Advertisement

Flavonoid biosynthesis and degradation play a role in early defence responses of bilberry (Vaccinium myrtillus) against biotic stress

  • Janne J. Koskimäki
  • Juho Hokkanen
  • Laura Jaakola
  • Marja Suorsa
  • Ari Tolonen
  • Sampo Mattila
  • Anna Maria Pirttilä
  • Anja Hohtola
Original Research

Abstract

Bilberry (Vaccinium myrtillus) represents one of the richest flavonoid sources among plants. Flavonoids play variable, species-dependent roles in plant defences. In bilberry, flavonoid metabolism is activated in response to solar radiation but not against mechanical injury. In this paper, the defence reaction and biosynthesis of phenolic compounds of bilberry was studied after infection by a fungal endophyte (Paraphaeosphaeria sp.) and a pathogen (Botrytis cinerea). The defence response of bilberry was faster against the endophyte than the pathogen. All flavonoid biosynthesis genes tested were activated by each infection. Biosynthesis and accumulation of phenolic acids, flavan-3-ols and oligomeric proanthocyanidins were clearly elevated in both infected samples. Infection by the pathogen promoted specifically accumulation of epigallocatechin, quercetin-3-glucoside, quercetin-3-O-α-rhamnoside, quercetin-3-O-(4”-HMG)-R-rhamnoside, chlorogenic acid and coumaroyl quinic acid. The endophyte-infected plants had a higher content of quercetin-3-glucuronide and coumaroyl iridoid. Therefore, accumulation of individual phenolic compounds could be specific for each infection. Quantity of insoluble proanthocyanidins was the highest in control plants, suggesting that they might act as storage compounds and become activated by degradation upon infection.

Keywords

Vaccinium myrtillus Gene expression LC-MS Flavonoid biosynthesis Proanthocyanidin Pathogenesis-related 

Abbreviations

CHS

chalcone synthase

DFR

dihydroflavonol 4-reductase

ANS

anthocyanidin synthase

ANR

anthocyanidin reductase

PR4

pathogenesis-related protein 4

MEA

malt-extract agar

Notes

Acknowledgements

We thank Dr. P. J. Fisher (University of Portsmouth, Portsmouth, UK) for the advice on endophyte isolation from the Ericaceae. This work was supported by the Ella and Georg Ehrnrooth Foundation and Academy of Finland (No. 118569), and is part of the Endis Network (Discovery and Development of Antibacterials from Endophytes) at the University of Oulu.

References

  1. Carlsen, S. C. K., Understrup, A., Fomsgaard, I. S., Mortensen, A. G., & Ravnskov, S. (2008). Flavonoids in roots of white clover: interaction of arbuscular mycorrhizal fungi and a pathogenic fungus. Plant Soil, 302, 33–43.CrossRefGoogle Scholar
  2. Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, D. G., et al. (2003). Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Research, 31, 497–500.CrossRefGoogle Scholar
  3. de Colmenares, N. G., Ramirez-Martinez, J. R., Aldana, J. O., Ramos-Nino, M. E., Clifford, M. N., Pekerar, S., et al. (1998). Isolation, characterisation and determination of biological activity of coffee proanthocyanidins. Journal of the Science of Food and Agriculture, 77, 368–372.CrossRefGoogle Scholar
  4. Dixon, R. A., & Paiva, N. L. (1995). Stress-Induced Phenylpropanoid Metabolism. Plant Cell, 7, 1085–1097.CrossRefPubMedGoogle Scholar
  5. Dixon, R. A., Achnine, L., Kota, P., Liu, C.-J., Reddy, M. S. S., & Wang, L. (2002). The phenylpropanoid pathway and plant defence—a genomics perspective. Molecular Plant Pathology, 3, 371–390.CrossRefGoogle Scholar
  6. Felsenstein, J. (1989). PHYLIP — Phylogeny Inference Package (Version 3.2). Cladistics, 5, 164–166.Google Scholar
  7. Fukuhara, M. (2002). Three Phaeosphaeria species and Paraphaeosphaeria michotii isolated from Phragmites leaves in Osaka, Japan. Mycoscience, 43, 375–382.CrossRefGoogle Scholar
  8. Ganley, R. J., & Newcombe, G. (2006). Fungal endophytes in seeds and needles of Pinus monticola. Mycological Research, 110, 318–327.CrossRefPubMedGoogle Scholar
  9. Goetz, G., Fkyerat, A., Metais, N., Kunz, M., Tabacchi, R., Pezet, R., et al. (1999). Resistance factors to grey mould in grape berries: identification of some phenolics inhibitors of Botrytis cinerea stilbene oxidase. Phytochemistry, 52, 759–767.CrossRefGoogle Scholar
  10. Hébert, C., Charles, M. T., Gauthier, L., Willemot, C., Khanizadeh, S., & Cousineau, J. (2002). Strawberry proanthocyanidins: Biochemical markers for Botrytis cinerea resistance and shelf-life predictability. Acta Horticulturae, 567, 659–662.Google Scholar
  11. Hildebrandt, P. D., McRae, K. B., & Lu, X. (2001). Factors affecting flower infection and disease severity of lowbush blueberry by Botrytis cinerea. Canadian Journal of Plant Pathology, 23, 364–370.Google Scholar
  12. Jaakola, L., Tolvanen, A., Laine, K., & Hohtola, A. (2001a). Effect of N6-isopentenyladenine concentration on growth initiation in vitro and rooting of bilberry and lingonberry microshoots. Plant Cell Tissue and Organ Culture, 66, 73–77.CrossRefGoogle Scholar
  13. Jaakola, L., Pirttilä, A. M., & Hohtola, A. (2001b). Isolation of RNA from bilberry (Vaccinium myrtillus). Molecular Biotechnology, 19, 201–203.CrossRefGoogle Scholar
  14. Jaakola, L., Määttä-Riihinen, K., Kärenlampi, S., & Hohtola, A. (2004). Activation of flavonoid biosynthesis by solar radiation in bilberry (Vaccinium myrtillus) leaves. Planta, 218, 721–728.CrossRefPubMedGoogle Scholar
  15. Jaakola, L., Koskimäki, J. J., Riihinen, K., Tolvanen, A., & Hohtola, A. (2008). Effect of wounding on chalcone synthase and pathogenesis related PR10 gene expression and content of phenolic compounds in bilberry leaves. Biologia Plantarum, 52, 391–395.CrossRefGoogle Scholar
  16. Larose, G., Chenevert, R., Moutoglis, P., Gagne, S., Piche, Y., & Vierheilig, H. (2002). Flavonoid levels in roots of Medicago sativa are modulated by the developmental stage of the symbiosis and the root colonizing arbuscular mycorrhizal fungus. Journal of Plant Physiology, 159, 1329–1339.CrossRefGoogle Scholar
  17. Laukkanen, H., Soini, H., Kontunen-Soppela, S., Hohtola, A., & Viljanen, M. (2000). A mycobacterium isolated from tissue cultures of mature Pinus sylvestris interferes with growth of Scots pine seedlings. Tree Physiology, 20, 915–920.PubMedGoogle Scholar
  18. Lee, M. H., & Bostock, R. M. (2007). Fruit exocarp phenols in relation to quiescence and development of Monilinia fructicola infections in Prunus spp.: A role for cellular redox? Phytopathology, 97, 269–277.CrossRefPubMedGoogle Scholar
  19. Logemann, E., & Hahlbrock, K. (2002). Crosstalk among stress responses in plants: Pathogen defence overrides UV protection through an inversely regulated ACE/ACE type of light-responsive gene promoter unit. Proceedings of the National Academy of Sciences, 99, 2428–2432.CrossRefGoogle Scholar
  20. Määttä, K., Kamal-Eldin, A., & Törrönen, R. (2001). Phenolic compounds in berries of black, red, green, and white currants (Ribes sp.). Antioxidants & Redox Signaling, 3, 981–993.CrossRefGoogle Scholar
  21. McKhann, H. I., Paiva, N. L., Dixon, R. A., & Hirsch, A. M. (1997). Chalcone synthase transcripts are detected in alfalfa root hairs following inoculation with wild-type Rhizobium meliloti. Molecular Plant-Microbe Interactions, 10, 50–58.CrossRefGoogle Scholar
  22. Miranda, M., Ralph, S. G., Mellway, R., White, R., Heath, M. C., Bohlmann, J., et al. (2007). The transcriptional response of hybrid poplar (Populus trichocarpa x P-deltoides) to infection by Melampsora medusae leaf rust involves induction of flavonoid pathway genes leading to the accumulation of proanthocyanidins. Molecular Plant-Microbe Interactions, 20, 816–831.CrossRefPubMedGoogle Scholar
  23. Mohr, U., Lange, J., Boller, T., Wiemken, A., & Vogeli-Lange, R. (1998). Plant defence genes are induced in the pathogenic interaction between bean roots and Fusarium solani, but not in the symbiotic interaction with the arbuscular mycorrhizal fungus Glomus mosseae. New Phytologist, 138, 589–598.CrossRefGoogle Scholar
  24. Morazzoni, P., & Bombardelli, E. (1996). Vaccinium myrtillus L. Fitoterapia, 67, 3–29.Google Scholar
  25. Muthuswamy, S., & Vasantha, R. H. P. (2007). Fruit phenolics as natural antimicrobial agents : Selective antimicrobial activity of catechin, chlorogenic acid and phloridzin. International Journal of Food, Agriculture and Environment, 5, 81–85.Google Scholar
  26. Pandey, M. K., Sarma, B. K., Singh, D. P., & Singh, U. P. (2007). Biochemical investigations of sclerotial exudates of Sclerotium rolfsii and their antifungal activity. Journal of Phytopathology, 155, 84–89.CrossRefGoogle Scholar
  27. Pehkonen, T., Koskimäki, J. J., Riihinen, K., Pirttilä, A. M., Hohtola, A., Jaakola, L., et al. (2008). Artificial infection of Vaccinium vitis-idaea L. and defence responses to Exobasidium species. Physiological and Molecular Plant Pathology, 12, 146–150.Google Scholar
  28. Pirttilä, A. M., Kämäräinen, T., Hirsikorpi, M., Jaakola, L., & Hohtola, A. (2001). DNA isolation methods for medicinal and aromatic plants. Plant Molecular Biology Reporter, 19, 273a–f.CrossRefGoogle Scholar
  29. Polya, G. M., & Foo, L. Y. (1994). Inhibition of eukaryote signal-regulated protein kinases by plant-derived catechin-related compounds. Phytochemistry, 35, 1399–1405.CrossRefPubMedGoogle Scholar
  30. Pozo, M. J., Azcón-Aguilar, C., Dumas-Gaudot, E., & Barea, J. M. (1999). β-1, 3-Glucanase activities in tomato roots inoculated with arbuscular mycorrhizal fungi and/or Phytophthora parasitica and their possible involvement in bioprotection. Plant Science, 141, 149–157.CrossRefGoogle Scholar
  31. Punyasiri, P. A. N., Tanner, G. J., Abeysinghe, S. B., Kumar, V., Campbell, P. M., & Pradeepa, N. H. L. (2004). Exobasidium vexans infection of Camellia sinensis increased 2, 3-cis isomerization and gallate esterification of proanthocyanidins. Phytochemistry, 65, 2987–2994.CrossRefGoogle Scholar
  32. Salzer, P., Feddermann, N., Wiemken, A., Boller, T., & Staehelin, C. (2004). Sinorhizobium meliloti-induced chitinase gene expression in Medicago truncatula ecotype R108-1: a comparison between symbiosis-specific class V and defence-related class IV chitinases. Planta, 219, 626–638.CrossRefPubMedGoogle Scholar
  33. Sauer, M., Lu, P., Sangar, R., Kennedy, S., Polishook, J., Bills, G., et al. (2002). Estimating polyketide metaholic potential among non-sporulating fungal endophytes of Vaccinium macrocarpon. Mycological Research, 106, 460–470.CrossRefGoogle Scholar
  34. Schulz, B., Römmert, A.-K., Dammann, U., Aust, H.-J., & Strack, D. (1999). The endophyte-host interaction: a balanced antagonism? Mycological Research, 103, 1275–1283.CrossRefGoogle Scholar
  35. Tolonen, A., & Uusitalo, J. (2004). Fast screening method for the analysis of total flavonoid content in plants and foodstuffs by high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry with polarity switching. Rapid Communications in Mass Spectrometry, 18, 3113–3122.CrossRefPubMedGoogle Scholar
  36. Treutter, D. (2005). Significance of flavonoids in plant resistance and enhancement of their biosynthesis. Plant Biology, 7, 581–591.CrossRefPubMedGoogle Scholar
  37. Veluri, R., Weir, T. L., Bais, H. P., Termitz, F. R. S., & Ivanco, J. M. V. (2004). Phytotoxic and antimicrobial activities of catechin derivatives. Journal of Agricultural and Food Chemistry, 52, 1077–1082.CrossRefPubMedGoogle Scholar
  38. Vlassova, T., Likhachev, A. & Blintsov, A. (2000). Accumulation of abscisic acid in culture filtrates of selected species of genus Botrytis. (Paper presented at the XIIth International Botrytis symposium (p. 23) Reims, France)Google Scholar
  39. White, T. J., Burns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M. A. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White (Eds.), PCR Protocols (pp. 315–322). San Diego: Academic.Google Scholar
  40. Witzell, J., & Shevtsova, A. (2004). Nitrogen-induced changes in phenolics of Vaccinium myrtillus - implications for interaction with a parasitic fungus. Journal of Chemical Ecology, 30, 1937–1956.CrossRefPubMedGoogle Scholar

Copyright information

© KNPV 2009

Authors and Affiliations

  • Janne J. Koskimäki
    • 1
  • Juho Hokkanen
    • 2
    • 3
  • Laura Jaakola
    • 1
  • Marja Suorsa
    • 1
  • Ari Tolonen
    • 3
  • Sampo Mattila
    • 2
  • Anna Maria Pirttilä
    • 1
  • Anja Hohtola
    • 1
  1. 1.Department of BiologyUniversity of OuluOuluFinland
  2. 2.Department of ChemistryUniversity of OuluOuluFinland
  3. 3.Novamass LtdOuluFinland

Personalised recommendations