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Localization of ergot alkaloids in sclerotia of Claviceps purpurea by matrix-assisted laser desorption/ionization mass spectrometry imaging

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Abstract

The fungus Claviceps purpurea produces highly toxic ergot alkaloids and accumulates these in the hardened bodies of fungal mycelium. These so-called sclerotia, or ergot bodies, replace the crop seed of infected plants, which can include numerous important food- and feedstuff such as rye and wheat. While several studies have explored details of the infection process and development of ergot bodies, little information is available on the spatial distribution of the mycotoxins in the sclerotia. Here we used matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) at a lateral resolution of 35 μm to visualize the distribution of two representative alkaloids, ergocristine and ergometrine, produced by Ecc93 and Gal 310 variants of C. purpurea, respectively, after infection of rye. To improve cryosectioning of this fragile biological material tissue with complex texture, we developed a practical embedding protocol based on cellulose polymers. The MALDI-MS images recorded from the so produced intact tissues sections revealed that ergometrine exhibited a relatively homogeneous distribution throughout the ergot body, whereas ergocristine was found to be enriched in the proximal region. This finding can be correlated to the morphological development of sclerotia as ergot alkaloids are only produced in the sphacelial stage. The ability to localize toxins and other secondary metabolites in intact sections of crop-infecting fungi with high lateral resolution renders MALDI-MSI a powerful tool for investigating biosynthetic pathways and for obtaining a deeper understanding of the parasite-host interaction.

Workflow for identification and spatial localization of ergot alkaloids in infected rye grains

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References

  1. Bové FJ. The story of ergot. Basel: S. Karger; 1970.

    Google Scholar 

  2. Hanosová H, Koprna R, Valík J, Knoppová L, Frébort I, Dzurová L, et al. Improving field production of ergot alkaloids by application of gametocide on rye host plants. N Biotechnol. 2015;32:739–46.

  3. Coufal-Majewski S, Stanford K, McAllister T, Blakley B, McKinnon J, Chaves AV, et al. Impacts of cereal ergot in food animal production. Front Vet Sci. 2016;3:15.

    Article  Google Scholar 

  4. Menzies JG, Turkington TK. An overview of the ergot (Claviceps purpurea) issue in western Canada: challenges and solutions. Can J Plant Pathol. 2015;1:40–51.

    Article  Google Scholar 

  5. Tittlemier SA, Drul D, Roscoe M, McKendry T. Occurrence of ergot and ergot alkaloids in Western Canadian wheat and other cereals. J Agric Food Chem. 2015;63:6644–50.

  6. Wäli PP, Wäli PR, Saikkonen K, Tuomi J. Is the pathogenic ergot fungus a conditional defensive mutualist for its host grass? PLoS ONE. 2013;8:e69249.

  7. Wegulo S, Carlson M. Ergot of small grain cereals and grasses and its health effects on humans and livestock. Lincoln: University of Nebraska-Lincoln Extension; 2011.

    Google Scholar 

  8. Kren V, Cvak L. Ergot. The genus Claviceps. Medicinal and aromatic plants: industrial profiles, vol. 6. Australia: Harwood Academic Publishers; 1999.

  9. Schiff PL. Ergot and its alkaloids. Am J Pharm Educ. 2006;70:98.

  10. Haarmann T, Rolke Y, Giesbert S, Tudzynski P. Ergot: from witchcraft to biotechnology. Mol Plant Pathol. 2009;10:563–77.

  11. European Food Safety Authority (EFSA). Scientific opinion on ergot alkaloids in food and feed. Parma: Opinion of the Scientific Committee/Scientific Panel; EFSA Journal 2012;10:2798.

  12. Shaw BI, Mantle PG. Parasitic differentiation of Claviceps purpurea. Trans Br Mycol Soc. 1980;75:117–21.

  13. Corbett K, Dickerson AG, Mantle PG. Metabolic studies on Claviceps purpurea during parasitic development on rye. J Gen Microbiol. 1974;84:39–58.

  14. Luttrell ES. Host–parasite relationships and development of the ergot sclerotium in Claviceps purpurea. Can J Bot. 1980;58:942–58.

  15. Lorenz K. Ergot on cereal grains. Crit Rev Food Sci Nutr. 1979;11:311–54.

  16. Young JC. Variability in the content and composition of alkaloids found in Canadian ergot. II. Wheat. J Environ Sci Health B. 1981;16:381–93.

  17. Negard M, Uhlig S, Kauserud H, Andersen T, Hoiland K, Vralstad T. Links between genetic groups, indole alkaloid profiles and ecology within the grass-parasitic Claviceps purpurea species complex. Toxins 2015;5:1431–56.

  18. Pazoutova S, Pesicova K, Chudickova M, Srutka P, Kolarik M. Delimitation of cryptic species inside Claviceps purpurea. Fungal Biol. 2015;119:7–26.

  19. Silber A, Bischoff W. Stability of alkaloid content in various species of ergot. Die Pharmazie. 1954;9:46–61.

  20. Young JC. Variability in the content and composition of alkaloids found in Canadian ergot. I. Rye. J Environ Sci Health B. 1981;16:83–110.

  21. Mantle PG, Tonolo A. Relationship between the morphology of yosh and the production of alkaloids. Trans Br Mycol Soc. 1968;51:499–505.

  22. Gessel MM, Norris JL, Caprioli RM. MALDI imaging mass spectrometry: spatial molecular analysis to enable a new age of discovery. J Proteomics. 2014;107:71–82.

    Article  CAS  Google Scholar 

  23. Kaspar S, Peukert M, Svatos A, Matros A, Mock H. MALDI-imaging mass spectrometry—an emerging technique in plant biology. Proteomics. 2011;11:1840–50.

  24. Takahashi K, Kozuka T, Anegawa A, Nagatani A, Mimura T. Development and application of a high-resolution imaging mass spectrometer for the study of plant tissues. Plant Cell Physiol. 2015;56:1329–38.

  25. Dreisewerd K. Recent methodological advances in MALDI mass spectrometry. Anal Bioanal Chem. 2014;406:2261–78.

  26. Soltwisch J, Kettling H, Vens-Cappell S, Wiegelmann M, Muthing J, Dreisewerd K. Mass spectrometry imaging with laser-induced postionization. Science. 2015;348:211–5.

  27. Boughton BA, Thinagaran D, Sarabia D, Bacic A, Roessner U. Mass spectrometry imaging for plant biology. A review. Phytochem Rev. 2016;15:445–88.

  28. Nielen MWF, van Beek TA. Macroscopic and microscopic spatially-resolved analysis of food contaminants and constituents using laser-ablation electrospray ionization mass spectrometry imaging. Anal Bioanal Chem. 2014;406:6805–15.

  29. Berisha A, Dold S, Guenther S, Desbenoit N, Takats Z, Spengler B, et al. A comprehensive high-resolution mass spectrometry approach for characterization of metabolites by combination of ambient ionization, chromatography and imaging methods. Rapid Commun Mass Spectrom. 2014;28:1779–91.

  30. Hickert S, Cramer B, Letzel MC, Humpf H. MALDI-imaging mass spectrometry of ochratoxin A and fumonisins in mold-infected food. Rapid Commun Mass Spectrom. 2016;30:2508-2516.

  31. Dong Y, Li B, Malitsky S, Rogachev I, Aharoni A, Kaftan F, et al. Sample preparation for mass spectrometry imaging of plant tissues. A review. Front Plant Sci. 2016;7:60.

  32. Heyman HM, Dubery IA. The potential of mass spectrometry imaging in plant metabolomics. A review. Phytochem Rev. 2016;15:297–316.

  33. Velickovic D, Ropartz D, Guillon F, Saulnier L, Rogniaux H. New insights into the structural and spatial variability of cell-wall polysaccharides during wheat grain development, as revealed through MALDI mass spectrometry imaging. J Exp Bot. 2014;65:2079–91.

  34. Zaima N, Goto-Inoue N, Hayasaka T, Setou M. Application of imaging mass spectrometry for the analysis of Oryza sativa rice. Rapid Commun Mass Spectrom. 2010;24:2723–9.

  35. Yoshimura Y, Zaima N, Moriyama T, Kawamura Y. Different localization patterns of anthocyanin species in the pericarp of black rice revealed by imaging mass spectrometry. PLoS ONE. 2012;7:e31285.

  36. Burrell M, Earnshaw C, Clench MR. Imaging matrix assisted laser desorption ionization mass spectrometry: a technique to map plant metabolites within tissues at high spatial resolution. J Exp Bot. 2007;58:757–63.

  37. Majeská Čudejková M, Vojta P, Valík J, Galuszka P. Quantitative and qualitative transcriptome analysis of four industrial strains of Claviceps purpurea with respect to ergot alkaloid production. N Biotechnol. 2016;33:743–54.

    Article  Google Scholar 

  38. Haarmann T, Machado C, Lübbe Y, Correia T, Schardl C, Panaccione DG, et al. The ergot alkaloid gene cluster in Claviceps purpurea: extension of the cluster sequence and intra species evolution. Phytochemistry. 2005;66:1312–20.

  39. EPA’s Office of Pollution Prevention Toxics and Syracus Research Corporation. EPI Suite: U.S. Environmental Protection Agency; 2012.

  40. Smit R, Tudzynski P. Efficient transformation of Claviceps purpurea using pyrimidine auxotrophic mutants—cloning of the omp decarboxylase gene. Mol Gen Genet. 1992;234:297–305.

  41. Keller U, Han M, Stoeffler-Meilicke M. D-Lysergic acid activation and cell-free synthesis of D-lysergyl peptides in an enzyme fraction from the ergot fungus Claviceps purpurea. Biochemistry. 1988;27:6164–70.

  42. Mantle PG, Nisbet LJ. Differentiation of Claviceps purpurea in axenic culture. J Gen Microbiol. 1976;93:321–34.

  43. Tenberge KB, Homann V, Oeser B, Tudzynski P. Structure and expression of two polygalacturonase genes of Claviceps purpurea oriented in tandem and cytological evidence for pectinolytic enzyme activity during infection of rye. Phytopathology. 1996;86:1084–97.

  44. Pirkl A, Meier M, Popkova Y, Letzel M, Schnapp A, Schiller J, et al. Analysis of free fatty acids by ultraviolet laser desorption ionization mass spectrometry using insect wings as hydrophobic sample substrates. Anal Chem. 2014;86:10763–71.

  45. Kettling H, Vens-Cappell S, Soltwisch J, Pirkl A, Haier J, Müthing J, et al. MALDI mass spectrometry imaging of bioactive lipids in mouse brain with a Synapt G2-S mass spectrometer operated at elevated pressure: improving the analytical sensitivity and the lateral resolution to ten micrometers. Anal Chem. 2014;86:7798–805.

  46. Lehner AF, Craig M, Fannin N, Bush L, Tobin T. Fragmentation patterns of selected ergot alkaloids by electrospray ionization tandem quadrupole mass spectrometry. J Mass Spectrom. 2004;39:1275–86.

  47. Uhlig S, Petersen D. Lactam ergot alkaloids (ergopeptams) as predominant alkaloids in sclerotia of Claviceps purpurea from Norwegian wild grasses. Toxicon. 2008;52:175–85.

  48. Lösecke W, Neumann D, Gröger D, Schmauder H. Changes of the cytoplasmic ultrastructure during development of sclerotia in Claviceps purpurea (Fr.) Tul. Arch Microbiol. 1980;125:251–7.

    Article  Google Scholar 

  49. Mantle PG. Fatty acid composition of sphacelial and sclerotial growth forms of Claviceps purpurea in relation to the production of ergoline alkaloids in culture. Trans Br Mycol Soc. 1969;53:441–7.

  50. Lehner AF, Craig M, Fannin N, Bush L, Tobin T. Electrospray[+] tandem quadrupole mass spectrometry in the elucidation of ergot alkaloids chromatographed by HPLC: screening of grass or forage samples for novel toxic compounds. J Mass Spectrom. 2005;40:1484–502.

  51. Crews C. Analysis of ergot alkaloids. Toxins. 2015;7:2024–50.

  52. Krska R, Crews C. Significance, chemistry and determination of ergot alkaloids: a review. Food Addit Contam Part A, Chem Anal Control Risk Asses. 2008;25:722–31.

  53. Merkel S, Köppen R, Koch M, Emmerling F, Nehls I. Lumi-ergometrine—structural identification and occurrence in sclerotia. Mycotox Res. 2012;28:59–66.

  54. Soltwisch J, Souady J, Berkenkamp S, Dreisewerd K. Effect of gas pressure and gas type on the fragmentation of peptide and oligosaccharide ions generated in an elevated pressure UV/IR-MALDI ion source coupled to an orthogonal time-of-flight mass spectrometer. Anal Chem. 2009;81:2921–34.

  55. Mulinti P, Allen NA, Coyle CM, Gravelat FN, Sheppard DC, Panaccione DG. Accumulation of ergot alkaloids during conidiophore development in Aspergillus fumigatus. Curr Microbiol. 2014;68:1–5.

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Acknowledgments

We thank Uwe Karst and Ann-Christin Niehoff for use of the cryotome. This research was supported by the German Science Foundation (DFG) by grants TU 50/18-1 (to P.T.), HU 730/11-1 (to H.-U.H.), and DR 416/9-1 (to K.D.), and by the Interdisciplinary Center for Clinical Research (IZKF) Münster by grant Z03 (to K.D.).

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    Authors and Affiliations

    1. Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstr. 45, 48149, Münster, Germany

      Julian Dopstadt, Benedikt Cramer & Hans-Ulrich Humpf

    2. Institute for Hygiene, Westfälische Wilhelms-Universität Münster, Robert-Koch-Str. 41, 48149, Münster, Germany

      Simeon Vens-Cappell & Klaus Dreisewerd

    3. Interdisciplinary Center for Clinical Research (IZKF), Westfälische Wilhelms-Universität Münster, Domagkstr. 3, 48149, Münster, Germany

      Simeon Vens-Cappell & Klaus Dreisewerd

    4. Institute of Plant Biology and Biotechnology, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, 48143, Münster, Germany

      Lisa Neubauer & Paul Tudzynski

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    1. Julian Dopstadt
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    Correspondence to Klaus Dreisewerd or Hans-Ulrich Humpf.

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    Julian Dopstadt and Simeon Vens-Cappell contributed equally to this work.

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    Dopstadt, J., Vens-Cappell, S., Neubauer, L. et al. Localization of ergot alkaloids in sclerotia of Claviceps purpurea by matrix-assisted laser desorption/ionization mass spectrometry imaging. Anal Bioanal Chem 409, 1221–1230 (2017). https://doi.org/10.1007/s00216-016-0047-2

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