their raw stress. We set the maximum number of iterations to interactions that is differences between amounts of com- 100. pound produced depending on factor combinations and not just single factors. The four major null hypotheses were: no differences Results between media, elicitors, or concentrations of elicitors in the time course of compounds produced; no difference E. coca forms calli able to produce cocaine between media in the amounts of cocaine or other com- pounds produced; no difference between elicitors in the The fully expanded leaves of a young plant were used as degree to which they elicit the production of any com- explants for callus induction on three different media: pound; and no difference between levels of the same Anderson’s Rhododendron medium (ARM), Gamborg B5 elicitor in the degree to which they elicit the production of (GB5) and modified Murashige-Tucker medium (MMT) any compound. Before analysis, the values were stan- (Supplementary Table 1). Within 15 days of growth at 24° dardized so that each amount of a compound was expressed in the dark, pale-yellow colored calli emerged on all three in units of the standard deviation of all amounts of that media (Fig. 2). particular compound. This is necessary to avoid a dispro- Calli growing on each type of medium in darkness portionate effect on the analysis by chemicals present in accumulate cocaine, the major TA, as determined by LC- considerably larger absolute amounts than the others (So- MS analysis (Fig. 2). Sub-culturing every 4 weeks pro- kal and Rohlf 1982). Analyses were carried out in SPSS 21. vided sufficient material for a range of further experiments. Significance levels of a \ 0.005 were sought to offset the experimental pseudoreplication (Hairston 1989). The Medium composition is the major factor controlling E. pseudoreplication arises from the relatedness of calli, all of coca callus metabolism which came from a single original plant. RMMA analysis showed that all four null hypotheses could be rejected After RMMA established the significance of the overall (Supplemental Table 2). design, the effects of medium, elicitor type, elicitor level, Where the RMMA showed significant effects, univariate and day were examined by univariate statistical analysis. As tests were used to locate the origin of the significance shown in Fig. 3, all variables tested significantly affected the within the design. Where univariate tests indicated signif- content of at least one metabolite. However, while medium icant differences between amounts of compound produced, composition affected virtually every compound examined, we applied Scheffe post hoc tests to identify the size, both elicitor and day were significant for only a subset of direction and significance of the differences. metabolites, and many of these effects were only marginally In addition to the RMMA, we also carried out multidi- significant. Surprisingly, treatment with the defense-related mensional scaling (MDS) in order to determine which elicitors, SA and coronalon, did not affect TAs, but did 1 metabolites responded similarly to experimental condi- affect CGA (F101 = 14.355, p  0.001). Thus, medium tions. MDS is a data visualization technique that finds the was by far the dominant factor controlling natural product structure in a set of similarity measures between objects. metabolism in these experiments.

123 Plant Cell Tiss Organ Cult (2015) 120:1061–1075 1067

Fig. 2 E. coca calli growth on the three media a ARM medium b GB5 medium c MMT medium. The right hand side of each panel shows chromatograms from an LC MS/MS experiment in which multiple reaction monitoring was used for the 304.3 m/ z ? 182.0 m/z transition specific for cocaine

Calli cultured on ARM have high levels of tropane alkaloids and low levels of hydroxycinnamate-quinate esters

Amounts of cocaine, cinnamoylcocaine, chlorogenic acid (CGA) and 4-coumaroyl quinate (CQA) were significantly 2 affected by the culture medium (F104 [ 35.00, p  0.001). Cocaine production was nearly an order of magnitude greater on ARM than on either MMT or GB5 (average concentration over the 7 day time course for ARM: 0.28 nmol g-1 DW; MMT: 0.04 nmol g-1 DW; GB5: 0.06 nmol g-1 DW; Scheffe test p  0.001; Fig. 4). Fur- thermore, the amounts produced on MMT and GB5 were not significantly different from each other. Cinnamoylco- caine was affected in the same way (Scheffe test p  0.001). In regards to CGA, the media were all sig- nificantly different from each other with lowest production on ARM and highest on MMT (average concentration for ARM: 0.34 nmol g-1 DW; MMT: 0.85 nmol g-1 DW; Fig. 3 Heat map of the RMMA univariate significance values for all Fig. 5). Less CQA was produced on ARM than on either of compounds tested in respect of the main experimental factors. Red p \ 0.005, orange p \ 0.01, yellow p \ 0.05. With this design and data the other two media which did not differ from each other p [ 0.005 should not be considered significant. (Color figure online) (Scheffe test p  0.001). Elicitor type and elicitor level interacted significantly in p  0.001). Elicitor effects were significant only for CGA, controlling production of one or more secondary metabo- with SA suppressing accumulation of this compound 4 lites (between-subjects factors Wilks’ Lambda101 = 0.501, *60 % (Fig. 5). Both cinnamoylcocaine and CGA

123 1068 Plant Cell Tiss Organ Cult (2015) 120:1061–1075

Fig. 4 Cocaine and cinnamoylcocaine production by E. coca calli. Medium, elicitor, elicitor level and time effects. Calli were grown on three different media—black columns ARM, gray GB5, white MMT. Media were assigned to coronalon (panels a, c)or salycilic acid b, d elicitor treatments. Elicitor treatments included either zero elicitor (solvent only; solid columns)or 100 lM(hatched columns). Measurements were made 1, 3 and 7 days after placing calli on the different media

amounts changed significantly over time (cinnamoylco- tryptophan (F [ 8.677, p \ 0.004) with an overall 2 caine; Huynh–Feldt208 = 3.763, p = 0.005, CGA; Huynh– inducing effect on amino acid production after elicitor 2 Feldt208 = 15.541, p  0.001; Figs. 4, 5). There were no treatment. The amounts of alanine, valine, isoleucine, significant interaction effects, indicating that where cin- aspartate, histidine, and tryptophan changed significantly namoylcocaine and CGA changed with time, they changed over time (Huynh–Feldt [ 2.643, p \ 0.004). Valine, similarly under all combinations of experimental factors. threonine, isoleucine, aspartate and histidine changed Interestingly, while cinnamoylcocaine increased with time, differently over time depending on which medium the CGA generally decreased, an inverse correlation also calli were on because there were significant day–medium observed with changes in medium. interactions (Huynh–Feldt [ 2.562, p \ 0.003). In par- ticular, the amount of these amino acids increased over Amino acids involved in cocaine biosynthesis also have time in calli grown on GB5. maximum accumulation in calli grown on ARM Elicitor treatment does not affect phytohormone Medium significantly and strongly affected the amounts of production in E.coca calli all amino acids (F [ 17.165, p  0.001) except threonine, methionine, histidine, and lysine (Supplemental Table 3). Medium significantly affected the amounts of all phyto- Of particular interest to this study, concentrations of the hormones except JA (all F [ 7.882, p \ 0.001). Signifi- putative cocaine precursors proline, glutamate, phenylala- cantly less SA was found in calli grown on ARM than on the nine and arginine were two to sixfold higher on ARM other two media and the SA amounts in calli on these two (Scheffe test, p \ 0.001; Fig. 6; average concentration other media were not different from each other (Scheffe over the 7 day time course: Pro: 4.77 nmol g-1 DW; Glu: test, p = 0.002; average SA concentration over time on 33.58 nmol g-1 DW; Phe: 1.34 nmol g-1 DW; Arg: ARM: 12.76 nmol g-1 DW; BG5: 12.46 nmol g-1 DW; 98.41 nmol g-1 DW), similar to the trend observed for MMT: 6.98 nmol g-1 DW). The highest concentrations of TAs. The same increase was observed for serine, alanine, JA and the JA-Ile were found in calli growing on ARM isoleucine, leucine, tyrosine, tryptophan, and asparagine medium with levels reaching as high as 0.11 nmol g-1 DW (Supplemental Table 3). The amount of valine produced on and JA-Ile 0.09 nmol g-1 DW respectively. Calli grown on each of the media was significantly different from each GB5 medium produced significantly less JA and JA-Ile with other with GB5 (1.93 nmol g-1 DW) \ MMT maximum levels reaching 0.10 nmol g-1 DW and (2.56 nmol g-1 DW) \ ARM (3.35 nmol g-1 DW). 0.01 nmol g-1 DW respectively (Scheffe test, p \ 0.002 in Elicitor type had a significant effect on proline, valine, both cases). The highest levels of abscisic acid were pro- isoleucine, and phenylalanine (F [ 8.659, p \ 0.004), duced in calli grown on ARM with maximum levels mea- whereas elicitor level significantly affected production of sured on day one with a concentration of 0.26 nmol g-1 valine, threonine, isoleucine, leucine, phenylalanine, DW. These levels were significantly greater than those in

123 Plant Cell Tiss Organ Cult (2015) 120:1061–1075 1069

Fig. 5 Chlorogenic acid a and 4-coumaroyl quinate b production by included either zero elicitor (solvent only; open symbols) or 100 lM E. coca calli. Medium, elicitor, elicitor level and time effects. Calli (solid symbols). Measurements were made 1, 3 and 7 days after were grown on three different media—solid lines ARM, dashed GB5, placing calli on the different media. Each point is the mean of five dotted MMT. Media were assigned to coronalon open circle or replicates, error bars SD/10 salycilic acid open triangle elicitor treatments. Elicitor treatments

Fig. 6 Amino acid production by E. coca calli phenylalanine a arginine b proline c glutamine d. Medium, elicitor, elicitor level and time effects. Calli were grown on three different media—solid lines ARM, dashed GB5, dotted MMT. Media were assigned to coronalon open circle or salycilic acid open triangle elicitor treatments. Elicitor treatments included either zero elicitor (solvent only; open symbols) or 100 lM(solid symbols). Measurements were made 1, 3 and 7 days after placing calli on the different media. Each point is the mean of ten replicates, error bars SD/ 10

123 1070 Plant Cell Tiss Organ Cult (2015) 120:1061–1075

Fig. 7 Plant hormone production by E. coca calli salicylic acid elicitor treatments. Elicitor treatments included either zero elicitor a jasmonic acid b jasmonic acid–isoleucine conjugate c. Medium, (solvent only; open symbols) or 100 lM(solid symbols). Measure- elicitor, elicitor level and time effects. Calli were grown on three ments were made 1, 3 and 7 days after placing calli on the different different media—solid lines ARM, dashed GB5, dotted MMT. Media media. Each point is the mean of ten replicates, error bars SD/10 were assigned to coronalon open circle or salycilic acid open triangle

calli growing on GB5 (maximum on day 1: 0.2 nmol g-1 amino acids. Asparagine, aspartate and glutamine also DW; Scheffe test, p = 0.003). Altogether, there was no follow the same pattern. CGA, CQA and p-coumaric acid consistent effect of media on hormone production. (Fig. 7). do not plot near cocaine or cinnamoylcocaine. In fact, these The amounts of all hormones except the JA-Ile conju- compounds are very strongly differentiated from the TAs gate differed among elicitor types (all F \ 9.703, in dimension one of the plot, indicating a negative corre- p \ 0.002), but only SA and JA differed among elicitor lation between these groups of compounds. This is sur- levels (SA F = 375.93, p \ 0.001, JA F = 32.691, prising since CGA and CQA have been discussed as p \ 0.001). The amounts of SA changed significantly over 2 time (Huynh–Feldt = 1.499107, p \ 0.001) with an overall decline after SA treatment. This compound was responsible for the significant effect of day on hormone production. The amounts of the other hormones did not change sig- nificantly over time (Fig. 7).

Cocaine and its amino acid precursors show similar patterns of change in the overall experiment

In order to determine which compounds included in this study behave similarly in the overall experimental setup, a multidimensional scaling plot was constructed. Multidi- mensional scaling is a data visualization technique that finds the structure in a set of similarity measures between objects. The initial normalized raw stress was 0.65. The final minimized stress was\0.04 with congruence after 36 iterations. The total dispersion accounted for Fig. 8 Multidimensional scaling (MDS) plot of all metabolites measured for this study in calli of E. coca. The more similar the (DAF) [ 80 % in these two dimensions (see ‘‘Materials responses of metabolites to experimental conditions the closer they and methods’’ for more details). appear in the plot. Proximities between pairs of metabolites were Cocaine and cinnamoylcocaine plot near to the proposed calculated as squared euclidian distance. Simplex start with intial amino acid precursors arginine, proline and glutamate normalized raw stress 0.65. Interval transformation. Final minimized stress \0.04 with congruence after 36 iterations using congruence (Fig. 8). Thus cocaine and cinnamoylcocaine responded to criterion, stress improvement = 0.0001. Total dispersion accounted experimental conditions in the same way as these three for (DAF) [ 80 % in these two dimensions 123 Plant Cell Tiss Organ Cult (2015) 120:1061–1075 1071 complexing agents involved in vacuolar storage of E. coca 2012; Leete 1980) and the immunolocalization of pathway TAs (Pardo Torre et al. 2013). Interestingly, JA also enzymes (Jirschitzka et al. 2012). However, the accumu- showed a strong negative association with TAs, despite the lation of cocaine in our E. coca calli is several orders of fact that elicitors did not affect TA accumulation. JA and magnitude lower than in intact plants, so specialized tissues JA-Ile are strongly separated from SA and ABA along may be an important factor in TA production and storage in dimension two, in agreement with the well-described non-Solanaceous species as well. antagonism between these two groups of hormones (Anderson et al. 2004; Takahashi et al. 2004). ARM medium may promote tropane alkaloid accumulation by its low salt, low nitrate or high adenine content Discussion Commonly used media for plant cell culture, such as Undifferentiated E. coca calli cultures derived from leaf Murashige and Skoog (MS) and GB5, are broadly appli- explants accumulate the tropane alkaloid, cocaine cable in inducing calli, but may not supply the proper nutrients for the growth of specific plant cell cultures. For Plant cell cultures frequently exhibit altered metabolism on example, MS medium, originally created for tobacco cell various media or in comparison to intact plants. These culture, is suitable for many plant species (Murashige and differences have helped in the discovery of genes con- Skoog 1962). However, in order to obtain viable cell cul- trolling metabolism and new aspects of pathway regulation. ture for the micropropagation of Citrus, modification of the Here we describe the first protocol for callus induction for medium was necessary (Murashige and Tucker 1962). The an Erythroxylum species, and the first evidence for accu- original MS medium is even toxic to Rhododendron spe- mulation of the TA cocaine in cell culture. There have been cies (Hsia and Korban 1997). A low salt medium (ARM) no previous reports on cell cultures from any TA producing was developed, which supports efficient micropropagation species not belonging to the Solanaceae. The establishment and calli production in Rhododendron (Ericaceae) and of a TA-producing E. coca callus system may facilitate Phyllanthus urinaria (Euphorbiaceae) (Anderson 1978; discovery of new TA biosynthetic genes and a better Catapan et al. 2002). As another example of medium ver- understanding of how TA formation is controlled. satility, the B5 medium, which was originally optimized Plant cell cultures often lack the ability to produce the for soybean cell cultures, supported vigorous growth of cell secondary metabolites found in the intact plant because cultures of Allium (Liliaceae) and Oryza (Poaceae) undifferentiated calli lack specific tissues necessary for (Greenway et al. 2012). biosynthesis or sequestration (Facchini 2001; Guo et al. Moreover, for pharmaceutically important species where 2013; Hashimoto and Yamada 1994; Palacio et al. 2012; not only growth but production of specific metabolites is Palazon et al. 1995; Ziegler and Facchini 2008). For desired, the choice of the medium is even more important. example, calli formed from leaf explants of Cannabis sa- For example, a major factor affecting secondary metabolite tiva are unable to produce cannabinoids, which are nor- production in plant tissue culture is the salt strength of the mally synthesized and stored in highly differentiated medium (Coste et al. 2011). Solasadine production in hairy glandular hairs (Flores-Sanchez et al. 2009; Pec et al. root cultures of Solanum khasianum was inversely pro- 2010). In addition, cells from TA-producing Solanaceae portional to the salt strength of the growth medium (Jacob species, which form TAs in the roots of intact plants and Malpathak 2005). Similarly, adventitious root cultures (Hashimoto and Yamada 1994; Oksman-Caldentey 2000) of Echinacea angustifolia required low levels of salt in the do not typically produce TAs in culture. However, large growth medium in order to achieve higher levels of bio- amounts of the TAs scopolamine, hyoscyamine and atro- active secondary metabolites (Wu et al. 2006). Similar pine are found in hairy root cultures of species, such as results were obtained for Panax ginseng and Morinda ci- Atropa belladonna, Datura metel, Hyoscyamus muticus trifolia (Baque et al. 2010; Huang et al. 2010). and Duboisia leichhardtii, that retain differentiated root The major factor controlling TA accumulation was tissues (Hartmann et al. 1986; Palazon et al. 2008; Yamada medium composition, with cocaine levels on ARM being and Endo 1984). nearly an order of magnitude greater than on the other In this study, E. coca calli were capable of making media (Fig. 3). To gain insight into which medium ingre- cocaine even in the absence of specialized tissues (Figs. 3, dients might influence this, we compared components of 4). This ability may be due to the origin of these calli from the various media. Many nutrients, including cobalt, cop- young leaf explants. Cocaine biosynthesis has been dem- per, molybdenum, calcium, magnesium, iron, boron, onstrated to take place in leaves independent of root tissues iodine, manganese, zinc and myo-inositol, and the growth based on precursor feeding experiments (Docimo et al. regulators and the ammonium:nitrate ratio are at equivalent 123 1072 Plant Cell Tiss Organ Cult (2015) 120:1061–1075 levels in ARM as in one of the other media, and can of nitrogen channeled into formation of the TA hyoscya- therefore be excluded as factors promoting TA accumula- mine in Datura stramonium, (Demeyer and Dejaegere tion. However, a number of factors differed between ARM 1989, 1992). The concentrations of these two nutrients in and the other media (Table 1), and might be responsible for E. coca calli might also play an important role in the the elevated TA content. Total ion concentration is lowest regulation of cocaine biosynthesis. in ARM, and could be an important factor given the As well as low salt and low nitrate, ARM also differs importance of salt content in controlling secondary from the other media tested by containing a high level of metabolism as discussed above. adenine. This compound is a cytokinin precursor that affects Nitrate concentration was also lower in ARM, and there cell, tissue and organ development in plant culture experi- are numerous reports in the literature of an inverse rela- ments (Merrett and Handoll 1967; Premkumar et al. 2011). tionship between nitrate availability and accumulation of It is therefore possible that adenine acted as a growth reg- secondary metabolites in many plant species including ulator in our experiments, resulting in developmental Arabidopsis thaliana (Scheible et al. 2004), Hordeum induction of the TA biosynthetic pathway. If so, adenine- vulgare (Kova´cˇik et al. 2014) and Nicotiana tabacum (Fritz treated calli could be an excellent source of samples enri- et al. 2006). Similarly, the reduction of nitrate concentra- ched in mRNAs and proteins involved in TA biosynthesis. tion in the culture medium of Atropa belladonna hairy roots increases alkaloid content (Bensaddek et al. 2001). The ARM culture medium also promoted Nitrate uptake in plant cells requires the reduction to nitrite the accumulation of various amino acids - (NO2 ) and its subsequent reduction to ammonium, a and jasmonates, but not hydroxycinnamate derivatives process which is energetically costly (Behrend and Mateles 1975, 1976). The negative effects of nitrate may be alle- The three different growth media tested in this study also viated by replacement with ammonium, which can be taken yielded different quantities of primary metabolites. The up directly and immediately assimilated into the amino amino acids suggested to be precursors of the tropane ring of acid glutamate thorough the Krebs cycle. However, when cocaine, arginine, proline and glutamate, all increased in ammonium exceeds a certain threshold it can disturb the quantity when the calli were grown on ARM medium regulation of the Krebs cycle. This causes a reduction in instead of GB5 and MMT (Fig. 6) following the pattern of pyridine nucleotides and conversely creates high levels of cocaine abundance. Unexpectedly, three other amino acids, a-ketoglutarate, thus limiting the rate of glutamate forma- aspartate, asparagine and glutamine behaved very similarly tion (Gamborg and Shyluk 1970). Since the ammonium in the different media, as they clustered near cocaine in the concentration of MMT is four-fold higher than that of multidimensional scaling plot (Fig. 8). While these amino ARM and tenfold that of GB5, the resulting changes in acids are not known to participate in TA synthesis, gluta- glutamate content could explain the inability of ARM to mine could potentially play a role as an amino-group donor. support high levels of TA biosynthesis. Nevertheless, the It is also of interest that the growth medium influenced - ? replacement of 20 % NO3 with NH4 raised the amount the endogenous pools of the phytohormones jasmonic acid, its isoleucine conjugate, and salicylic acid. The ARM Table 1 Characteristics of different culture media investigated that medium, which promoted TA accumulation, also showed may control differential accumulation of TAs relatively high amounts of jasmonic acid and its conjugate with a concomitant decrease in salicylic acid (Fig. 7). ARM Gamborg B5 MMT These differences may also arise from the low salt or high Major components (mM) adenine content of ARM. Nitrate levels especially have Ammonium 5.0 2.0 20.6 profound effects on phytohormones and other components Potassium 4.8 24.8 20.1 of plant signaling networks, although their impact on Sodium 2.8 1.2 0.4 defense hormones and the resulting defense responses Nitrate 9.8 24.8 39.4 differs from species to species (Castaings et al. 2011; Phosphate 2.4 1.0 1.3 Krouk and Gojon 2011; Krouk et al. 2010). In tobacco Total ions 36.1 59.4 92.3 plants, nitrate feeding stimulates both the nitric oxide and Total nitrogen 14.8 26.8 60.1 salicylic acid pathways and augments defense responses Minor components (lM) (Gupta et al. 2013), while in arabidopsis high ammo- Thiamine-HCl 1.3 33.2 16.6 nium:nitrate ratios in the growth medium contribute to the Pyridoxine-HCl 0 4.9 24.3 autoimmune response (Wang et al. 2013). Nicotinic acid 0 8.1 20.3 In addition to amino acids and phytohormones, we also Adenine sulfate 434.8 0 0 observed the effects of medium on two non-TA secondary metabolites chlorogenic acid (CGA) and 4-coumaroyl 123 Plant Cell Tiss Organ Cult (2015) 120:1061–1075 1073 quinate (CQA). These hydroxycinnamate-quinate esters Thus, by correlating cocaine accumulation with transcript were found in higher quantities in calli growing on GB5 levels, calli cultures can serve as a powerful tool for the and MMT medium (Fig. 5). Just as multidimensional discovery of novel genes involved in the biosynthesis of scaling (Fig. 8) showed that cocaine and cinnamoylcocaine TAs. Second, studying the differential accumulation of cluster with their amino acid precursors, CGA and CQA TAs on diverse media with varying levels of nutrients, cluster similarly with their precursor, the hydroxycinna- growth regulators and primary metabolites can shed new mate p-coumaric acid (Fig. 8). The relatively large distance light on the regulation of the pathway. Finally, the ability between CGA/CQA and TAs is surprising since the hy- to produce large quantities of genetically and develop- droxycinnamate-quinate esters may well be involved in the mentally uniform plant material with a functioning TA complexation and storing of TAs in the vacuoles of E. coca pathway is a tremendous advantage to in vivo biosynthetic leaves (Pardo Torre et al. 2013). But, since the concen- studies based on feeding candidate precursors or interme- tration of CQA at least is much higher than the concen- diates in isotopically-labeled forms. tration of either TA in the plant, a negative correlation may not prevent effective complexation. Acknowledgments This work was supported by the Max Planck Society and an Alexander von Humboldt Foundation postdoctoral fellowship to J.D. We thank Dr. Tamara Kru¨gel for her advice The lack of tropane alkaloid induction in E. coca callus regarding tissue culture methods and practices, Andreas Weber and the in response to jasmonates and salicylate can be rest of the gardening staff of the MPI-ICE for their help in plant an advantage in biosynthetic investigations maintenance, Dr. Axel Mitho¨fer for providing the coronalon used in our elicitation experiments, and Jan Jirschitzka for technical assistance. Treatment of E. coca calli with either the JA-Ile analogue coronalon or with salicylic acid did not enhance TA pro- duction. In contrast, in the cell cultures of a large range of References plant species, jasmonates or salicylate treatment elicits increases in secondary metabolite levels (Karuppusamy Anderson WC (1978) Tissue culture propogation of . 2009; Zhao et al. 2005). 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