Analytical and Bioanalytical Chemistry

, Volume 407, Issue 21, pp 6357–6368 | Cite as

Metabolomic analysis of wild and transgenic Nicotiana langsdorffii plants exposed to abiotic stresses: unraveling metabolic responses

  • Elisa ScalabrinEmail author
  • Marta Radaelli
  • Giovanni Rizzato
  • Patrizia Bogani
  • Marcello Buiatti
  • Andrea Gambaro
  • Gabriele Capodaglio
Research Paper
Part of the following topical collections:
  1. High-Resolution Mass Spectrometry in Food and Environmental Analysis


Nicotiana langsdorffii plants, wild and transgenic for the Agrobacterium rhizogenes rol C gene and the rat glucocorticoid receptor (GR) gene, were exposed to different abiotic stresses (high temperature, water deficit, and high chromium concentrations). An untargeted metabolomic analysis was carried out in order to investigate the metabolic effects of the inserted genes in response to the applied stresses and to obtain a comprehensive profiling of metabolites induced during abiotic stresses. High-performance liquid chromatography separation (HPLC) coupled to high-resolution mass spectrometry (HRMS) enabled the identification of more than 200 metabolites, and statistical analysis highlighted the most relevant compounds for each plant treatment. The plants exposed to heat stress showed a unique set of induced secondary metabolites, some of which were known while others were not previously reported for this kind of stress; significant changes were observed especially in lipid composition. The role of trichome, as a protection against heat stress, is here suggested by the induction of both acylsugars and glykoalkaloids. Water deficit and Cr(VI) stresses resulted mainly in enhanced antioxidant (HCAs, polyamine) levels and in the damage of lipids, probably as a consequence of reactive oxygen species (ROS) production. Moreover, the ability of rol C expression to prevent oxidative burst was confirmed. The results highlighted a clear influence of GR modification on plant stress response, especially to water deficiency—a phenomenon whose applications should be further investigated. This study provides new insights into the field of system biology and demonstrates the importance of metabolomics in the study of plant functioning.

Graphical Abstract

Untargeted metabolomic analysis was applied to wild type, GR and RolC modified Nicotiana Langsdorffii plants exposed to heat, water and Cr(VI) stresses. The key metabolites, highly affected by stress application, were identified, allowing to outline the main metabolic responses to stress in each plant genotype.


Metabolomic HPLC-HRMS Nicotiana langsdorffii Environmental stress GR rol C 



This work was supported by the PRIN grant number 20098TN4CY from the Italian Ministry of Education, University and Research (MIUR). The authors thank Dr. Daniela Almansi for improving English language.

Supplementary material

216_2015_8770_MOESM1_ESM.pdf (676 kb)
ESM 1 (PDF 676 kb)


  1. 1.
    Bartwal A, Mall R, Lohani P, Guru SK, Arora S (2013) Role of secondary metabolites and brassinosteroids in plant defense against environmental stresses. J Plant Growth Regul 32:216–232. doi: 10.1007/s00344-012-9272-x CrossRefGoogle Scholar
  2. 2.
    Larkindale J, Huang B (2004) Changes of lipid composition and saturation level in leaves and roots for heat-stressed and heat-acclimated creeping bentgrass (Agrostis stolonifera). Environ Exp Bot 51:57–67. doi: 10.1016/S0098-8472(03)00060-1 CrossRefGoogle Scholar
  3. 3.
    Yordanov I, Velikova V, Tsonev T (2000) Plant responses to drought, acclimation and stress tolerance. Photosynthetica 38:171–186. doi: 10.1023/A:1007201411474 CrossRefGoogle Scholar
  4. 4.
    Obata T, Fernie AR (2012) The use of metabolomics to dissect plant responses to abiotic stresses. Cell Mol Life Sci 69:3225–3243. doi: 10.1007/s00018-012-1091-5 CrossRefGoogle Scholar
  5. 5.
    Ganesh KS, Baskaran L, Rajasekaran S, Sumathi K, Chidambaram AL, Sundaramoorthy P (2008) Chromium stress induced alterations in biochemical and enzyme metabolism in aquatic and terrestrial plants. Colloids Surf B Biointerfaces 63:159–163. doi: 10.1016/j.colsurfb.2007.11.016 CrossRefGoogle Scholar
  6. 6.
    Lipiec J, Doussan C, Nosalewicz A, Kondracka K (2013) Effect of drought and heat stresses on plant growth and yield: a review. Int Agrophysics 27:463–477. doi: 10.2478/intag-2013-0017 CrossRefGoogle Scholar
  7. 7.
    Giannarelli S, Muscatello B, Bogani P, Spiriti MM, Buiatti M, Fuoco R (2010) Comparative determination of some phytohormones in wild-type and genetically modified plants by gas chromatography–mass spectrometry and high-performance liquid chromatography-tandem mass spectrometry. Anal Biochem 398:60–68. doi: 10.1016/j.ab.2009.10.038 CrossRefGoogle Scholar
  8. 8.
    Irdani T, Caroppo S, Ambrogioni L (2003) Response of Nicotiana tabacum plants overexpressing a glucocorticoid receptor to Meloidogyne incognita (Nematoda tylenchida) infestation. Redia 86:35–38Google Scholar
  9. 9.
    Bettini P, Michelotti S, Bindi D, Giannini R, Capuana M, Buiatti M (2003) Pleiotropic effect of the insertion of the Agrobacterium rhizogenes rolD gene in tomato (Lycopersicon esculentum Mill.). Theor Appl Genet 107:831–836. doi: 10.1007/s00122-003-1322-0 CrossRefGoogle Scholar
  10. 10.
    Palazón J, Cusidó RM, Roig C, Piñol MT (1998) Expression of the rol C gene and nicotine production in transgenic roots and their regenerated plants. Plant Cell Rep 17:384–390. doi: 10.1007/s002990050411 CrossRefGoogle Scholar
  11. 11.
    Del Bubba M, Ancillotti C, Checchini L, Ciofi L, Fibbi D, Gonnelli C, Mosti S (2013) Chromium accumulation and changes in plant growth, selected phenolics and sugars of wild type and genetically modified Nicotiana langsdorffii. J Hazard Mater 262:394–403. doi: 10.1016/j.jhazmat.2013.08.073 CrossRefGoogle Scholar
  12. 12.
    Intrieri MC, Buiatti M (2001) The horizontal transfer of Agrobacterium rhizogenes genes and the evolution of the genus Nicotiana. Mol Phylogenet Evol 20:100–110. doi: 10.1006/mpev.2001.0927 CrossRefGoogle Scholar
  13. 13.
    Fuoco R, Bogani P, Capodaglio G, Del Bubba M, Abollino O, Giannarelli S, Spiriti MM, Muscatello B, Doumett S, Turetta C, Zangrando R, Zelano V, Buiatti M (2013) Response to metal stress of Nicotiana langsdorffii plants wild-type and transgenic for the rat glucocorticoid receptor gene. J Plant Physiol 170:668–675. doi: 10.1016/j.jplph.2012.12.009 CrossRefGoogle Scholar
  14. 14.
    Dunn WB, Erban A, Weber RJM, Creek DJ, Brown M, Breitling R, Hankemeier T, Goodacre R, Neumann S, Kopka J, Viant MR (2013) Mass appeal: metabolite identification in mass spectrometry-focused untargeted metabolomics. Metabolomics 9:44–66. doi: 10.1007/s11306-012-0434-4 CrossRefGoogle Scholar
  15. 15.
    De Vos RCH, Moco S, Lommen A, Keurentjes JJB, Bino RJ, Hall RD (2007) Untargeted large-scale plant metabolomics using liquid chromatography coupled to mass spectrometry. Nat Protoc 2:778–791. doi: 10.1038/nprot.2007.95 CrossRefGoogle Scholar
  16. 16.
    Marti G, Erb M, Boccard J, Glauser G, Doyen GR, Villard N, Christelle RAM, Turlings TCJ, Rudaz S, Wolfender J-L (2013) Metabolomics reveals herbivore-induced metabolites of resistance and susceptibility in maize leaves and roots. Plant Cell Environ 36:621–639. doi: 10.1111/pce.12002 CrossRefGoogle Scholar
  17. 17.
    Lommen A, Kools HJ (2012) MetAlign 3.0: performance enhancement by efficient use of advances in computer hardware. Metabolomics 8:719–726. doi: 10.1007/s11306-011-0369-1 CrossRefGoogle Scholar
  18. 18.
    Lommen A (2009) MetAlign: interface-driven, versatile metabolomics tool for hyphenated full-scan mass spectrometry data preprocessing. Anal Chem 81:3079–3086. doi: 10.1021/ac900036d CrossRefGoogle Scholar
  19. 19.
    Tikunov YM, Laptenok S, Hall RD, Bovy A, De Vos RCH (2012) MSClust: a tool for unsupervised mass spectra extraction of chromatography-mass spectrometry ion-wise aligned data. Metabolomics 8:714–718. doi: 10.1007/s11306-011-0368-2 CrossRefGoogle Scholar
  20. 20.
    Sumner LW, Amberg A, Barrett D, Beale MH, Beger R, Daykin CA, Fan TW-M, Fiehn O, Goodacre R, Griffin JL, Hankemeier T, Hardy N, Harnly J, Higashi R, Kopka J, Lane AN, Lindon JC, Marriott P, Nicholls AW, Reily MD, Thaden JJ, Viant MR (2007) Proposed minimum reporting standards for chemical analysis. Metabolomics 3:211–221. doi: 10.1007/s11306-007-0082-2 CrossRefGoogle Scholar
  21. 21.
    Sato N, Aoki M, Maru Y, Sonoike K, Minoda A, Tsuzuki M (2003) Involvement of sulfoquinovosyl diacylglycerol in the structural integrity and heat-tolerance of photosystem II. Planta 217:245–251. doi: 10.1007/s00425-003-0992-9 Google Scholar
  22. 22.
    Chen J, Burke JJ, Xin Z, Xu C, Velten J (2006) Characterization of the Arabidopsis thermosensitive mutant atts02 reveals an important role for galactolipids in thermotolerance. Plant Cell Environ 29:1437–1448. doi: 10.1111/j.1365-3040.2006.01527.x CrossRefGoogle Scholar
  23. 23.
    Burgos A, Szymanski J, Seiwert B, Degenkolbe T, Hannah MA, Giavalisco P, Willmitzer L (2011) Analysis of short-term changes in the Arabidopsis thaliana glycerolipidome in response to temperature and light. Plant J 66:656–668. doi: 10.1111/j.1365-313X.2011.04531.x CrossRefGoogle Scholar
  24. 24.
    Otsuru M, Yu Y, Mizoi J, Kawamoto-Fujioka M, Wang J, Fujiki Y, Nishida I (2013) Mitochondrial phosphatidylethanolamine level modulates Cyt c oxidase activity to maintain respiration capacity in Arabidopsis thaliana rosette leaves. Plant Cell Physiol 54:1612–1619. doi: 10.1093/pcp/pct104 CrossRefGoogle Scholar
  25. 25.
    Kim J, Kang K, Gonzales-Vigil E, Shi F, Jones AD, Barry CS, Last RL (2012) Striking natural diversity in glandular trichome acylsugar composition is shaped by variation at the acyltransferase2 Locus in the wild tomato Solanum habrochaites. Plant Physiol 160:1854–1870. doi: 10.1104/pp. 112.204735 CrossRefGoogle Scholar
  26. 26.
    Ghosh B, Westbrook TC, Jones AD (2013) Comparative structural profiling of trichome specialized metabolites in tomato (Solanum lycopersicum) and S. habrochaites: acylsugar profiles revealed by UHPLC / MS and NMR. Metabolomics 10:496–507. doi: 10.1007/s11306-013-0585-y CrossRefGoogle Scholar
  27. 27.
    Schilmiller A, Shi F, Kim J, Charbonneau AL, Holmes D, Jones AD, Last RL (2010) Mass spectrometry screening reveals widespread diversity in trichome specialized metabolites of tomato chromosomal substitution lines. Plant J 62:391–403. doi: 10.1111/j.1365-313X.2010.04154.x CrossRefGoogle Scholar
  28. 28.
    Pérez-estrada LB, Cano-santana Z, Oyama K (2000) Variation in leaf trichomes of Wigandia urens: environmental factors and physiological consequences. Tree Physiol 20:629–632. doi: 10.1093/treephys/20.9.629 CrossRefGoogle Scholar
  29. 29.
    Kuc J (1984) Steroid glykoalkaloids and related compounds as potato quality factors. Am Potato J 61:123–139. doi: 10.1007/BF02854034 CrossRefGoogle Scholar
  30. 30.
    Udalova ZV, Zinov SV, Vasil IS, Paseshnichenko VA (2004) Correlation between the structure of plant steroids and their effects on phytoparasitic nematodes. Appl Biochem Microbiol 40:109–113. doi: 10.1023/B:ABIM.0000010362.79928.77 CrossRefGoogle Scholar
  31. 31.
    Coria NA, Sarquı JI, Penalosa I, Urzua M (1998) Heat-induced damage in potato (Solanum tuberosum) tubers: membrane stability, tissue viability, and accumulation of glycoalkaloids. J Agric Food Chem 46:4524–4528. doi: 10.1021/jf980151+ CrossRefGoogle Scholar
  32. 32.
    Dimenstein L, Lisker N, Kedar N, Levy D (1997) Changes in the content of steroidal glycoalkaloids in potato tubers grown in the field and in the greenhouse under different conditions of light, temperature and daylength. Physiol Mol Plant Pathol 50:391–402. doi: 10.1006/pmpp.1997.0098 CrossRefGoogle Scholar
  33. 33.
    Nitithamyong A, Vonelbe JH, Wheeler RM, Tibbitts TW (1999) Glycoalkaloids in potato tubers grown under controlled environments. Am J Potato Res 76:337–343. doi: 10.1007/BF02910006 CrossRefGoogle Scholar
  34. 34.
    Torras-Claveria L, Jáuregui O, Codina C, Tiburcio AF, Bastida J, Viladomat F (2012) Analysis of phenolic compounds by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry in senescent and water-stressed tobacco. Plant Sci 182:71–78. doi: 10.1016/j.plantsci.2011.02.009 CrossRefGoogle Scholar
  35. 35.
    Van de Mortel J, Schat H, Moerland PD, Van Themaat VEL, Van der Ent S, Blankestijn H, Ghandilyan A, Tsiatsiani S, Aarts MGM (2008) Expression differences for genes involved in lignin, glutathione and sulphate metabolism in response to cadmium in Arabidopsis thaliana and the related Zn / Cd-hyperaccumulator Thlaspi caerulescens. Plant Cell Environ 31:301–324. doi: 10.1111/j.1365-3040.2007.01764.x CrossRefGoogle Scholar
  36. 36.
    Mithöfer A, Schulze B, Boland W (2004) Biotic and heavy metal stress response in plants: evidence for common signals. FEBS Lett 566:1–5. doi: 10.1016/j.febslet.2004.04.011 CrossRefGoogle Scholar
  37. 37.
    Ryu SB, Wang X (1998) Increase in free linolenic and linoleic acids associated with phospholipase D-mediated hydrolysis of phospholipids in wounded castor bean leaves. Biochim Biophys Acta Lipids Lipid Metab 1393:193–202. doi: 10.1016/S0005-2760(98)00048-4 CrossRefGoogle Scholar
  38. 38.
    Bulgakov VP, Aminin DL, Shkryl YN, Gorpenchenko TY, Veremeichik GN, Dmitrenok PS, Zhuravlev YN (2008) Suppression of reactive oxygen species and enhanced stress tolerance in Rubia cordifolia cells expressing the rolC oncogene. Mol Plant Microbe Interact 21:1561–1570. doi: 10.1094/MPMI-21-12-1561 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Elisa Scalabrin
    • 1
    Email author
  • Marta Radaelli
    • 2
  • Giovanni Rizzato
    • 1
  • Patrizia Bogani
    • 3
  • Marcello Buiatti
    • 3
  • Andrea Gambaro
    • 1
    • 2
  • Gabriele Capodaglio
    • 1
    • 2
  1. 1.Department of Environmental Sciences, Informatics and StatisticsUniversity of VeniceVeniceItaly
  2. 2.Institute for the Dynamics of Environmental Processes-CNRUniversity of VeniceVeniceItaly
  3. 3.Department of Evolutionary BiologyFlorence UniversityFlorenceItaly

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