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

An MS-based metabolomic approach was used to profile the secondary metabolite of the ornamental plant Erythrina lysistemon via ultra-performance liquid chromatography coupled to photodiode array detection and high resolution q-TOF mass spectrometry (UPLC-PDA-MS). Cultures maintained the capacity to produce E. lysistemon flavonoid subclasses with pterocarpans 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 detected at trace levels in suspension cultures. Methyl jasmonate (MeJA), phytohormone, was further supplied to culture with the aim of increasing secondary metabolites production and with metabolite profiles subjected to multivariate data analysis to evaluate its effect. Results revealed that triterpene i.e. oleanolic acid and fatty acid i.e. hydroxy-octadecadienoic acid were elicited in response to methyl jasmonate, whereas pterocarpans i.e., isoneorautenol showed a decline in response to elicitation suggesting for the induction of terpenoid biosynthetic pathway and concurrent with a down regulation of pterocarpans. In conclusion, a total of 53 secondary metabolites including 3 flavones, 12 isoflavones, 4 isoflavanones, 4 alkaloids, 11 pterocarpans, and 5 phenolic acids were identified.

Journal of Advanced Research (2016) 7, 681–689 Cairo University Journal of Advanced Research ORIGINAL ARTICLE Metabolomics driven analysis of Erythrina lysistemon cell suspension culture in response to methyl jasmonate elicitation Mohamed A Farag a,*, Hattem Mekky b, Sawsan El-Masry b a b Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Kasr El-Einy Street, 11562 Cairo, Egypt Pharmacognosy Department, Faculty of Pharmacy, Alexandria University, El Khartoum Square, 21521 Alexandria, Egypt G R A P H I C A L A B S T R A C T A R T I C L E I N F O Article history: Received 29 April 2016 Received in revised form July 2016 Accepted July 2016 Available online 14 July 2016 A B S T R A C T An MS-based metabolomic approach was used to profile the secondary metabolite of the ornamental plant Erythrina lysistemon via ultra-performance liquid chromatography coupled to photodiode array detection and high resolution q-TOF mass spectrometry (UPLC-PDA-MS) Cultures maintained the capacity to produce E lysistemon flavonoid subclasses with pterocarpans 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: Mohamed.farag@pharma.cu.edu.eg (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 Keywords: Cell culture Erythrina lysistemon Metabolomics Methyl jasmonate Oleanolic acid Ultra-performance liquid chromatography M.A Farag et al detected at trace levels in suspension cultures Methyl jasmonate (MeJA), phytohormone, was further supplied to culture with the aim of increasing secondary metabolites production and with metabolite profiles subjected to multivariate data analysis to evaluate its effect Results revealed that triterpene i.e oleanolic acid and fatty acid i.e hydroxy-octadecadienoic acid were elicited in response to methyl jasmonate, whereas pterocarpans i.e., isoneorautenol showed a decline in response to elicitation suggesting for the induction of terpenoid biosynthetic pathway and concurrent with a down regulation of pterocarpans In conclusion, a total of 53 secondary metabolites including flavones, 12 isoflavones, isoflavanones, alkaloids, 11 pterocarpans, and 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 pea family ‘‘Fabaceae” which are distributed worldwide in tropical and subtropical regions growing as trees, often recognized in agriculture for its bright red flowers as coral or flame trees [1] Alkaloids and phenolics are among the most widely distributed constituents in these flowering trees mostly localized in stem bark [2,3], roots [4] and seeds [5,6] Erythrina alkaloids are tetracyclic spiroamines possessing an erythrinane skeleton Over 90 Erythrina alkaloids have been isolated [7,8], often classified as dienoid or lactonic alkaloids Interest in Erythrina alkaloids is mostly driven by its curare-like neuromuscular blocking effect Moreover, Erythrina spp possess a broadspectrum of physiological activities such as anti-plasmodial activity due to the flavonoids and isoflavonoids [9], antioxidant and anti-inflammatory activities due to pterocarpans [10] and fungicidal activity associated with its alkaloidal content [11] Erythrina genus has been extensively examined in terms of its taxonomy and chemical composition However, very little information is available concerning biotechnological attempts for natural products production within that genus Garcia-Mateos et al., showed that an unexpected profile of oxygenated alkaloids was observed in undifferentiated callus of Erythrina Coralloides and Erythrina americana [12] Furthermore, San Miguel-Chavez et al., showed that jasmonic acid elicited E americana cell culture has led to reduction in alkaloid accumulation [13] Among the most common and effective elicitors used for stimulating secondary metabolites 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 as transducers of elicitor signals for the production of plant secondary metabolites Application of elicitors results in the induction of signaling compounds, including jasmonic acid and or MeJA, as well as the downstream up regulation of secondary metabolites In contrast to salicylic acid being an elicitor of limited secondary metabolite classes, jasmonates seem to be general natural products inducer via the activation and de novo biosynthesis of transcription factors that up regulate genes involved in secondary metabolites production [14] For example, jasmonates induce the accumulation of terpenoids, flavonoids, alkaloids and phenylpropanoids [15] The aim of this work was to examine MeJA elicitation effect on cell suspension culture of Erythrina lysistemon regarding the accumulation of alkaloids, flavonoids, pterocarpans and phenolic acids using an MS-based metabolomic approach Plant material Seeds of E lysistemon were collected in January 2012 from trees previously authenticated by Professor Nabil El Hadidi, College of Science, Cairo University, Egypt Voucher specimens of the flowers and seeds are deposited at Faculty of Pharmacy, Alexandria University, Egypt Callus initiation Seeds were surface sterilized in 20% sodium hypochlorite solution for 30 min, washed three times in sterile purified water and placed on agarized Murashige and Skoog (MS; Caisson, Smithfield, USA) germination medium (1962) and incubated under 12 h light period and a temperature of 23 °C ± °C in a culture room [16] Leaves were excised from 28 d-old seedlings The leaves were then scored on their abaxial sides with a sterile scalpel blade and cut into cm2 pieces Explants were cultured on 25 mL aliquots of MS supplemented with either mg LÀ1 or mg LÀ1 of each of kinetin (Kin; Acros, Geel, Belgium) and 2,4-dichlorophenoxyacetic acid (2,4D; Acros, Belgium) in addition to 30 g LÀ1 sucrose (El-Nasr, Alexandria, Egypt), and semi-solidified with 0.8% (w/v) agar (Roko, Llanera – Asturias, Spain), pH 5.6, in a cm diameter Petri dishes The explants were transferred onto fresh media, until callus was produced [16] Cell suspension culture initiation Cell suspensions were established by transferring 1–2 g fresh wt of callus and maintained in 100 mL MS liquid medium supplemented with same growth regulators, but no agar was added, pH 5.6 in 100 mL Erlenmeyer flasks Cultures were maintained on a rotary shaker at 100 rpm and incubated under a 12 h photoperiod, with day and night temperatures of 23 °C ± °C until the stock suspension was produced Elicitation Aliquots of 2.5 mL packed cell volume (PCV) with 2.5 mL spent medium of the stock suspension were transferred to six 100 mL Erlenmeyer flasks, each containing 45 mL of fresh media and maintained under same conditions for two weeks Methyl jasmonate (MeJA; Sigma Aldrich, Poole, UK) was then added into five flasks to produce a concentration Erythrina lysistemon cell culture metabolomics mM LÀ1 MeJA The dose mM LÀ1 MeJA was previously optimized to elicit secondary metabolic pathways in cell cultures [17,18] Furthermore, an increase in the concentration of MeJA resulted in retarded callus growth The remaining flask was used as control by the addition of the same volume of sterile water Cultures were kept at 23 °C ± °C, with a 12 h photoperiod and maintained on a rotary shaker at 100 rpm Cell culture samples were harvested at 0, 6, 12, 24 and 48 h post elicitation and kept at À80 °C until being analyzed Extraction and UPLC-MS analysis of cell culture extracts Metabolites extraction followed the protocol developed for similar metabolite classes [18,19] Briefly, lyophilized E lysistemon cultures (20 ± 0.06 mg) were extracted with 1.8 mL aq.80% MeOH for 10 h using an orbital shaker in the dark Extracts were centrifuged at 10,000g for 15 and 1.4 mL of the supernatant was aliquot and evaporated under nitrogen till complete dryness The dried residue was resuspended in 300 lL 45% aq MeOH For comparative analysis, the extracts were spiked with lg umbelliferone as an internal standard (IS) and quantifications were determined from peak areas normalized based on the amount of recovered IS peak The residue was re-suspended in 300 lL methanol and used for UPLC-MS analysis following the exact chromatographic conditions described by Farag et al [20] Identification and quantification of metabolites and MS data multivariate analysis File Converter tool in X-Calibur software was used to convert UPLC–MS files to NetCDF file format and then further processed by AMDIS software for background subtraction and peak deconvolution Metabolite identification was done via UV-VIS spectra (220–600 nm), retention times relative to external standards, mass spectra, and comparison to both the reference literature and phytochemical dictionary of natural product database Quantification of alkaloids was calculated from the calibration curve of erythraline, pterocarpans using medicarpin standard, and for oleanolic acid using that of oleanolic acid standard detected using MS detector Standard calibration curves were constructed for each standard using concentrations spanning from 0.1, 1, 10 and 200 lg/mL Assays were carried out in triplicate MS data processing for multivariate analysis Relative quantification and comparison of metabolites profiles after UPLC-MS were performed using XCMS data analysis software, which can be downloaded for free as an R package from the Metlin Metabolite Database (http://137.131.20 83/download/) [21] Results and discussion E lysistemon cell culture metabolite profile 683 for the elution of cinnamates, flavonoids, alkaloids and fatty acids within 13 (ca 800 s) The elution order of secondary metabolites followed a sequence of decreasing polarity, whereby cinnamates and alkaloids eluted first, followed by flavonoid glycosides, free aglycones, prenylated aglycones and finally triterpenes and fatty acids Simultaneously acquired UPLC–PDA and UPLC–MS total ion chromatograms of E lysistemon cell culture extracts in positive and negative ionization mode are presented in Fig The identities, retention times, UV and MS spectral data observed for secondary metabolites are presented in Table with a total of 53 identified metabolites It is worth noting that this is the first comprehensive metabolite profile of E lysistemon plant Identified metabolites belonged to various classes (Table 1, Suppl Fig 1) including phenolic acids (cinnamates) i.e N-caffeoyl aspartic acid (2), alkaloids i.e erysotrine (6), pterocarpans i.e isoneorautenol (30), isoflavones i.e lysisteisoflavone (44), triterpenes i.e oleanolic acid (53) and fatty acid i.e hydroxy-9,11octadecadienoic acid (45), with isoflavones and pterocarpans as the most abundant classes in cell culture extract The structures of major metabolites identified in E lysistemon and discussed throughout the manuscript are shown in Suppl Fig Flavonoids Photodiode array detection provided an overview of the main flavonoid constituents (Fig 1A) UV spectra (200–600 nm) were measured for flavonoid sub-classes including 12 isoflavones, flavones, isoflavanones and 11 pterocarpans Each sub-class exhibits a characteristic UV spectrum For example, flavones have a maximum absorbance near 265 nm with a second maximum between 320 and 340 nm (peak 9), whereas pterocarpans have k max around 280–290 nm (42) Extracts were analyzed in positive and negative ion electrospray ionization (ESI) MS modes to provide a comprehensive overview of the metabolite composition Compared to the positive-ion ESI mode (Fig 1C), negative-ion MS spectra (Fig 1B) revealed better sensitivity than in positive mode, especially in the elution range of flavonoids (200–500 s) In addition, negativeion MS spectral characteristics showed strong [M À H]À ions and lower chemical noise and consequently better sensitivity [22] The positive ion ESI mass spectra were characterized by cations corresponding to [M + H]+, [M + Na]+ and fragment ions attributed to the sequential losses of isoprenyl (69 amu), malonyl (86 amu) and hexosyl (162 amu) groups Few minor isoflavone peaks 13, 15, 18, 32 and 43 were only detected in positive ionization mode warranting the importance of acquiring data in both ionization modes Two major flavone glycosides including dihydroxyflavone hexoside (m/z 415.102, [M À H]À peak 9) and apigenin hexosylmalonate (m/z 517.1702, [M À H]À peak 11) were identified in cell culture With regard to isoflavanone subclass, vogelin A (m/z 369.0999, [M À H]À peak 25) and 5-deoxyglyasperin F/5-deoxylicoisoflavanone (m/z 337.1085, [M À H]À peak 27) exhibiting UV max around 310–320 nm typical for flavanones were measured Pterocarpans Callus was produced from cut ends of scored E lysistemon explants after weeks Chemical constituents of callus extracts were analyzed via UPLC/PDA/(À)ESI-qTOF-MS that allowed Among flavonoid subclasses, pterocarpans amounted for the major forms in cell culture (11 peaks), exhibiting k max around 684 M.A Farag et al UV response mAU 500 400 (A) 32 IS 300 200 42 100 x105 (B) IS MS response 30 33 17 23 34 42 53 27 35 31 (C) 42 33 x105 18 47 34 11 13 52 22 200 300 400 500 600 700 800 rt (sec) Fig 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 and represent UV spectra of peak (dihydroxyflavone hexoside) and peak 42 (sandwicensin, a pterocarpan), respectively The identities, rt-value, UV and MS spectra of all peaks are listed in Table IS = spiked internal standard (umbelliferone) Chromatographic conditions followed that were described in [20] 280–290 nm with isoneorautenol (m/z 321.1147, [MÀH]À peak 30) as the most abundant (Table 1) Other identified pterocarpans include erythribyssin B (m/z 283.0598, [M À H]À peak 19), eryvarin D (m/z 335.1264, [M À H]À peak 20), dihydroisoneorautenol (m/z 323.1288, [M À H]À peak 31) and sandwicensin (m/z 337.1445, [M À H]À peak 42) The predominant loss of 69 amu (–C5H9, prenyl group) in the MSn spectrum of pterocarpans is diagnostic for the presence of the isoprenyl group; a total of peaks showed this pattern For example, erystagallin B (m/z 437.1993, [M À H]À peak 38) showed mass fragments at m/z 368 and 299 indicative for isoprenyl losses (À2 Â 69 amu) The abundance of isoprenylated pterocarpans in cell culture suggests for the presence of isoprenyl transferase enzyme with higher affinity toward pterocarpans This is the first report for the accumulation of pterocarpans in E lysistemon cell culture and suggests that it could provide a resource of that flavonoid subclass Alkaloids With an increased sensitivity for detection of nitrogenous metabolites in positive mode, alkaloids could only be detected in that mode Alkaloids that are known to predominate E lysistemon plant extracts were almost absent in cell culture, except for few alkaloid peaks present at trace levels including erysotrine (m/z 314.1756 [M + H]+, peak 6), erythrartine/11methoxyerysodine (m/z 330.1696, [M + H]+, peak 10) and erysotramidine (m/z 328.1534 [M + H]+, peak 12) In contrast, DOPA methyl ether (m/z 226.1073 [M + H]+, peak 8) was present as the major nitrogenous secondary metabolite identified in culture No UV absorbance could be traced for alkaloid peaks, except for DOPA methyl ether showing distinct UV max at 270 nm In tandem MS, alkaloids showed methyl losses from methoxy group (À15 Da) Phenolic acid (cinnamates) The most abundant nitrogenous compounds detected in cell culture were amino acyl hydroxycinnamic acid conjugates A total of peaks (2–5, 16) not previously reported in E lysistemon plant tissue were identified in cell culture suggesting for an activation toward the production of acylcinnamates in cell culture The predominant fragment of cinnamic acid derivatives in the MSn spectrum and characteristic UV max values at 298 and 325 nm are diagnostic for cinnamates; a total of peaks showed similar UV (Table 1) MS/MS analysis confirmed the structure of N-p-coumaroylaspartic acid (3) m/z 278 and N-feruloylaspartic acid (5) m/z 308 from their respective product ions at m/z 163 and 193 indicative of a p-coumaroyl and feruloyl moieties, respectively, whereas N-caffeoylaspartic acid (2) gave a [M–H]À at m/z 294 with product ions m/z 132 for the aspartic acid moiety Differences in metabolites composition observed in E lysistemon callus from its native plant are likely to be the result of genetic variation and/or lack of differentiation [22,23] It is worth mentioning that there was no obvious qualitative or quantitative difference in the metabolite profile of the different treatments of the calli (1 mg lÀ1 or mg lÀ1 of Kinetin and 2,4D), results not shown PCA of E lysistemon MeJA elicited and control suspension culture observed in negative ionization mode Cell culture was further subjected to MeJA treatment to determine its impact on reprogramming of secondary metabolites as revealed via UPLC-MS analysis To assess for changes in metabolite composition in response to elicitation as monitored via UPLC-MS traces of the different callus samples harvested at 0, 12 and h post MeJA elicitation (Suppl Fig 2), principal Erythrina lysistemon cell culture metabolomics 685 Table 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 122 285 Phenolic acid 447.1139[M À H]À 161 198 209 294, 325 294, 326 287, 312 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 220 237 238 238 245 252 262 263 277 278 289 292 350 350 357 369 380 395 408 485 485 507 510 294, 282 272, 280 270, 325 nd nd 282, 280, 282, 272, 262, 282, 232, 280, 284 280 280, 280, 230, nd 270, 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 531 531 537 552 562 567 596 601 621 630 642 655 659 665 678 680 689 691 714 721 748 762 779 287, 330 287 320 287, 311 286, 323 287,322 282 287, 304 285, 340 282 281 280 283 272 286 281, 287 287,322 285, 280 280 280 280 nd 282 51 52 53 785 806 838 287,330 nd nd 325 340 332 286 308 286 319 308 286 285 335 335 310 287 307 Dihydroxybenzoic acid pentosylhexoside N-Caffeoylaspartic acid N-p-Coumaroylaspartic acid N-(Hydroxycinnamoyl) tyraminehexoside N-Feruloylaspartic acid Erysotrine Apigeninpentosyl hexoside DOPA methyl ether Dihydroxyflavone hexoside Erythrartine/11-Methoxyerysodine Apigeninhexosylmalonate Erysotramidine Demethylmedicarpin hexosylmalonate Diacetoxy benzoic acid Demethylmedicarpin hexosylmalonate N-Cinnamoyl-Aspartic acid Dihydroxyisoflavone Demethylmedicarpin Erythribyssin B Eryvarin D Unknown isoflavone Unknown isoflavone Apigenin Unknown isoflavone Vogelin A Oleanolic acid trihexoside 5-Deoxyglyasperin F/5-Deoxylicoisoflavanone Unknown isoflavanone Licoisoflavanone/Ficuisoflavone Isoneorautenol Dihydroisoneorautenol Eryvarin I/Erypoegin B Erythrabissin I 5-Deoxylicoisoflavanone Erysubin A Unknown Unknown Erystagallin B Unknown isoflavone Unknown Unknown triterpene Sandwicensin Dimethoxyisoflavone Lysisteisoflavone Hydroxy-9,11-octadecadienoic acid Unknown 40 -O-Methylalpinumisoflavone Erylysin A Unknown sterol Hydroxy-9,11-octadecadienoic acid isomer Unknown Unk307.2/878 299.1/419 527.2/922 528.8/559 508.3/769 283.3/936 281.2/877 295.2/828 295.1/542 398.2/444 398.2/937 415.2/878 447.2/683 391.2/826 391/258 449.1/223 257.2/919 277.1/309 780.5/551 670.4/919 718.4/877 351.2/444 348.2/877 349.2/877 351.1/505 353.1/438 187.1/309 209.1/309 163/259 617.3/361 634.4/571 627.4/877 651.4/877 650.4/877 583.5/877 603.4/919 635.4/856 635.4/882 863.4/524 797.5/524 880.7/919 911.5/524 925.5/565 885.7/877 993.5/527 994.5/527 928.5/527 969.5/603 973.5/510 971.5/527 990.5/509 974.5/510 945.5/509 958.5/425 957.5/509 956.5/426 947.6/919 324.2/919 535.5/919 293.2/562 471.3/826 278.2/684 369.1/381 276.2/639 271.2/919 669.4/919 737.4/920 717.4/877 211.1/877 171.1/878 235.2/638 586.5/936 602.4/919 879.7/919 889.7/936 924.5/565 957.5/426 955.5/426 337.1/441 338.1/587 315.2/704 309.2/643 281.2/936 568.4/769 397.2/444 379.2/683 431.2/684 361.2/639 633.4/571 649.4/877 347.2/877 796.5/524 923.5/565 884.7/877 927.5/527 939.5/498 989.5/509 941.5/545 323.2/919 317.2/683 297.1/555 812.7/919 883.7/877 940.5/498 327.2/476 409.2/857 275.2/639 633.4/861 795.5/524 601.4/919 614.4/683 944.5/509 330.2/444 410.2/857 254.1/351 585.5/936 285/353 364.2/684 582.5/877 811.7/919 926.5/527 988.5/509 967.5/561 324.1/555 534.5/919 256.2/919 280.2/877 940.5/545 295.2/782 277.2/684 613.4/683 987.5/509 925.5/527 329.2/444329.2/478 581.5/877 283/447 939.5/545 943.5/509 295.2/695 533.5/919 363.2/684 293.2/713 296.2/683 279.2/877 253/351 255.2/919 942.5/509 0.0 -0.4 (B) 337.1/684 161/259 -0.2 PC2 0.2 0.4 PC1 (92%) 322.1/542 323.1/555 353.1/534 337.1/587 Erythrabissin I 321.1/542 Isoneorautenol Hydroxy-octadecadienoic acid 295.2/683 271.2/816 -0.2 0.0 -0.2 -0.4 -0.6 PC1 Fig 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 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 (PC1 = 92%) and principal component (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 12 h PC2 (6%) 2000 (A) 0h -2000 24 h -50000 5000 0.0 -0.2 -0.1 PC2 0.1 0.2 PC1 (93%) 439.4/841 425.3/891 713.5/919 283.1/683 Dimethoxy isoflavone Unknown sterol 425.4/859 379.3/886 274.3/479 403.2/758 439.4/853 337.1/510 476.2/211 361.1/683 562.2/238 393.1/398 377.3/825 565.2/244 440.4/841 503.1/290 426.3/891 426.4/859 163/398 407.4/859 671.4/741 377.1/510 671.4/781 495.3/747 393.4/841 321.1/337 445.2/683 479.4/852 631.4/741 459.3/894 335.1/555 380.3/886 284.3/938 519.1/263 522.4/705 393.3/638 284.1/683 358.2/292 177.1/221 191.1/398 440.4/853 339.3/637 302.3/548 299.1/230 404.2/758 205.2/859 322.1/337 593.3/919 271.1/263 437.3/671 411.4/936 299.2/509 437.3/742 226.1/240353.3/865 340.2/302 563.2/238 935.7/841 246.2/401 275.3/479 672.4/741 479.4/840 339.3/886 217.2/859 885.8/859 631.4/781 457.2/799 179/683 437.3/781 393.4/884 455.1/204 504.1/290 313.3/852 282.1/241 547.1/244 494.2/212 566.2/244 338.2/510 621.3/810 290.3/487 177.1/178 378.3/825 519.1/325 477.2/211 335.2/877 587.1/244 265.3/886 672.4/781 659.3/904 377.3/807 495.1/568 353.1/398 886.8/859 460.3/894 411.4/883 408.4/859 632.4/741 373.7/742 541.2/337 351.2/826 394.1/398 191.2/860 203.1/510 936.7/841 322.1/306 429.2/826 419.3/895 362.1/683 495.4/671 577.3/261 895.7/841 913.7/841 580.2/238 461.2/683 226.1/292 314.2/264 943.5/350 342.3/669 480.3/698 335.1/683 218.2/305 457.4/841 441.4/841 302.1/683 179.2/841 379.3/937 378.1/510 480.4/852 496.4/747 332.1/221 339.2/683 295.7/841 391.2/592 581.3/810 259.2/841 595.2/435 637.3/904 443.4/859 281.1/316 281.1/287 368.4/686 366.3/645 278.2/842 541.3/761 375.1/554 477.2/193 304.2/877 337.1/434 629.4/730 296.3/479 405.2/683 203.1/670 896.7/841 907.8/859 446.2/683 359.1/670 223.1/398 594.3/919 425.4/909 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507.7/426 229.2/828 498.2/545 426.4/547 408.2/281 419.2/509 227.2/936 701.5/936 424.4/508 734.2/333 276.1/292 600.4/508 606.5/508 751.4/868 244.3/503 237.2/919 239.2/764 427.4/527 415.2/865 503.2/575 505.3/537 422.4/841 411.2/223 227.1/302 229.2/883 261.2/515 722.2/281 721.2/281 273.2/828 600.4/877 601.4/547 609.3/898 751.2/261 237.1/882 237.1/858 242.2/308 508.1/445 428.3/419 231.1/925 507.1/566 507.3/821 500.2/387 406.8/682 421.3/884 422.9/919 427.1/344 227.1/308 229.1/221 224.1/485 225.1/545 408.4/574 417.1/432 425.1/510 701.5/919 705.5/877 597.2/628 276.2/826 601.2/420 602.2/626 610.2/440 747.2/359 750.5/922 602.5/877 243.2/826 429.1/522 430.1/566 234.1/803 407.2/385 229.1/316 499.2/334 699.2/472 701.5/877 600.2/597 601.3/734 607.2/430 431.1/587 504.3/758 407.2/605 261.2/826 502.2/338 243.1/356 410.1/366 730.3/566 594.2/509 595.2/546 749.3/566 509.2/409 223/258 225/258 409.1/566 274/258 732.3/566 274/350 598.3/363 748.3/566 223/605 501.2/338 593.2/509 731.3/566 408.2/683 229.1/683 593.2/571 224.1/534 641.4/760 641.4/728 634.4/544 647.5/919 641.4/921 652.4/838 654.4/733 643.3/779 647.2/466 645.2/419 647.3/683 646.3/683 645.3/644 645.3/683 657.2/683 440.3/877 442.4/508 440.1/566 441.1/587 291.2/502 289.3/501 287.2/874 283.3/575 283.3/553 291.1/619 288.1/292 291.2/667 292.1/733 287.1/248 289.2/658 289.2/826 285.1/522 291.1/598 285.1/294 291/463 283.3/866 315.2/841 315.2/919 313.1/683 313.1/587 314.3/675 314.1/238 301.2/765 300.1/534 299.2/707 301.1/484 299.1/566 302.2/803 299.1/631 962.5/508 963.5/508 947.5/508 955.5/508 959.5/425 968.5/508 957.5/426 965.5/544 957.5/508 964/508 269.1/176 269.2/680 266.3/590 267.2/293 269.1/215 267.1/339 267.1/263 271.2/482 269.1/510 269.1/684 267.1/461 267.7/681 267/258 279.5/258 280.2/919 694.5/918 677.5/936 686.5/937 685.5/937 185.5/245 178/566 180/541 327.2/935 331.2/460 521.4/919 327.1/627 326.1/616 523.2/338 521.1/607 519.3/681 353.3/877 356.1/484 356.1/566 356.3/920 362.3/585 363.1/415 367.1/520 369.1/764 362.1/554 371.1/380 373.1/522 436.3/674 282/463 381.3/571 387.2/443 543.8/490 387.2/477 176.1/477 342.2/683 543.1/526 544.3/490 991.5/560 530.1/463 551.1/292 345.2/797 550.5/918 546.4/763 341.2/877 531.2/938 992.5/560 175.1/838 540.4/920 342.3/792 543.5/877 531.1/489 537.4/844 379.1/362 544.2/218 173.1/788 539.1/462 377.1/484 661.2/582 537.1/484 343.1/535 346.3/765 347.1/541 375.1/605 165/350 344.1/588 341.1/483 341.1/587 551.2/566 171/541 669.3/750 340.1/510 377.1/525 484.3/508 492.3/508 798.5/523 819.5/523 492.8/508 309.2/365 484.8/508 513.3/537 514.2/602 324.3/936 571.2/547 473.8/544 517.3/581 335.2/515 132.1/292 148.5/258 309.2/919 761.5/896 335.3/836 511.2/571 784.6/919 787.5/835 819.2/358 577.4/841 471.4/920 339.1/669 513.8/537 495.4/834 305.2/840 562.5/877 124.5/292 809.7/919 809.4/902 133.5/292 479.3/515 467.4/904 161.1/878 515.2/777 123.1/919 307.1/802 337.1/748 485.2/427 575.2/628 163.5/554 311.1/566 337.1/317 307.1/361 573.2/419 338.1/573 134/265 577.2/655 110/258 132/258 156/292 478.3/681 144.1/183 335.1/535 336.1/566 339.2/566 123/554 149/734 631.5/936 351.2/543 192.5/258 194.1/703 627.4/877 199.2/935 195.1/444 293.2/782 295.2/703 399.4/934 192/258 351.2/877 351.3/852 189.2/877 613.4/855 350.3/790 395.1/547 395.1/261 401.3/764 629.4/877 188.2/789 610.3/882 298.2/791 631.4/808 618.4/683 625.3/868 395.1/411 613.4/880 297.1/580 617.4/653 352.1/541 615.2/571 197/587 297.1/541 393.1/554 391.1/566 353.1/487 616.4/653 925.5/508 943.5/544 213.1/443 580.4/938 584.4/840 583.1/560 213.2/841 205.1/734 580.4/910 589.4/860 579.2/626 592.2/584 211.6/541 207.1/587 591.2/744 208.6/541 219/289 205/566 319.2/743 318.2/816 319.2/877 318.2/638 323.1/684 317.2/686 321.1/554 322.1/535 321.1/416 318/200 322.1/588 461.2/614 257.1/354 251.2/749 255.1/435 258.1/449 256.1/244 257.1/825 258/292 359.1/659 359.1/551 425.4/526 500.2/544 508.8/425 499.7/508 509.3/547 421.3/772 423.4/581 733.2/333 599.4/508 502.7/508 277.2/887 275.2/668 275.2/619 241.2/919 423.4/775 601.5/877 605.5/935 749.2/344 425.3/445 427.1/280 704.5/877 608.3/868 609.2/440 509.1/561 409.1/468 425.1/566 225.1/587 226.1/764 605.4/919 412.1/344 424.1/566 607.2/651 509.1/522 234/219 703.5/877 720.3/557 608.2/599 699.2/586 276.7/680 408.3/931 719.3/557 409.3/565 423.1/566 498.4/681 409.1/366 607.2/599 647.2/304 658.5/752 645.5/877 632.4/808 633.4/826 646.3/717 645.3/717 440.1/244 440.8/877 441.4/877 284.3/935 288.3/501 293.2/476 285.3/590 288.2/842 288.6/554 290.7/768 293/350 316.3/570 316.2/264 316.2/789 317.1/616 317.1/607 313.3/675 299.2/645 301.3/553 303.2/919 299.1/379 299.1/547 963/508 944.5/526 264.6/258 269.2/789 264.1/237 269.1/442 187.2/919 281.1/362 281.1/200 185.1/477 680.5/920 689.5/919 679.3/558 331.2/502 327.2/877 522.4/905 325.3/895 330.1/243 369.2/444 361.2/803 365.1/560 369.2/477 371.2/877 363.1/554 437.2/921 433.1/273 282.2/250 348.3/827 343.2/829 533.2/563 527.2/559 539.4/920 165.1/877 547.1/289 557.2/768 175.1/877 659.4/921 553.4/912 379.1/437 547.4/767 483.8/508 573.4/762 482.3/544 332.2/763 473.3/545 490.2/545 339.2/703 482.8/545 565.1/292 782.5/508 787.4/903 514.9/841 337.2/936 339.3/531 463.3/860 479.4/884 333.2/541 333.2/649 161.1/707 309.2/731 575.2/481 487.4/826 119/258 107/258 339.1/387 126/292 307.1/422 156/200 337.1/659 295.1/291 618.4/508 351.2/703 352.2/476 298.1/263 393.3/703 293.2/649 617.5/936 612.4/860 200.1/554 393.3/691 617.4/683 199.1/541 351.1/566 193.1/566 395.2/442 209.5/258 588.2/440 583.3/863 579.4/911 206.1/566 581.1/200 321.3/551 318.3/531 323.2/773 324.1/389 321.1/588 459.1/212 255.1/290 259.3/919 257.2/837 361.1/541 361.1/422 272.5/258 597.4/762 277.2/443 499.2/508 425.4/547 427.2/523 507.2/426 504.3/560 261.2/683 245.1/308 603.5/936 235.1/877 410.4/860 409.3/826 605.3/928 241.2/772 426.2/803 221.2/877 597.3/355 594.2/419 239.1/686 425.1/541 406.2/385 409.1/554 409.1/415 599.2/597 409.1/409 411.1/566 409.3/553 729.3/566 728.8/566 747.3/566 728.3/566 637.4/683 648.3/750 441.4/508 291.2/877 291.1/788 284.1/587 285.1/361 315.2/766 315.2/619 317.1/733 299.3/530 303.1/200 299.1/534 967.5/508 964.5/544 266.1/264 179.1/703 679.5/919 689.5/935 181/554 329.1/308 327.1/566 353.2/489 355.1/318 362.1/587 278.2/712 282.3/574 283.1/566 534.7/527 986.5/537 377.1/555 539.2/768 539.2/788 173.1/765 172.5/554 174/350 376.3/877 341.1/468 531.2/557 377.1/534 343.1/588 375.1/683 491.2/508 797.5/523 781.5/508 798.5/508 337.2/877 484.4/903 491.2/559 480.4/536 336.2/607 510.1/489 565.4/843 465.3/920 339.1/441 465.3/731 154/554 463.3/683 336/200 296.2/816 351.1/560 201.2/841 353.1/391 400.3/812 297.1/554 351.1/541 393.1/469 209.2/683 580.4/919 319.2/762 319.2/782 321.2/683 321.1/535 454.3/675 246.2/877 257.1/877 359.1/573 499.2/497 421.3/765 499.7/497 423.4/508 427.3/419 605.4/508 508.1/463 599.4/877 242.3/581 277.2/855 509.1/412 715.4/919 608.3/888 241.1/566 409.4/860 273.2/279 601.2/626 245.1/514 503.7/541 633.4/544 636.4/508 632.4/683 442.3/755 442.3/778 443.4/874 286.1/292 283.2/249 293.2/919 283.1/485 315.2/250 316.1/554 963.5/544 946.5/508 271.1/317 264.2/877 271.1/554 268.1/541 280.2/683 281.1/382 688.5/920 187/566 326.2/877 326.2/845 354.2/443 355.1/566 356.1/534 278.1/350 279.1/484 543.3/490 529.1/463 345.2/804 347.1/788 350.3/877 535.4/937 376.3/772 483.3/508 308.2/365 485.4/765 565.3/762 324.3/590 766.4/903 561.5/877 464.4/566 467.3/802 333.2/566 164/554 148/200 628.3/394 191.1/554 455.3/675 250.2/484 501.3/508 508.2/425 277.2/646 277.2/638 221.2/919 425.1/423 607.3/868 507.2/389 411.1/374 595.2/569 411.1/344 597.1/200 443.4/832 302.2/683 326.2/554 354.2/477 356.3/811 282.3/553 281.3/877 340.3/553 553.4/863 544.2/271 529.2/240 341.3/792 348.1/554 546.4/768 342.1/541 333.2/803 137/258 758.6/919 576.5/919 485.2/389 337.1/356 325.1/541 617.4/508 403.1/424 192.6/789 611.4/860 297.2/791 395.2/450 942.5/545 207.1/468 216.1/587 324.1/419 237.1/733 288.1/554 271.2/765 183.1/232 353.2/506 355.1/484 431.2/626 283.1/587 985.5/537 347.3/918 171.1/192 797.5/508 465.3/874 469.4/885 510.4/925 467.3/789 577.2/597 518.3/681 351.2/476 587.2/440 319.3/553 455.4/896 257.1/449 256.1/350 509.3/765 507.1/463 275.3/919 609.3/882 291.1/928 293.2/868 312.3/811 525.4/767 433.2/856 553.4/764 534.2/527 529.4/764 535.5/919 668.2/541 512.8/537 196/566 209/258 579.4/919 579.4/937 317.2/638 361.1/554 499.7/545 225.1/788 596.3/355 291.1/733 302.2/919 265/258 325.2/802 439.1/244 557.5/683 379.1/383 378.1/566 765.4/903 337.2/789 191.1/686 353.1/605 208.1/541 227.2/816 593.2/419 640.4/761 293.2/888 293.2/720 300.3/553 966.5/508 279.2/919 280.1/200 185/258 281.3/551 348.3/842 572.2/419 337.1/683 615.5/936 941.5/544 214.3/507 319.2/751 245.2/877 635.4/508 443.3/811 303.2/877 302.1/733 269.1/275 269.1/381 535.3/527 552.4/863 335.2/595 478.3/515 163/554 318.2/712 320.3/550 323.1/389 607.3/888 599.2/616 315.2/789 302.3/590 355.3/827 347.3/853 512.3/537 483.4/903 335.2/607 574.5/921 338.2/566 191.2/852 631.4/683 596.4/762 509.1/489 647.3/750 441.3/754 285/446 349.2/877 497.3/682 339.1/510 318.2/703 319.2/695 639.4/761 441.3/764 Oleanolic acid 341.2/683 346.1/541 325.2/845 575.5/919 499.2/544 425.2/802 284.3/590 965.5/508 339.1/422 255.1/244 265/262 187.2/789 331.2/763 331.2/829 317.2/646 361.1/587 163/258 282.1/566 340.1/587 317.2/816 311.3/827 263.2/877 460.3/531 277.2/712 667.2/541 298.3/530 500.8/508 509.4/925 325.2/825 277.1/350 338.3/531 469.4/866 945.5/508 240.2/919 552.4/763 346.2/869 534.5/919 131.1/292 399.3/812 317.3/531 441.3/778 301.2/683 524.4/767 353.2/443 528.2/240 225.1/764 315.2/669 323.1/419 267.1/541 172/554 500.3/508 302.1/200 347.1/554 295.2/816 258.3/919 355.3/797 463.3/777 320.2/683 464.3/765 325.1/554 353.2/477 420.3/765 311.3/811 270.1/554 508.3/765 279.2/683 280.3/550 571.2/419 317.2/722 301.1/733 355.1/534 377.1/566 147/200 355.3/811 325.2/877 496.3/681 318.3/553 trihexoside 595.4/762 376.3/763 324.1/541 255.1/350 301.2/919 944.5/508 347.3/842 337.1/566 345.1/541 317.2/712 317.2/703 339.1/587 551.4/764375.3/772 345.2/869 316.3/531 239.2/919 507.3/765 943.5/508 419.3/765 319.2/683 463.3/765 257.3/919 Unknown pterocrapan 281.1/566 Dihydroisoneorautenol 269.1/554 -0.2 0.0 (B) 323.1/541 -0.2 -0.4 PC1 Fig 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 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 (PC1 = 93%) and principal component (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 Callus iniƟaƟon R H3C H3C O MeJA H 3C O CH3 H3C O CH3 O Isoneorautenol CH3 Erythrabissin N O CH H3 CH3 H3C HO O H3C OH O OH R OH CH3 CH3 O HO Pterocarpans H 3C CH H3 Oleanolic acid O R=CH2, R1=OCH3 Erythristemine Erysotramidine R=CO, R1=H Triterpenes Alkaloids Fig 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 Table Quantification of metabolites identified in E lysistemon cell suspension at 0, 12 and 24 h post 1.0 mM MeJA elicitation using MS detection in positivea and negativeb ionization modes Values are expressed in lg gÀ1 as average for biological replicates Metabolites (lg gÀ1) a Erysotrine Erysotramidinea ErylysinAb Sandwicensinb Oleanolic acidb ErythrabissinIb Isoneorautenolb Christoph Boăttcher, Leibniz Institute of Plant Biochemistry, Germany, for assistance with the UPLC-MS We are grateful to Dr Tilo Luăbcken, University of Dresden, Germany, for providing R scripts for UPLC-MS data analysis Appendix A Supplementary material E lysistemon cell suspension 0h 12 h 24 h 2.8 ± 0.9 10.8 ± 3.8 36.0 ± 8.2 10.7 ± 2.9 406 ± 32.1 1268 ± 85 2217 ± 89 3.4 ± 2.1 8.9 ± 2.5 22.2 ± 5.4 3.4 ± 0.5 4907 ± 133 268 ± 59 473 ± 16 0.6 ± 0.5 6.0 ± 1.4 12.7 ± 2.6 2.2 ± 0.5 4838 ± 237 243 ± 18 564 ± 47 elicitation will help affirm induction hypothesis derived via metabolite profiling It should be noted that oleanolic acid tri-hexoside conjugate was not detected by visual examination of unelicited cell culture chromatograms, suggesting that coupling of metabolomics for analysis of elicited samples presents a powerful methodology for identification of novel metabolites Quantification of the major differential metabolites in elicited cell culture is presented in Table Conclusions This study provides the first report on E lysistemon cell suspension culture metabolite fingerprint via UPLC-MS A metabolomic approach was used to investigate secondary metabolites viz alkaloids, flavonoids and triterpenes and their reprogramming in response to MeJA elicitation The results confirm MeJA elicitation effect on terpenoid accumulation and extend our knowledge base concerning secondary metabolism in other legume species [27] Comparative metabolic profiling of E lysistemon cell suspension culture and in response to elicitation using MeJA, revealed an activation in sterol/triterpenes formation, see model depicted in Fig The effect of other elicitors on secondary metabolites accumulation in Erythrina cell culture could also provide more holistic insight into elicitation effect within that genus and how it can reprogram its different secondary metabolite pathways Conflict of Interest The authors declare that they have no conflict of interest Compliance with Ethics Requirements This article does not contain any studies with human or animal subjects Acknowledgments Dr Mohamed A Farag acknowledges the funding received by Science and Technology Development Fund STDF, Egypt (grant number 12594), and the support of the Alexander von Humboldt Foundation, Germany We also thank Dr Supplementary data associated with this article can 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Cell culture Erythrina lysistemon Metabolomics Methyl jasmonate Oleanolic acid Ultra-performance liquid chromatography M.A Farag et al detected at trace levels in suspension cultures Methyl jasmonate. .. tissue were identified in cell culture suggesting for an activation toward the production of acylcinnamates in cell culture The predominant fragment of cinnamic acid derivatives in the MSn spectrum... hydroxy-octadecadienoic acid were elicited in response to methyl jasmonate, whereas pterocarpans i.e., isoneorautenol showed a decline in response to elicitation suggesting for the induction of terpenoid biosynthetic