Journal of Advanced Research (2016) 7, 681–689

Cairo University Journal of Advanced Research

ORIGINAL ARTICLE Metabolomics driven analysis of lysistemon cell suspension culture in response to methyl jasmonate elicitation

Mohamed A. Farag a,*, Hattem Mekky b, Sawsan El-Masry b a Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Kasr El-Einy Street, 11562 Cairo, Egypt b Pharmacognosy Department, Faculty of Pharmacy, Alexandria University, El Khartoum Square, 21521 Alexandria, Egypt

GRAPHICAL ABSTRACT

ARTICLE INFO ABSTRACT

Article history: An MS-based metabolomic approach was used to profile the secondary metabolite of the orna- Received 29 April 2016 mental Erythrina lysistemon via ultra-performance liquid chromatography coupled to Received in revised form 6 July 2016 photodiode array detection and high resolution q-TOF mass spectrometry (UPLC-PDA-MS). Accepted 6 July 2016 Cultures maintained the capacity to produce E. lysistemon flavonoid subclasses with ptero- Available online 14 July 2016 carpans amounting for the most abundant ones suggesting that it could provide a resource of such flavonoid subclass. In contrast, alkaloids, major constituents of Erythrina genus, were

* Corresponding author. Fax: +20 11 202 25320005. E-mail address: [email protected] (M.A. Farag). Peer review under responsibility of Cairo University.

Production and hosting by Elsevier http://dx.doi.org/10.1016/j.jare.2016.07.002 2090-1232 Ó 2016 Production and hosting by Elsevier B.V. on behalf of Cairo University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 682 M.A. Farag et al.

detected at trace levels in suspension cultures. Methyl jasmonate (MeJA), phytohormone, was Keywords: further supplied to culture with the aim of increasing secondary metabolites production and Cell culture with metabolite profiles subjected to multivariate data analysis to evaluate its effect. Results Erythrina lysistemon revealed that triterpene i.e. oleanolic acid and fatty acid i.e. hydroxy-octadecadienoic acid were Metabolomics elicited in response to methyl jasmonate, whereas pterocarpans i.e., isoneorautenol showed a Methyl jasmonate decline in response to elicitation suggesting for the induction of terpenoid biosynthetic pathway Oleanolic acid and concurrent with a down regulation of pterocarpans. In conclusion, a total of 53 secondary Ultra-performance liquid metabolites including 3 flavones, 12 isoflavones, 4 isoflavanones, 4 alkaloids, 11 pterocarpans, chromatography and 5 phenolic acids were identified. Ó 2016 Production and hosting by Elsevier B.V. on behalf of Cairo University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/).

Introduction Material and methods

The genus Erythrina constitutes 115 species in the family Plant material ‘‘” which are distributed worldwide in tropical and subtropical regions growing as , often recognized in agri- Seeds of E. lysistemon were collected in January 2012 from culture for its bright red flowers as coral or flame trees [1]. trees previously authenticated by Professor Nabil El Hadidi, Alkaloids and phenolics are among the most widely distributed College of Science, Cairo University, Egypt. Voucher constituents in these flowering trees mostly localized in stem specimens of the flowers and seeds are deposited at Faculty bark [2,3], roots [4] and seeds [5,6]. Erythrina alkaloids are of Pharmacy, Alexandria University, Egypt. tetracyclic spiroamines possessing an erythrinane skeleton. Over 90 Erythrina alkaloids have been isolated [7,8], often clas- Callus initiation sified as dienoid or lactonic alkaloids. Interest in Erythrina alkaloids is mostly driven by its curare-like neuromuscular Seeds were surface sterilized in 20% sodium hypochlorite solu- blocking effect. Moreover, Erythrina spp. possess a broad- tion for 30 min, washed three times in sterile purified water and spectrum of physiological activities such as anti-plasmodial placed on agarized Murashige and Skoog (MS; Caisson, activity due to the flavonoids and isoflavonoids [9], anti- Smithfield, USA) germination medium (1962) and incubated oxidant and anti-inflammatory activities due to pterocarpans under 12 h light period and a temperature of 23 °C±1°C [10] and fungicidal activity associated with its alkaloidal con- in a culture room [16]. were excised from 28 d-old seed- tent [11]. lings. The leaves were then scored on their abaxial sides with a Erythrina genus has been extensively examined in terms of sterile scalpel blade and cut into 1 cm2 pieces. Explants were its and chemical composition. However, very little cultured on 25 mL aliquots of MS supplemented with either information is available concerning biotechnological attempts 1mgL1 or 2 mg L1 of each of kinetin (Kin; Acros, Geel, for natural products production within that genus. Belgium) and 2,4-dichlorophenoxyacetic acid (2,4D; Acros, Garcia-Mateos et al., showed that an unexpected profile of Belgium) in addition to 30 g L1 sucrose (El-Nasr, Alexandria, oxygenated alkaloids was observed in undifferentiated callus Egypt), and semi-solidified with 0.8% (w/v) agar (Roko, of Erythrina Coralloides and Erythrina americana [12]. Fur- Llanera – Asturias, Spain), pH 5.6, in a 9 cm diameter Petri thermore, San Miguel-Chavez et al., showed that jasmonic dishes. The explants were transferred onto fresh media, until acid elicited E. americana cell culture has led to reduction in callus was produced [16]. alkaloid accumulation [13]. Among the most common and effective elicitors used for stimulating secondary metabolites Cell suspension culture initiation production in plant cell culture are the carbohydrate fractions of fungal and plant cell walls, MeJA, chitosan and/or heavy metal salts. In particular, jasmonates have been long regarded Cell suspensions were established by transferring 1–2 g fresh as transducers of elicitor signals for the production of plant wt. of callus and maintained in 100 mL MS liquid medium secondary metabolites. Application of elicitors results in the supplemented with same growth regulators, but no agar was induction of signaling compounds, including jasmonic acid added, pH 5.6 in 100 mL Erlenmeyer flasks. Cultures were and or MeJA, as well as the downstream up regulation of sec- maintained on a rotary shaker at 100 rpm and incubated under ondary metabolites. In contrast to salicylic acid being an elic- a 12 h photoperiod, with day and night temperatures of ° ° itor of limited secondary metabolite classes, jasmonates seem 23 C±1 C until the stock suspension was produced. to be general natural products inducer via the activation and de novo biosynthesis of transcription factors that up regulate Elicitation genes involved in secondary metabolites production [14]. For example, jasmonates induce the accumulation of terpenoids, Aliquots of 2.5 mL packed cell volume (PCV) with 2.5 mL flavonoids, alkaloids and phenylpropanoids [15]. The aim of spent medium of the stock suspension were transferred to six this work was to examine MeJA elicitation effect on cell 100 mL Erlenmeyer flasks, each containing 45 mL of fresh suspension culture of Erythrina lysistemon regarding the accu- media and maintained under same conditions for two weeks. mulation of alkaloids, flavonoids, pterocarpans and phenolic Methyl jasmonate (MeJA; Sigma Aldrich, Poole, UK) was acids using an MS-based metabolomic approach. then added into five flasks to produce a concentration Erythrina lysistemon cell culture metabolomics 683

1mML1 MeJA. The dose 1 mM L1 MeJA was previously for the elution of cinnamates, flavonoids, alkaloids and fatty optimized to elicit secondary metabolic pathways in cell cul- acids within 13 min (ca. 800 s). The elution order of secondary tures [17,18]. Furthermore, an increase in the concentration metabolites followed a sequence of decreasing polarity, of MeJA resulted in retarded callus growth. The remaining whereby cinnamates and alkaloids eluted first, followed by fla- flask was used as control by the addition of the same volume vonoid glycosides, free aglycones, prenylated aglycones and of sterile water. Cultures were kept at 23 °C±1°C, with a finally triterpenes and fatty acids. Simultaneously acquired 12 h photoperiod and maintained on a rotary shaker at UPLC–PDA and UPLC–MS total ion chromatograms of 100 rpm. Cell culture samples were harvested at 0, 6, 12, 24 E. lysistemon cell culture extracts in positive and negative ion- and 48 h post elicitation and kept at 80 °C until being ization mode are presented in Fig. 1. The identities, retention analyzed. times, UV and MS spectral data observed for secondary metabolites are presented in Table 1 with a total of 53 identified Extraction and UPLC-MS analysis of cell culture extracts metabolites. It is worth noting that this is the first comprehen- sive metabolite profile of E. lysistemon plant. Identified metabo- Metabolites extraction followed the protocol developed for lites belonged to various classes (Table 1, Suppl. Fig. 1) similar metabolite classes [18,19]. Briefly, lyophilized E. lysiste- including phenolic acids (cinnamates) i.e. N-caffeoyl aspartic mon cultures (20 ± 0.06 mg) were extracted with 1.8 mL acid (2), alkaloids i.e. erysotrine (6), pterocarpans i.e. isoneo- aq.80% MeOH for 10 h using an orbital shaker in the dark. rautenol (30), isoflavones i.e. lysisteisoflavone (44), triterpenes Extracts were centrifuged at 10,000g for 15 min and 1.4 mL i.e. oleanolic acid (53) and fatty acid i.e. hydroxy-9,11- of the supernatant was aliquot and evaporated under nitrogen octadecadienoic acid (45), with isoflavones and pterocarpans till complete dryness. The dried residue was resuspended in as the most abundant classes in cell culture extract. The struc- 300 lL 45% aq. MeOH. For comparative analysis, the extracts tures of major metabolites identified in E. lysistemon and dis- were spiked with 2 lg umbelliferone as an internal standard cussed throughout the manuscript are shown in Suppl. Fig. 1. (IS) and quantifications were determined from peak areas nor- malized based on the amount of recovered IS peak. The resi- Flavonoids due was re-suspended in 300 lL methanol and used for UPLC-MS analysis following the exact chromatographic con- Photodiode array detection provided an overview of the main ditions described by Farag et al. [20]. flavonoid constituents (Fig. 1A). UV spectra (200–600 nm) were measured for flavonoid sub-classes including 12 isofla- Identification and quantification of metabolites and MS data vones, 3 flavones, 4 isoflavanones and 11 pterocarpans. Each multivariate analysis sub-class exhibits a characteristic UV spectrum. For example, flavones have a maximum absorbance near 265 nm with a sec- File Converter tool in X-Calibur software was used to convert ond maximum between 320 and 340 nm (peak 9), whereas pte- k UPLC–MS files to NetCDF file format and then further pro- rocarpans have max around 280–290 nm (42). Extracts were cessed by AMDIS software for background subtraction and analyzed in positive and negative ion electrospray ionization peak deconvolution. Metabolite identification was done via (ESI) MS modes to provide a comprehensive overview of the UV-VIS spectra (220–600 nm), retention times relative to exter- metabolite composition. Compared to the positive-ion ESI nal standards, mass spectra, and comparison to both the refer- mode (Fig. 1C), negative-ion MS spectra (Fig. 1B) revealed ence literature and phytochemical dictionary of natural better sensitivity than in positive mode, especially in the elu- product database. Quantification of alkaloids was calculated tion range of flavonoids (200–500 s). In addition, negative- from the calibration curve of erythraline, pterocarpans using ion MS spectral characteristics showed strong [M H] ions medicarpin standard, and for oleanolic acid using that of and lower chemical noise and consequently better sensitivity oleanolic acid standard detected using MS detector. Standard [22]. The positive ion ESI mass spectra were characterized by + + calibration curves were constructed for each standard using 4 cations corresponding to [M + H] , [M + Na] and frag- concentrations spanning from 0.1, 1, 10 and 200 lg/mL. Assays ment ions attributed to the sequential losses of isoprenyl were carried out in triplicate. (69 amu), malonyl (86 amu) and hexosyl (162 amu) groups. Few minor isoflavone peaks 13, 15, 18, 32 and 43 were only MS data processing for multivariate analysis detected in positive ionization mode warranting the impor- tance of acquiring data in both ionization modes. Two major Relative quantification and comparison of metabolites profiles flavone glycosides including dihydroxyflavone hexoside after UPLC-MS were performed using XCMS data analysis (m/z 415.102, [M H] peak 9) and apigenin hexosylmalonate software, which can be downloaded for free as an R package (m/z 517.1702, [M H] peak 11) were identified in cell from the Metlin Metabolite Database (http://137.131.20. culture. With regard to isoflavanone subclass, vogelin A 83/download/) [21]. (m/z 369.0999, [M H] peak 25) and 5-deoxyglyasperin F/5-deoxylicoisoflavanone (m/z 337.1085, [M H] peak 27) Results and discussion exhibiting UV max around 310–320 nm typical for flavanones were measured. E. lysistemon cell culture metabolite profile Pterocarpans Callus was produced from cut ends of scored E. lysistemon explants after 3 weeks. Chemical constituents of callus extracts Among flavonoid subclasses, pterocarpans amounted for the were analyzed via UPLC/PDA/( )ESI-qTOF-MS that allowed major forms in cell culture (11 peaks), exhibiting k max around 684 M.A. Farag et al.

mAU 500 (A) 32 2 400 IS 1 300 200 9 42 100

UV response 0 4 (B) 30 33 42 x105 3 34 IS 53 2 17 27 3 23 1 5 35

0 4 (C) 31 33 42 3 MS response 47 5 34 x10 2 11 13 52 1 18 22

0 200 300 400 500 600 700 800 rt (sec)

Fig. 1 UPLC–UV at 250 nm (A), UPLC–negative-ion ESI–MS (B) and UPLC–positive-ion ESI–MS chromatogram of E. lysistemon cell culture extracts (C). Chromatographic conditions are described under Material and methods. Insets 1 and 2 represent UV spectra of peak 9 (dihydroxyflavone hexoside) and peak 42 (sandwicensin, a pterocarpan), respectively. The identities, rt-value, UV and MS spectra of all peaks are listed in Table 1. IS = spiked internal standard (umbelliferone). Chromatographic conditions followed that were described in [20]. 280–290 nm with isoneorautenol (m/z 321.1147, [MH] peak Phenolic acid (cinnamates) 30) as the most abundant (Table 1). Other identified ptero- carpans include erythribyssin B (m/z 283.0598, [M H] The most abundant nitrogenous compounds detected in cell peak 19), eryvarin D (m/z 335.1264, [M H] peak 20), culture were amino acyl hydroxycinnamic acid conjugates. A dihydroisoneorautenol (m/z 323.1288, [M H] peak 31) total of 5 peaks (2–5, 16) not previously reported in E. lysiste- and sandwicensin (m/z 337.1445, [M H] peak 42). The pre- mon n plant tissue were identified in cell culture suggesting for an dominant loss of 69 amu (–C5H9, prenyl group) in the MS activation toward the production of acylcinnamates in cell cul- spectrum of pterocarpans is diagnostic for the presence of ture. The predominant fragment of cinnamic acid derivatives the isoprenyl group; a total of 6 peaks showed this pattern. in the MSn spectrum and characteristic UV max values at For example, erystagallin B (m/z 437.1993, [M H] peak 298 and 325 nm are diagnostic for cinnamates; a total of 5 38) showed 2 mass fragments at m/z 368 and 299 indicative peaks showed similar UV (Table 1). MS/MS analysis con- for 2 isoprenyl losses ( 2 69 amu). The abundance of firmed the structure of N-p-coumaroylaspartic acid (3) m/z isoprenylated pterocarpans in cell culture suggests for the pres- 278 and N-feruloylaspartic acid (5) m/z 308 from their respec- ence of isoprenyl transferase enzyme with higher affinity tive product ions at m/z 163 and 193 indicative of a toward pterocarpans. This is the first report for the accumula- p-coumaroyl and feruloyl moieties, respectively, whereas tion of pterocarpans in E. lysistemon cell culture and suggests N-caffeoylaspartic acid (2) gave a [M–H] at m/z 294 with that it could provide a resource of that flavonoid subclass. product ions m/z 132 for the aspartic acid moiety. Differences in metabolites composition observed in E. lysis- Alkaloids temon callus from its native plant are likely to be the result of genetic variation and/or lack of differentiation [22,23].Itis With an increased sensitivity for detection of nitrogenous worth mentioning that there was no obvious qualitative or metabolites in positive mode, alkaloids could only be detected quantitative difference in the metabolite profile of the 2 differ- in that mode. Alkaloids that are known to predominate ent treatments of the calli (1 mg l1 or2mgl1 of Kinetin and E. lysistemon plant extracts were almost absent in cell culture, 2,4D), results not shown. except for few alkaloid peaks present at trace levels including erysotrine (m/z 314.1756 [M + H]+, peak 6), erythrartine/11- PCA of E. lysistemon MeJA elicited and control suspension methoxyerysodine (m/z 330.1696, [M + H]+, peak 10) and culture observed in negative ionization mode erysotramidine (m/z 328.1534 [M + H]+, peak 12). In con- + trast, DOPA methyl ether (m/z 226.1073 [M + H] , peak 8) Cell culture was further subjected to MeJA treatment to deter- was present as the major nitrogenous secondary metabolite mine its impact on reprogramming of secondary metabolites as identified in culture. No UV absorbance could be traced for revealed via UPLC-MS analysis. To assess for changes in alkaloid peaks, except for DOPA methyl ether showing dis- metabolite composition in response to elicitation as monitored tinct UV max at 270 nm. In tandem MS, alkaloids showed via UPLC-MS traces of the different callus samples harvested methyl losses from methoxy group ( 15 Da). at 0, 12 and 2 h post MeJA elicitation (Suppl. Fig. 2), principal Erythrina lysistemon cell culture metabolomics 685

Table 1 Metabolites identified in E. lysistemon L. cell suspension methanol extract using UPLC–PDA–MS/MS in negative/positive ionization modes. Peak rt (s) UV (nm) Metabolite Class Molecular ion Error Composition MS/MS m/z (±) ppm 1 122 285 Dihydroxybenzoic acid Phenolic acid 447.1139[M H] 1.2 C18H23O13 378, 304 pentosylhexoside 2 161 294, 325 N-Caffeoylaspartic acid Phenolic acid 294.0592[M H] 9.1 C13H12NO7 175, 132 + 3 198 294, 326 N-p-Coumaroylaspartic acid Phenolic acid 278.0661[M + H] 3.2 C13H12NO6 163, 132 + 4 209 287, 312 N-(Hydroxycinnamoyl) Phenolic acid 476.187[M + H] 9.3 C24H30NO9 314 tyraminehexoside 5 220 294, 325 N-Feruloylaspartic acid Phenolic acid 308.0758[M H] 5.9 C14H14NO7 193, 132 + 6 237 282 Erysotrine Alkaloid 314.1756[M + H] 1.6 C19H24NO3 280 7 238 272, 340 Apigeninpentosyl hexoside Flavone 563.1423[M H] 3C26H27O14 269, 253 + 8 238 280 DOPA methyl ether Alkaloid 226.1073[M + H] 0.6 C11H16NO4 9 245 270, 332 Dihydroxyflavone hexoside Flavone 415.102 [M H] 3.5 C21H19O9 253 + 10 252 325 Erythrartine/11-Methoxyerysodine Alkaloid 330.1696[M + H] 1.3 C19H24NO4 312, 280 11 262 nd Apigeninhexosylmalonate Flavone 517.1702[M H] 4.0 C24H21O13 269, 253 + 12 263 nd Erysotramidine Alkaloid 328.1534[M + H] 2.9 C19H22NO4 313 + 13 277 282, 286 Demethylmedicarpin hexosylmalonate Pterocarpan 503.1158[M + H] 5.1 C24H23O12 255 14 278 280, 308 Diacetoxy benzoic acid – 237.0397[M H] 3.3 C11H9O6 215, 174 + 15 289 282, 286 Demethylmedicarpin hexosylmalonate Pterocarpan 503.1162[M + H] 5.1 C24H23O12 255 16 292 272, 319 N-Cinnamoyl-Aspartic acid – 262.0717[M H] 1.5 C13H12NO5 218, 146 17 350 262, 308 Dihydroxyisoflavone Isoflavone 253.0497[M H] 3.5 C15H9O4 + 18 350 282, 286 Demethylmedicarpin Pterocarpan 255.0637[M + H] 5.7 C15H11O4 174 19 357 232, 285 Erythribyssin B Pterocarpan 283.0598[M H] 4.9 C16H11O5 269, 253, 214 20 369 280, 335 Eryvarin D Pterocarpan 335.1264[M H] 7.3 C21H19O4 271, 266, 241 21 380 284 Unknown isoflavone Isoflavone 355.1173[M H] 4.0 C20H19O6 333, 267 22 395 280 Unknown isoflavone Isoflavone 369.1324[M H] 5.4 C21H21O6 321 23 408 280, 335 Apigenin Flavone 269.0448[M H] 2.9 C15H9O5 24 485 280, 310 Unknown isoflavone Isoflavone 397.1288[M H] 1.3 C22H21O7 353 25 485 230, 287 Vogelin A Isoflavanone 369.0999[M H] 5.2 C20H17O7 329, 269 + 26 507 nd Oleanolic acid trihexoside Triterpene 943.5253[M + H] 5.4 C48H79O18 457 27 510 270, 307 5-Deoxyglyasperin Isoflavanone 337.1085[M H] 1C20H17O5 F/5-Deoxylicoisoflavanone + 28 531 287, 330 Unknown isoflavanone Isoflavanone 353.1014[M + H] 1.6 C20H17O6 29 531 287. 320 Licoisoflavanone/Ficuisoflavone Isoflavone 353.104[M H] 2.6 C20H17O6 319 30 537 287, 311 Isoneorautenol Pterocarpan 321.1147[M H] 4.5 C20H17O4 269, 252, 174 31 552 286, 323 Dihydroisoneorautenol Pterocarpan 323.1288[M H] 0.3 C20H19O4 + 32 562 287,322 Eryvarin I/Erypoegin B Isoflavone 337.1419[M + H] 4.6 C21H21O4 33 567 282 Erythrabissin I Pterocarpan 353.1409[M H] 4.1 C21H21O5 338, 309, 269 34 596 287, 304 5-Deoxylicoisoflavanone Isoflavanone 337.1084[M H] 0.8 C20H17O5 35 601 285, 340 Erysubin A Isoflavone 351.0878[M H] 1.2 C20H15O6 311 36 621 282 Unknown – 339.1237[M H] 0.2 C20H19O5 315, 248 37 630 281 Unknown – 353.1392[M H] 0.6 C21H21O5 316, 239 38 642 280 Erystagallin B Pterocarpan 437.1993[M H] 5.4 C26H29O6 368, 299 39 655 283 Unknown isoflavone Isoflavone 351.1223[M H] 4.3 C21H19O5 316, 248, 174 40 659 272 Unknown – 335.0922[M H] 0.8 C20H15O5 316 41 665 286 Unknown triterpene Triterpene 471.3500[M H] 4.4 C30H47O4 316, 284 42 678 281, 287 Sandwicensin Pterocarpan 337.1445[M H] 0C21H21O4 295, 268, 112 + 43 680 287,322 Dimethoxyisoflavone Isoflavone 283.0962[M + H] 1C17H15O4 253 44 689 285, Lysisteisoflavone Isoflavone 421.1664[M H] 1.7 C25H25O6 337, 295, 293 45 691 280 Hydroxy-9,11-octadecadienoic acid Fatty acid 295.2269[M H] 3.4 C18H31O3 248, 174 46 714 280 Unknown – 643.2375[M H] 9.5 C36H35O11 471, 365, 297 0 47 721 280 4 -O-Methylalpinumisoflavone Isoflavone 349.108[M H] 0.5 C21H17O5 335, 297, 248 48 748 280 Erylysin A Pterocarpan 405.1709[M H] 0.5 C25H25O5 + 49 762 nd Unknown sterol Sterol 463.3013[M + H] 3.2 C27H43O6 50 779 282 Hydroxy-9,11-octadecadienoic Fatty acid 295.2274[M H] 1.8 C18H31O3 248, 180 acid isomer 51 785 287,330 Unknown – 311.1682[M H] 9.5 C20H23O3 + 52 806 nd Unknown – 421.2047[M + H] 0.2 C25H25O6 377 53 838 nd Oleanolic acid Triterpene 455.3551[M H] 4.4 C30H47O3 384, 297 rt, retention time; nd, not detected. 686 M.A. Farag et al. component analysis (PCA) was further adopted to classify analyzed in positive ionization MS condition. PCA score plots samples in a more holistic way. From all samples, a total of derived from MS peaks in positive ionization mode were com- 3152 mass signals were extracted by XCMS from the UPLC- parable to those in negative mode concerning segregation of MS data set acquired in negative ionization mode. The main samples at 0 h from 12 and 24 h. The PCA model (Fig. 3A) principal component (PC) to differentiate between samples, explained 93% of the total variance in the first component, i.e. PC1, accounted for 92% of the variance. The multivariate PC1, whereas the second principal component, PC2 presented data analysis performed on MS data revealed a significant sep- 6% of the variance. Although comparable score plots in PCA aration among samples (Fig. 2A) with cells harvested at 0 h were derived from both data sets, loading plots revealed a clearly distinguished (positive PC1 values) from cells treated slightly different set of metabolites contributing for sample with the MeJA at 12 and 24 h (negative PC1 values, Fig. 2A clustering. As revealed in Fig. 3B, the major group that stood right side of the score plot). Loading plot that exposes the most out in this plot corresponded to MS signals for dimethoxy- variant MS signals among samples revealed for enrichment of isoflavone (43), isoneorautenol (30) and an unknown ptero- pterocarpans, sandwicensin (42), isoneorautenol (30) and ery- carpan found more enriched in unelicited cell culture samples thrabissin I (33) in unelicited cultures. In contrast, cell culture harvested at 0 h. In contrast, negative loading plot results samples harvested at 12 and 24 h were found more enriched in along PC1 revealed that the triterpene glycoside ‘‘oleanolic triterpenes and fatty acids namely oleanolic acid (53) and acid tri-hexoside” (26) and an unknown sterol (49) (Fig. 3B) hydroxy-octadecadienoic acid (45) and suggestive for a sup- levels were higher in the MeJA treated samples and accounting pression effect on pterocarpan biosynthetic branch in E. lysis- for its segregation at 12 h and 24 h from 0 h time point. The temon cell culture. The induction of oleanolic acid is consistent enrichment of the major pterocarpan ‘‘isoneorautenol” (30) with reports on MeJA up regulation of terpenoid biosynthetic in the untreated control cell culture samples (Fig. 3B) concurs pathways in planta [24]. MeJA induction of b-amyrin synthase results derived from negative ionization mode and highlighting gene associated with oleanolic acid (54) production was also the negative impact of MeJA on pterocarpans biosynthetic previously reported in Gentiana straminea [25]. branch. The decrease in pterocarpan levels in response to MeJA treatment is contrary to previous reports in Medicago PCA of E. lysistemon MeJA elicited and control suspension truncatula cell culture [17] and lupines [26], suggesting that a culture observed in positive ionization mode differential response to MeJA exists in various legume species. This is the first report of MeJA differential effect on terpenoid To provide more overview on the effects of elicitation on accumulation versus pterocarpans in E. lysistemon cell culture E. lysistemon cell culture metabolome, samples were also (Fig. 4). Studies focused on the genetic bases of MeJA

(A) 12 h 0 h PC2 (7%) 2000 0 2000 6000 - 24 h 6000 - -20000 -15000 -10000 -5000 0 5000 10000 PC1 (92%)

455.3/842 (B) Oleanolic acid 523.3/841 Sandwicensin 563.1/244 337.1/684 161/259 653.3/351 653.8/351 433.2/800 654.3/351311.2/814571.3/767311.2/791595.3/725564.1/244 277.2/826 327.2/418645.3/359632.3/340655.3/340453.2/193467.2/229645.8/359687.3/351632.8/340431.1/205265.1/712459.2/921572.3/767596.3/725434.2/800353.1/467566.3/706483.3/857267/414 353.1/534 349.2/936282.2/936330.2/478655.8/340586.5/936162/259666.3/373687.8/351993.5/527675.3/352351.2/444654.8/351675.8/352327.2/543653.5/936666.8/373527.2/922350.2/936483.2/878499.1/205475.1/193527.2/559666.3/341633.3/340574.3/387187.1/309209.1/309624.3/348924.5/565889.7/936994.5/527398.2/444489.2/229527.7/559779.5/551666.8/341656.3/340331.2/444309.2/798624.8/348417.2/936485.2/936468.2/229669.4/919293.2/885654.5/936574.8/388928.5/527528.3/559391.2/920551.2/879353.2/847737.4/920721.5/936640.3/361633.8/340640.3/301324.2/919309.2/936278.2/684283.3/936133/259348.2/877647.3/348631.3/334617.3/361925.5/565911.5/524945.5/509958.5/425969.5/603973.5/510447.2/683476.1/193397.2/878535.5/919553.2/936780.5/551656.8/340667.3/341722.5/936738.4/919717.4/877634.4/571640.8/361640.8/302276.2/639194.1/577206.1/245789.4/936790.4/935647.8/348631.8/334625.3/348617.8/360621.2/937575.3/388879.7/919863.4/524602.4/919956.5/426957.5/509990.5/509971.5/527971.5/435415.2/878398.2/937484.2/879403.1/222481.2/825469.2/228528.8/559519.8/576527.2/349547.2/277551.3/363367.1/432555.3/826549.2/825670.4/919785.4/877786.4/877281.2/877718.4/877689.2/937321.2/877321.2/860313.2/919271.2/919257.2/919277.1/309307.2/878266.1/712267.2/728271.1/309277.1/636211.1/877293.2/562295.2/828227.2/828349.2/877797.5/524650.4/877651.4/877627.4/877577.2/276617.2/825250.1/216253.2/860925.4/936880.7/919873.4/920881.7/919583.5/877593.1/224947.6/919974.5/510407.2/826471.3/826457.1/291434.1/303391.2/826566.1/244571.3/737781.4/826783.1/244767.1/245700.1/245715.1/245171.1/878347.2/826653.1/245653.4/670619.2/879337.2/877235.2/638233.1/877885.7/877603.4/919969.5/391413.2/826699.1/244269/409275/259305.2/826273.1/826646.4/826632.1/244237/280405.2/750433.1/303565.1/244635.4/856713.4/826299.1/419211.1/826279.2/826346.2/826631.1/244499.2/359477.2/857661.1/221117/259579.1/229251.2/826681.3/904291/258163/259645.4/826229/258476.3/650270/409307/258134/259351.1/505353.1/318345/259340/216242.2/432391/258635.4/882484.3/857501.2/801369.1/381353.1/438595.3/701483.3/822578.4/826449.1/223504.3/656345.2/826564.3/656480.3/760355.1/534508.3/769476.3/711295.1/542278.2/826577.4/826480.3/682540.3/682354.1/534452.3/676 281.2/936329.2/478926.5/527330.2/444585.5/936397.2/444988.5/509323.2/919927.5/527327.2/476944.5/509939.5/498347.2/877795.5/524364.2/684923.5/565315.2/704256.2/919940.5/498941.5/545989.5/509275.2/639796.5/524601.4/919957.5/426431.2/684534.5/919361.2/639379.2/683317.2/683309.2/643614.4/683967.5/561280.2/877811.7/919633.4/571254.1/351649.4/877812.7/919883.7/877884.7/877582.5/877955.5/426337.1/441410.2/857285/353633.4/861297.1/555409.2/857568.4/769324.1/555338.1/587 322.1/542 PC2 940.5/545295.2/782 329.2/444 925.5/527943.5/509939.5/545295.2/695987.5/509277.2/684613.4/683283/447581.5/877 337.1/587 293.2/713363.2/684533.5/919 296.2/683 323.1/555 Erythrabissin I 321.1/542 255.2/919279.2/877253/351 942.5/509 Isoneorautenol Hydroxy-octadecadienoic acid

295.2/683 0.4 -0.2 0.0 0.2 0.4 - 271.2/816 -0.2 0.0 -0.2 -0.4 -0.6 PC1

Fig. 2 UPLC-qTOF-negative ionization MS (m/z 100–1000) principal component analyses of E. lysistemon unelicited cell culture (), cell cultures treated with 1.0 mM MeJA at 0 h (D), 12 h (D) and 24 h (+) (n = 3). The metabolome clusters are located at the distinct positions in two-dimensional space prescribed by two vectors of principal component 1 (PC1 = 92%) and principal component 2 (PC2 = 7%). (A) Score Plot of PC1 vs PC2 scores. (B) Loading plot for PC1 and PC2 contributing to mass peaks and their assignments, with each metabolite denoted by its mass/rt (s) pair. Erythrina lysistemon cell culture metabolomics 687

(A) 12 h

0 h PC2 (6%) 24 h -2000 0 2000

-50000 0 5000 PC1 (93%)

439.4/841 425.3/891 713.5/919 283.1/683 Unknown sterol Dimethoxy isoflavone 425.4/859 379.3/886 274.3/479 403.2/758 439.4/853 337.1/510 440.4/841476.2/211562.2/238393.1/398565.2/244 377.3/825361.1/683 503.1/290426.4/859426.3/891163/398 671.4/741671.4/781407.4/859377.1/510 631.4/741393.4/841479.4/852358.2/292440.4/853459.3/894495.3/747335.1/555191.1/398445.2/683321.1/337393.3/638519.1/263284.3/938380.3/886177.1/221522.4/705 284.1/683 226.1/240 437.3/742672.4/741631.4/781437.3/671437.3/781246.2/401479.4/840339.3/637340.2/302275.3/479563.2/238302.3/548455.1/204935.7/841322.1/337504.1/290299.1/230205.2/859217.2/859404.2/758885.8/859393.4/884299.2/509313.3/852593.3/919339.3/886411.4/936271.1/263457.2/799179/683 353.3/865672.4/781632.4/741373.7/742495.4/671218.2/305342.3/669351.2/826629.4/730519.1/325295.7/841457.4/841259.2/841429.2/826494.2/212477.2/211226.1/292441.4/841895.7/841943.5/350179.2/841480.4/852577.3/261461.2/683296.3/479913.7/841936.7/841580.2/238290.3/487621.3/810460.3/894368.4/686659.3/904477.2/193411.4/883896.7/841304.2/877394.1/398191.2/860408.4/859322.1/306377.3/807419.3/895886.8/859282.1/241637.3/904581.3/810443.4/859353.1/398496.4/747223.1/398907.8/859541.3/761541.2/337335.2/877379.3/937391.2/592973.5/434281.1/316337.1/434446.2/683281.1/287278.2/842366.3/645594.3/919314.2/264425.4/909177.1/178359.1/670375.1/554265.3/886566.2/244480.3/698587.1/244547.1/244405.2/683378.1/510203.1/670338.2/510203.1/510495.1/568290/382595.2/435332.1/221302.1/683378.3/825362.1/683335.1/683339.2/683 283.3/935683.4/759265.3/936272/258727.5/758200/258603.3/707351.2/417684.4/759247.2/935949.5/526227.1/240305.2/935321.2/724177.1/741547.5/935195.1/741351.2/455563.3/558354.3/865438.3/742328.2/935492.3/526632.4/781339.3/621264/258321.2/704455.4/741728.5/758649.4/741382.7/741771.5/756266.3/935373.7/781374.2/741264.1/292950.5/526288.3/514321.2/802673.4/741617.4/761275.2/417329.2/750177.1/781445.2/828185.1/444581.3/387188.1/176631.4/793271.1/325497.4/840394.4/841563.8/558525.1/290529.5/936397.2/757438.3/781438.3/671492.8/526411.4/841195.1/781437.2/587352.2/416453.2/587926.5/508572.3/387455.4/781469.3/858243.1/734383.2/741485.2/526339.1/526346.3/351347.1/258495.3/878304.7/841685.4/759194.1/444426.4/526367.2/417671.4/817306.2/671633.4/741673.4/781306.3/938581.8/387627.3/678650.4/742239.2/865491.2/229359.2/292548.5/936200.5/258772.5/756458.4/841370.4/734309.1/526641.4/635374.2/781189.1/826699.3/350248.2/935451.4/883287.2/840480.4/840321.2/842572.8/387455.4/671323.1/526577.8/261243.2/841313.3/865314.2/249382.7/781344.5/258350.3/867433.1/204355.2/413483.4/819296.2/841914.7/841286.7/840665.2/233501.3/852951.5/526954.5/508638.3/461403.2/826699.8/350456.4/742576.2/352541.1/325322.2/724598.2/352467.4/693625.3/636649.4/781343.3/669430.2/826328.1/352328.1/325349.2/445214.1/398407.2/826485.4/861495.2/212233.1/547232.1/229230.3/481515.4/841337.2/596330.2/257330.3/613554.3/558546.3/558643.2/413507.2/449375.2/443437.2/350217.1/936215.2/841409.4/741482.2/497352.2/826352.2/455352.3/936271.1/408271.1/283520.1/325947.5/564935.7/852943.5/387177.2/936365.7/741555.3/765638.8/461635.3/461341.2/302564.3/558191.2/936408.4/526525.2/581526.1/290525.4/841453.3/698289.2/841530.5/936306.3/935247.2/401247.2/671335.1/336306.7/671575.3/386573.3/387256.3/843137.1/841541.1/526474.3/558275.2/454601.4/526653.3/340429.3/792658.3/260343.2/802343.2/724413.3/936485.2/366493.3/526233.2/671313.3/766549.2/558503.2/527507.7/449349.2/510340.3/637427.4/865213.2/936407.4/396758.6/936483.4/782483.4/738353.1/308495.3/778495.4/896233.1/581269.2/842517.2/598512.2/325946.5/527339.3/810687.4/741546.8/558555.2/321642.4/636506.2/434498.4/840374.3/837350.3/783563.2/243189.2/859203.1/477434.2/856877.7/841867.8/859752.5/918419.8/755203.2/741409.4/671205.2/852481.2/895409.1/398423.4/558419.1/827355.2/559355.3/865123.1/841103.1/292225.1/193261.2/841299.1/399908.8/859948.5/564948.5/508947.5/526923.5/558927.5/527935.7/936937.7/841997.5/527181.2/936177.2/742320.2/751365.1/566371.2/443660.3/904306.2/747308.3/797632.5/936635.8/461497.4/861394.4/884564.3/396564.2/238615.3/400593.3/406712.5/918437.3/896249.2/852815.5/755463.2/936245.2/724786.6/919786.6/936580.3/379447.2/350971.5/602964.5/508283.3/669457.1/242288.1/264457.4/635289.3/514320.3/554571.2/300438.2/587247.2/860247.2/852273.1/404335.1/317573.8/387573.2/308733.7/558617.4/558597.3/581257.1/325256.1/290466.3/526465.4/859277.2/475277.7/852582.3/387581.2/238541.2/308322.3/802737.4/897627.2/347441.4/853441.3/860260.2/841474.8/558474.8/508477.3/896478.2/211470.3/858467.1/526443.4/937161.1/522153.1/936164/258654.3/340653.8/340655.2/379656.3/340659.2/330240.1/261313.1/209314.3/865310.1/352508.4/919381.3/939344.2/262343.2/936412.4/841412.4/883410.4/741493.3/701241.2/840233.2/841301.2/936517.7/599517.8/581514.2/390338.1/291338.3/581337.3/919337.1/258329.2/936330.1/413360.2/692554.8/558549.1/292549.8/558551.3/511503.3/796505.9/841503.4/841505.1/290507.1/366375.1/398348.1/258348.1/212198.2/204427.3/891208.5/258407.4/409407.4/575421.3/841407.4/910206.1/245407.1/486759.5/873761.5/558767.5/936759.6/919351.3/867207.2/859486.2/526353.1/424483.2/308495.3/537496.3/878225.2/671269.3/559271.2/859517.3/527511.7/571897.7/841899.8/859924.5/558915.7/841923.5/461460.2/563179.1/522317.3/581339.3/779337.2/587331.2/919369.2/865361.1/257369.3/693365.2/461369.4/686305.3/877666.2/233683.5/865309.1/558557.8/558558.3/559557.3/558557.2/394545.1/463546.3/461547.3/558546.8/460546.3/575502.3/852498.2/449506.7/434500.7/545563.3/810189.1/374195.1/936192.2/853431.2/692868.8/860215.2/860205.2/936763.5/508409.3/782757.7/387757.2/348779.5/544423.4/387423.4/916481.3/776496.4/896265.5/258267.3/936265.1/237268.2/401265.1/292298.3/936295.2/751509.8/547578.3/261878.7/841444.4/859959.5/442887.8/859579.3/723944.5/544928.5/527931.5/526936.7/936944.5/386452.4/883284.2/559284.2/461941.5/498287.1/353925.5/387462.3/461462.2/683987.5/508526.8/547982.5/508526.4/841173.1/919177.2/671177.2/859181.1/234332.2/501367.3/912679.2/272555.3/558636.3/461637.3/809638.3/904405.2/814395.3/749564.3/386615.8/401615.4/899711.4/836192.1/398218.2/859817.2/336247.1/193803.5/550245.2/704782.6/919272.1/325525.1/526445.2/690445.2/734972.5/602973.5/508970.5/508455.2/856283.2/859456.4/671456.1/205971.5/390455.2/730454.2/587974.5/434533.7/516542.2/337321.2/936321.2/877674.4/742307.1/229307.1/193307.7/841575.8/387617.3/663438.3/896255.2/841254.1/229463.4/916135.1/936541.2/287322.2/704322.1/400335.1/ 327.2/935286.1/292353.2/489483.3/508962.5/508501.3/508425.4/526553.4/764351.3/936534.2/527499.7/545963/508942.5/545509.3/765491.2/508354.2/443963.5/544308.2/365967.5/508272.5/258483.8/508963.5/508284.3/935529.4/764255.1/290209/258485.4/765499.2/497319.2/743573.4/762597.4/762946.5/508944.5/526925.5/508271.2/765369.2/444354.2/477797.5/508315.2/766543.3/490641.4/760565.3/762534.7/527302.2/919407.4/526512.8/537507.1/463351.2/476264.6/258319.2/762964.5/544288.3/501333.2/803617.4/508381.3/571421.3/765491.7/508484.3/508482.3/544985.5/537797.5/523631.5/936277.2/443179.1/703499.7/497499.2/508351.2/543529.1/463332.2/763579.4/919508.2/425387.2/443500.2/544943.5/544331.2/502781.5/508353.2/506176.1/444324.3/590661.5/759633.4/544473.3/545315.2/619348.3/827490.2/545492.3/508339.2/703543.8/490340.3/553425.4/547482.8/545947.5/508326.2/877501.8/508423.4/508295.2/443955.5/508565.1/292798.5/508455.4/698474.3/508278.2/712277.2/646316.3/570387.2/477347.3/918337.2/877427.2/523427.3/444407.4/547766.4/903369.2/477361.2/803365.1/560559.1/526502.2/508194.1/477526.2/547959.5/425293.2/476986.5/537636.4/508819.5/523798.5/523455.4/896968.5/508185.1/477257.1/449465.3/874137/258469.4/885278.2/703279.2/752275.3/919277.2/638508.8/425343.2/829492.8/508302.2/683309.2/365507.2/426381.3/618345.2/804491.2/559490.7/545484.4/903484.8/508513.3/537514.2/602499.7/508437.2/921427.3/419221.2/919282.3/553317.2/638332.2/250679.5/919632.4/683641.4/728373.2/936782.5/508295.1/291965.5/544957.5/426457.4/497533.2/563183.1/749176.1/477324.3/936672.4/817571.2/547618.4/508257.1/354256.1/350473.8/544441.4/508601.5/919605.4/508315.2/841315.2/250509.3/547344/258427.7/523504.3/560504.3/537508.1/463342.2/683349.3/887209.2/683209.5/258213.1/443209.2/936421.3/772483.3/544517.3/581326.3/827331.2/460305.1/527544.3/490543.1/526192.5/258195.2/936351.2/703423.4/581351.1/724352.2/476261.2/683266.1/264269.1/176296.2/816285.3/590991.5/560526.3/527975.5/508981.5/508173.1/683319.2/782319.2/936335.2/515634.4/544699.4/444194.1/703248.1/239820.5/522787.4/903132.1/292131.5/258579.4/937580.4/919283.2/249293.2/919291.2/502530.1/463533.2/516183.1/232255.1/435251.2/749795.5/544627.4/877733.2/333280.2/683599.4/877599.4/508743.4/903627.1/547148.5/258149.1/936163.1/936245.1/308243.2/874647.2/304647.5/919242.3/581329.2/536315.2/919310.3/514509.3/598345.2/936428.4/861514.9/841336.1/258336.6/258309.2/919551.1/292553.1/547549.5/935504.8/560504.8/537507.2/509507.7/426345.2/797199.2/935211.1/308758.6/919761.5/896765.5/526768.5/936482.7/497495.4/937229.2/828271.1/317299.3/530299.2/645517.2/547461.2/877527.2/559167.1/936318.2/816319.2/877339.3/531317.2/919326.2/845337.2/936335.3/836369.2/703362.3/585680.5/920683.5/920553.4/863641.4/921502.7/508498.2/545341.3/553561.3/937195.1/444426.4/547857.7/919858.7/919425.1/423351.1/560408.2/281419.2/509227.2/936109.1/936265/258264.2/877526.3/490298.1/263295.2/703511.2/571281.3/877293.2/782281.5/258282.3/574282.2/250580.4/938957.5/508453.2/816927.5/508929.5/506997.5/508987.5/537319.3/553303.3/590689.5/935637.4/683403.1/424399.4/934566.1/292615.4/497615.4/544192/258701.5/936819.2/358787.5/835424.4/508799.5/508784.6/919784.6/935525.7/581289.3/501535.8/520531.1/350185.1/703185.2/936321.2/683321.3/551334.1/292393.3/703576.5/919479.4/884463.3/860539.4/920537.3/490577.4/841734.2/333621.4/877466.3/874137.5/258140.5/258278.1/350277.2/887584.4/840440.3/877259.3/919471.4/920275.2/619275.2/668276.1/292277.2/855600.4/508751.4/868603.5/936606.5/508244.3/503301.3/553316.2/264654.4/733652.4/838509.1/412241.2/919237.2/919239.2/764339.1/669329.1/308329.2/554383.3/755386.1/333381.3/897427.4/527415.2/865484.4/782303.2/919513.8/537515.1/279327.2/877337.1/408327.3/602309.2/794544.2/271550.5/918545.2/271643.3/779505.3/537503.2/575377.1/555377.1/408347.1/788198.2/306411.2/223215.2/919351.2/877422.4/841351.3/852482.4/775495.4/834495.3/825227.1/302229.2/883269.2/680519.1/244519.4/918165.1/877169.1/211318.3/531318.2/638367.1/221373.1/724305.2/840694.5/918557.2/768557.5/919559.3/852560.1/526547.1/289546.4/763547.1/292341.2/877341.2/826341.2/782350.3/877561.5/877613.4/855562.5/877614.4/835563.1/442189.2/877193.2/877437.2/236248.2/852763.4/877423.4/775350.3/790481.3/643353.2/519124.5/292103.1/919261.2/515266.3/590267.2/293295.2/602520.2/537294.2/919294.7/671293.2/649964/508929.7/877930.7/877287.2/874457.4/893291.2/877992.5/560993.5/560529.2/240531.2/938175.1/877175.1/838181.2/877325.3/590659.4/921661.2/328678.5/935401.3/764396.3/749395.1/261395.1/547391.2/901566.4/842567.3/395715.4/919480.4/536249.1/579247.1/763820.2/358246.2/877809.4/902809.7/919219.2/919807.7/919788.4/903783.5/508133.5/292132.5/258522.4/905521.4/919580.5/878283.3/553283.3/575455.1/283291.1/788291.1/619535.4/937532.5/919534.2/490322.1/264333.2/541333.2/649677.5/936307.3/938571.3/355574.3/387575.3/592577.1/290617.5/936721.2/281722.2/281257.1/411479.3/515464.4/566273.2/828587.2/440588.2/440591.1/290455.4/537539.2/788539 Oleanolic acid 639.4/761965.5/508163/258596.4/762966.5/508640.4/761331.2/763499.2/544941.5/544331.2/829319.2/751615.5/936185/258318.2/703317.2/646512.3/537319.2/695318.2/712191.2/852635.4/508483.4/903341.2/683765.4/903284.3/590303.2/877347.3/853348.3/842535.3/527631.4/683302.3/590557.5/683320.3/550279.2/919441.3/754300.3/553335.2/595227.2/816269.1/275245.2/877441.3/764552.4/863349.2/877265/262439.1/244478.3/515335.2/607281.3/551509.1/489214.3/507285/446607.3/888337.2/789325.2/845315.2/789325.2/802293.2/720293.2/888575.5/919187.2/789443.3/811379.1/383425.2/802255.1/244269.1/381593.2/419599.2/616574.5/921323.1/389191.1/686572.2/419355.3/827163/554302.1/733647.3/750208.1/541337.1/683361.1/587346.1/541339.1/422280.1/200378.1/566497.3/682338.2/566353.1/605339.1/510 500.3/508353.2/443552.4/763500.8/508945.5/508277.2/712131.1/292301.2/683346.2/869258.3/919338.3/531295.2/816315.2/669240.2/919263.2/877317.2/816298.3/530317.3/531460.3/531469.4/866277.1/350311.3/827325.2/825441.3/778528.2/240225.1/764355.3/797509.4/925399.3/812534.5/919323.1/419172/554347.1/554340.1/587524.4/767302.1/200267.1/541667.2/541282.1/566 508.3/765464.3/765353.2/477420.3/765463.3/777280.3/550320.2/683279.2/683311.3/811270.1/554325.1/554 355.1/534 PC2 317.2/722325.2/877355.3/811571.2/419147/200301.1/733 377.1/566496.3/681 trihexoside 595.4/762944.5/508 301.2/919376.3/763347.3/842255.1/350318.3/553 324.1/541 337.1/566 317.2/712 345.1/541 Unknown pterocrapan 551.4/764375.3/772317.2/703 339.1/587 345.2/869316.3/531 281.1/566 943.5/508507.3/765419.3/765 239.2/919 463.3/765 319.2/683 Dihydroisoneorautenol 257.3/919 (B) 0.2 0.2 -0.1 0.0 0.1 0.2 - 269.1/554 323.1/541 -0.2 0.0 -0.2 -0.4 PC1

Fig. 3 UPLC-qTOF-positive ionization MS (m/z 100–1000) principal component analyses of E. lysistemon unelicited cell culture samples (), cell cultures treated with 1.0 mM MeJA at 0 h (o), 12 h (D) and 24 h (+) (n = 3). The metabolome clusters are located at the distinct positions in two-dimensional space prescribed by two vectors of principal component 1 (PC1 = 93%) and principal component 2 (PC2 = 6%). (A) Score Plot of PC1 versus PC2 scores. (B) Loading plot for PC1 and PC2 contributing to mass peaks and their assignments, with each metabolite denoted by its mass/rt (s) pair.

E. lysistemon plant E. lysistemon callus E. Lysistemon MeJA elicited cell culture

O OH MeJA

OH O H3CHCH3 Callus iniaon O O CH3 O Isoneorautenol H3C O CH 1 CH 3 R 3 OH H3C CH3 CH3 O HO Erythrabissin O H3C CH3 H3C N R HO O Oleanolic acid H CHCH Pterocarpans 3 3 H3C O R=CH , R1=OCH Erythristemine 2 3 Triterpenes R=CO, R1=H Erysotramidine

Alkaloids

Fig. 4 Diagram showing major secondary metabolite pathways with represented major structures that are up regulated in E. lysistemon plant, cell culture and in response to MeJA elicitation. 688 M.A. Farag et al.

Christoph Bo¨ ttcher, Leibniz Institute of Plant Biochemistry, Table 2 Quantification of metabolites identified in E. lysiste- Germany, for assistance with the UPLC-MS. We are grateful mon cell suspension at 0, 12 and 24 h post 1.0 mM MeJA to Dr. Tilo Lu¨ bcken, University of Dresden, Germany, for elicitation using MS detection in positivea and negativeb providing R scripts for UPLC-MS data analysis. ionization modes. Values are expressed in lgg 1 as average for 3 biological replicates. Appendix A. Supplementary material Metabolites (lgg1) E. lysistemon cell suspension 0h 12h 24h Supplementary data associated with this article can be found, Erysotrinea 2.8 ± 0.9 3.4 ± 2.1 0.6 ± 0.5 in the online version, at http://dx.doi.org/10.1016/j.jare.2016. Erysotramidinea 10.8 ± 3.8 8.9 ± 2.5 6.0 ± 1.4 07.002. ErylysinAb 36.0 ± 8.2 22.2 ± 5.4 12.7 ± 2.6 Sandwicensinb 10.7 ± 2.9 3.4 ± 0.5 2.2 ± 0.5 References Oleanolic acidb 406 ± 32.1 4907 ± 133 4838 ± 237 ErythrabissinIb 1268 ± 85 268 ± 59 243 ± 18 Isoneorautenolb 2217 ± 89 473 ± 16 564 ± 47 [1] Gledhill D. The names of . 4th ed. Cambridge, UK: Cambridge University Press; 2008. p. 157. [2] El-Masry S, Amer ME, Abdel-Kader MS, Zaatout HH. elicitation will help affirm induction hypothesis derived via Prenylated flavonoids of Erythrina lysistemon grown in Egypt. metabolite profiling. It should be noted that oleanolic acid Phytochemistry 2002;60(8):783–7. [3] El-Masry S, Hamoda HM, Zaatout HH, Abdel-Kader MS. tri-hexoside conjugate was not detected by visual examination Constituents of stem bark grown in Egypt. of unelicited cell culture chromatograms, suggesting that cou- Natural Product Sciences 2010;16(4):211–6. pling of metabolomics for analysis of elicited samples presents [4] El-Masry S, Amer ME, Dawood HM, Radwan MM, ElSohly a powerful methodology for identification of novel metabo- MA, Abou-Karam M, et al. Bioassay-guided isolation of lites. Quantification of the major differential metabolites in eli- cytotoxic agents from Erythrina caffra root bark. Planta Med cited cell culture is presented in Table 2. 2014;80(10):807–8. [5] Amer ME, El-Masry S, Shamma M, Freyer AJ. Three novel Conclusions glycodienoid alkaloids from Erythrina lysistemon. J Nat Prod 1990;54(1):161–6. [6] Iranshahi M, Vu H, Pham N, Zencak D, Forster P, Quinn RJ. This study provides the first report on E. lysistemon cell sus- Cytotoxic evaluation of alkaloids and isoflavonoids from the pension culture metabolite fingerprint via UPLC-MS. A meta- Australian Erythrina vespertilio. Planta Med 2012;78 bolomic approach was used to investigate secondary (7):730–6. metabolites viz. alkaloids, flavonoids and triterpenes and their [7] Wanjala CCW, Majinda RRT. Two novel glucodienoid reprogramming in response to MeJA elicitation. The results alkaloids from Erythrina latissima seeds. J Nat Prod confirm MeJA elicitation effect on terpenoid accumulation 2000;63:871–3. [8] Tanaka H, Etoh H, Shimizu H, Oh-Uchil T, Terada Y, Tateishi and extend our knowledge base concerning secondary Y. Erythrina alkaloids and isoflavonoids from Erythrina metabolism in other legume species [27]. Comparative meta- poeppigiana. Planta Med 2001;67:871–2. bolic profiling of E. lysistemon cell suspension culture and in [9] Yenesew A, Derese S, Irungu B, Midiwo JO, Waters NC, Liyala response to elicitation using MeJA, revealed an activation in P, et al. Flavonoids and isoflavonoids with antiplasmodial sterol/triterpenes formation, see model depicted in Fig. 4. activities from the root bark of Erythrina abyssinica. Planta Med The effect of other elicitors on secondary metabolites accumu- 2003;69:658–61. lation in Erythrina cell culture could also provide more holistic [10] Njamen D, Talla E, Mbafor JT, Fomum ZT, Kamanyi A, insight into elicitation effect within that genus and how it can Mbanya JC, Cerda, et al. Anti-inflammatory activity of reprogram its different secondary metabolite pathways. erycristagallin, a pterocarpene from Erythrina mildbraedii. Eur J Pharmacol 2003;468:67–74. [11] San Miguel-Cha´vez R, Soto-Herna´ndez M. Antifungal activity Conflict of Interest of the alkaloid extract of Erythrina americana miller seedlings. In: Vinciery F, editor. Proceedings of the 53rd annual congress The authors declare that they have no conflict of interest. of medicinal plant research, Florence. p. 326. [12] Garcı´ a-Mateos R, Soto-Herna´ ndez M, Martı´ nez-Va´ zquez M, Compliance with Ethics Requirements Villegas-Monter A. Isolation of alkaloids of Erythrina from tissue culture. Phytochem Anal 1999;10(1):12–6. [13] San Miguel-Cha´ vez R, Soto-Herna´ ndez M, Ramos-Valdivia This article does not contain any studies with human or animal AC, Kite G. Alkaloid production in elicited cell suspension subjects. cultures of Erythrina americana miller. Phytochem Rev 2007;6:167–73. [14] Baenas N, Garcı´ a-Viguera C, Moreno DA. Elicitation: a tool for enriching the bioactive composition of foods. Molecules Acknowledgments 2014;19:13541–63. [15] Zhao J, Davisb LC, Verpoorte R. Elicitor signal transduction Dr. Mohamed A. Farag acknowledges the funding received by leading to production of plant secondary metabolites. Science and Technology Development Fund STDF, Egypt Biotechnol Adv 2005;23:283–333. (grant number 12594), and the support of the Alexander von [16] Mekky H, Mohamed M, Lazarus C, Power JB, Davey MR. Biosynthesis of very long chain polyunsaturated fatty acids in Humboldt Foundation, Germany. We also thank Dr. the leafy vegetable chicory. Agric Food Sci 2011;20:327–40. Erythrina lysistemon cell culture metabolomics 689

[17] Farag MA, Huhman DV, Dixon RA, Sumner LW. isoflavonoids in roots and cell suspension cultures of Medicago Metabolomics reveals novel pathways and differential truncatula using HPLC-UV-ESIMS and GC-MS. mechanistic and elicitor specific responses in phenylpropanoid Phytochemistry 2007;68:342–54. and isoflavonoid biosynthesis in Medicago truncatula cell [23] Jalal MAF, Collin HA. Polyphenols of mature plant, seedling cultures. Plant Physiol 2008;146(2):387–402. and tissue cultures of Theobroma cacao. Phytochemistry [18] Farag MA, El Sayed AM, El Banna A. Metabolomics reveals 1977;16:1377–80. distinct methylation reaction in MeJA elicited Nigella sativa [24] Scholz M, Lipinski M, Leupold M, Luftmann H, Harig L, Ofir callus via UPLC-MS and chemometrics. Plant Cell Tiss Org R, et al. Methyl jasmonate induced accumulation of 2015;122:453–63. kalopanaxsaponin I in Nigella sativa. Phytochemistry 2009;70 [19] El Senousy AS, Farag MA, Al-Mahdy DA, Wessjohann LA. (4):517–22. Developmental changes in phenolics composition from [25] Liu Y, Cai Y, Zhao Z, Wang J, Li J, Xin W, et al. Cloning and three artichoke cvs. (Cynara scolymus) as determined via functional analysis of a beta-amyrin synthase gene associated UHPLC-MS and chemometrics. Phytochemistry with oleanolic acid biosynthesis in Gentiana straminea MAXIM. 2014;108:67–76. Biol Pharm Bull 2009;32(5):818–24. [20] Farag MA, EL-Ahmady S, Alian F, Wessjohann LA. [26] Katagiri Y, Hashidoko Y, Ibrahim RK, Tahara S. Activation of Metabolomics driven analysis of artichoke leaf and its isoflavone biosynthesis in excised cotyledons of Lupinus commercial products via UHPLC-q-TOF-MS. Phytochemistry seedlings by jasmonoids and excess light. Z Naturforsch C 2013;95:177–87. 2001;56(11–12):1038–46. [21] Smith CA, Want EJ, O’Maille G, Abagyan R, Siuzdak G. [27] Dixon RA, Sumner LW. Legume natural products. XCMS: processing mass spectrometry data for metabolite Understanding and manipulating complex pathways for profiling using nonlinear peak alignment, matching, and human and animal health. Plant Physiol 2003;131:878–85. identification. Anal Chem 2006;78:779–87. [22] Farag MA, Huhman DV, Lei Z, Sumner LW. Metabolic profiling and systematic identification of flavonoids and