, Volume 70, Issue 8, pp 1011–1018 | Cite as

Effects of low-temperature hardening on the biochemical response of winter oilseed rape seedlings inoculated with the spores of Leptosphaeria maculans

  • Katarzyna HuraEmail author
  • Tomasz Hura
  • Marcin Rapacz
  • Agnieszka Płażek


The aim of the study was to assess the effects of low-temperature hardening (2°C) on the biochemical compounds and processes that can increase resistance of winter rape to inoculation with Leptosphaeria maculans spores. The study involved an evaluation of the entire pool of phenolic compounds, L-phenylalanine ammonia lyase (PAL) activity, excitation intensity for blue and green fluorescence, catalase (CAT) activity, respiration intensity and heat emission from leaf tissues. All the measurements were performed 24 and 72 hours after the inoculation. Low-temperature hardening, which preceded the inoculation of rape seedlings with spores of L. maculans, caused a significant increase in CAT activity and the level of phenolic compounds. The observed changes in PAL activity reflected the changes in phenolics content. The hardened plants showed a significantly higher intensity of blue fluorescence excitation at 24 and 72 hours after the inoculation, as compared to the non-hardened seedlings. Increased content of phenolic compounds and PAL and catalase activity triggered by the temperature of 2 °C and maintained for 24 hours after the inoculation, may confirm the stimulating effect of the hardening temperature. Intensified emission of blue fluorescence indicating saturation of a cell wall with phenolic compounds makes the cell wall structure less stretchy, more tight and leakproof, and thereby hinders fungal growth through plant tissue.

Key words

cold respiration intensity heat emission catalase phenolics phenylalanine ammonia lyase blue-green fluorescence 





blue fluorescence


green fluorescence


heat emission


leaf water content


phenylalanine ammonia lyase


photosynthetic photon flux density


isolate of L. maculans


respiration intensity


reactive oxygen species


soluble phenolics


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  1. Aebi H. 1984. Catalase in vitro. Methods Enzymol. 105: 121–126.PubMedCrossRefGoogle Scholar
  2. Anekonda T.S., Criddle R.S. & Libby W.J. 1994. Calorimetric evidence for site-adapted biosynthetic metabolism in coast redwood. Can. J. Forest Res. 24: 380–389.CrossRefGoogle Scholar
  3. Atkinson N.J. & Urwin P.E. 2012. The interaction of plant biotic and abiotic stresses: from genes to the field. J. Exp. Bot. 63: 3523–3543.PubMedCrossRefGoogle Scholar
  4. Baena-González E. & Sheen J. 2008. Convergent energy and stress signaling. Trends Plant Sci. 13: 474–482.PubMedPubMedCentralCrossRefGoogle Scholar
  5. Baker C.J., Mock N.M., Deahl K. & Domek J. 1997. Monitoring the rate of oxygen consumption in plant cell suspensions. Plant Cell Tissue Organ Cult. 51: 111–117.CrossRefGoogle Scholar
  6. Balesdent M.H., Jędryczka M., Jain L., Mendes-Pereira E., Bertrandy J. & Rouxel T. 1998. Conidia, substrate for internal transcribed spacer-based PCR identification of component of the Leptosphaeria maculans species complex. Phytopathology 88: 12–17.CrossRefGoogle Scholar
  7. Ben Hamed K., Chibani F., Abdelly C. & Magne C. 2014. Growth, sodium uptake and antioxidant responses of coastal plants differing in their ecological status under increasing salinity. Biologia 69: 193–201.CrossRefGoogle Scholar
  8. Bradford M. 1976. A rapid and sensitive method for the quantitation of microgram quantitaties of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.CrossRefPubMedGoogle Scholar
  9. Criddle R.S., Fontana A.J., Rank D.R., Paige D., Hansen L.D. & Breidenbach R.W. 1991. Simultaneous measurement of metabolic heat rate, CO2 production, and O2 consumption by microcalorimetry. Anal. Biochem. 194: 413–417.PubMedCrossRefPubMedCentralGoogle Scholar
  10. Davar R., Darvishzadeh R. & Majd A. 2013. Changes in antioxidant systems in sunflower partial resistant and susceptible lines as affected by Sclerotinia sclerotiorum. Biologia 68: 821–829.CrossRefGoogle Scholar
  11. De Ascensao A.R.F.D.C. & Dubery LA. 2003. Soluble and wall-bound phenolics and phenolic polymers in Musa acuminata roots exposed to elicitors from Fusarium oxysporum, f.sp. cubense. Phytochemistry 63: 679–686.PubMedCrossRefPubMedCentralGoogle Scholar
  12. De Gara L., de Pinto M.C. & Tommasi F. 2003. The antioxidant systems vis-f-vis reactive oxygen species during plantpathogen interaction. Plant Physiol. Biochem. 41: 863–870.CrossRefGoogle Scholar
  13. El Modafar C. & El Boustani E. 2001. Cell wall-bound phenolic acid and lignin contents in date palm as related to its resistance to Fusarium, oxysporum,. Biol. Plantarum 44: 125–130.CrossRefGoogle Scholar
  14. Ergon A. & Tronsmo A.M. 2006. Components of pink snow mould resistance in winter wheat are expressed prior to cold hardening and in detached leaves. J. Phytopathol. 154: 134–142.CrossRefGoogle Scholar
  15. Fitt B.D.L., Brun H., Barbetti M.J. & Rimmer S.R. 2006. Worldwide importance of phoma stem canker (Leptosphaeria maculans and L. biglobosa) on oilseed rape (Brassica napus). Eur. J. Plant Pathol. 114: 3–15.CrossRefGoogle Scholar
  16. Fornalé S., Lopez E., Salazar-Henao J.E., Fernández-Nohales P., Rigau J. & Caparros-Ruiz D. 2014. AtMYB7, a new player in the regulation of UV-sunscreens in Arabidopsis thaliana. Plant Cell Physiol. 55: 507–516.PubMedCrossRefPubMedCentralGoogle Scholar
  17. Friedt W. & Snowdon R. 2009. Oilseed rape, pp. 91–126. In: Vollmann J. & Rajcanpp I. (eds), Oil Crops, Handbook of Plant Breeding 4, Springer, Dordrecht, Heidelberg, London, New York.Google Scholar
  18. Fry S.C. 1982. Phenolic components of the primary cell wall. Biochem. J. 203: 493–504.PubMedPubMedCentralCrossRefGoogle Scholar
  19. Fry S.C. 1987. Intercellular feruloylation of pectic polysaccharides. Planta 171: 205–211.PubMedCrossRefPubMedCentralGoogle Scholar
  20. Gomes M.P. & Garcia Q.S. 2013. Reactive oxygen species and seed germination. Biologia 68: 351–357.Google Scholar
  21. Gugel R.K. & Petrie G.A. 1992. History, occurrence, impact, and control of blackleg of rapeseed. Can. J. Plant Pathol. 14: 36–45.CrossRefGoogle Scholar
  22. Hura K., Hura T., Bączek-Kwinta R., Grzesiak M. & Płażek A. 2014a. Induction of defense mechanisms in seedlings of oilseed winter rape inoculated with Phoma lingam (Leptosphaeria maculans). Phytoparasitica 42: 145–154.CrossRefGoogle Scholar
  23. Hura K., Hura T., Dziurka K. & Dziurka M. 2014b. Biochemical defense mechanisms induced in winter oilseed rape seedlings with different susceptibility to infection with Leptosphaeria maculans. Physiol. Mol. Plant Pathol. 87: 42–50.CrossRefGoogle Scholar
  24. Hura K., Hura T., Dziurka K., Dziurka M. 2015. Carbohydrate, phenolic and antioxidant level in relation to chlorophyll a content in oilseed winter rape (Brassica napus L.) inoculated with Leptosphaeria maculans. Eur. J. Plant Pathol, (in press) DOI: 10.1007/s10658-015-0680-1.Google Scholar
  25. Hura K., Hura T. & Grzesiak M. 2014c. Function of the photo-synthetic apparatus of oilseed winter rape under elicitation by Phoma lingam phytotoxins in relation to carotenoid and phenolic levels. Acta Physiol. Plant. 36: 295–305.CrossRefGoogle Scholar
  26. Hura K., Hura T., Grzesiak M. & Rapacz M. 2014d. Early detection of Phoma lingam infection in oilseed winter rape before visible symptoms appear. Acta Biol. Cracov. Ser. Bot. 56: 59–65.Google Scholar
  27. Hura T., Hura K., Dziurka K., Ostrowska A., Bączek-Kwinta R. & Grzesiak M.T. 2012. An increase in the content of cell wall-bound phenolics correlates with the productivity of triticale under soil drought. J. Plant Physiol. 169: 1728–1736.PubMedCrossRefPubMedCentralGoogle Scholar
  28. Hura T., Hura K., Ostrowska A., Grzesiak M. & Dziurka K. 2013. The cell wall-bound phenolics as a biochemical indicator of soil drought resistance in winter triticale. Plant Soil Environ. 59: 189–195.CrossRefGoogle Scholar
  29. Jesus C., Meijón M., Monteiro P., Correia B., Amaral J., Escandón M., Cañal M.J. & Pinto G. 2015. Salicylic acid application modulates physiological and hormonal changes in Eucalyptus globulus under water deficit. Environ. Exp. Bot. 118: 56–66.CrossRefGoogle Scholar
  30. Jędryczka M., Rouxel T., Balesdent M.H., Mendes Pereira E. & Bertrandy J. 1997. Molecular characterization of Polish Phoma lingam isolates. Cereal Res. Commun. 25: 279–283.Google Scholar
  31. Kamisaka S., Takeda S., Takahashi K. & Shibata K. 1990. Diferulic and ferulic acid in the cell wall of Avena coleoptiles: their relationships to mechanical properties of the cell wall. Physiol. Plant. 78: 1–7.CrossRefGoogle Scholar
  32. Lang M., Lichtenthaler H.K., Sowinska M., Summ P. & Heisel F. 1994. Blue, green and red fluorescence signatures and images of tobacco leaves. Plant Biol. 107: 230–236.Google Scholar
  33. Lang M., Siffel P., Braunova Z. & Lichtenthaler H.K. 1992. Investigations of the blue-green fluorescence emission of plant leaves. Plant Biol. 105: 435–440.Google Scholar
  34. Lichtenthaler H.K. & Schweiger J. 1998. Cell wall bound ferulic acid, the major substance of the blue-green ?uorescence emission of plants. J. Plant Physiol. 152: 272–282.CrossRefGoogle Scholar
  35. Mandal S., Mitra A. & Mallick N. 2009. Time course study on accumulation of cell wall-bound phenolics and activities of defense enzymes in tomato roots in relation to Fusarium, wilt. World J. Microbiol. Biotechnol. 25: 795–802.CrossRefGoogle Scholar
  36. Mittler R. 2002. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7: 405–410.CrossRefGoogle Scholar
  37. Payri R., Salvador F.J., Gimeno J. & Bracho G. 2011. The effect of temperature and pressure on thermodynamic properties of diesel and biodiesel fuels. Fuel 90: 1172–1180.CrossRefGoogle Scholar
  38. Peltonen S. & Karjalainen R. 1995. Phenylalanine ammonia-lyase activity in barley after infection with Bipolaris sorokiniana or treatment with its purified xylanase. J. Phytopathol. 143: 239–245.CrossRefGoogle Scholar
  39. Płażek A., Dubert F. & Marzec K. 2009. Cell membrane permeability and antioxidant activities in the rootstocks of Miscanthus x giganteus as an effect of cold and frost treatment. J. Appl. Bot. Food Qual. 82: 158–162.Google Scholar
  40. Płażek A., Hura K. & Żur I. 2003a. Reaction of winter oilseed rape callus to different concentrations of elicitors: pectinase or chitosan. Acta Physiol. Plant. 25: 83–89.CrossRefGoogle Scholar
  41. Płażek A., Hura K., Żur I. & Niemczyk E. 2003b. Relationship between frost tolerance and cold-induced resistance of spring barley, meadow fescue and winter oilseed rape to fungal pathogens. J. Agron. Crop Sci. 189: 333–340.CrossRefGoogle Scholar
  42. Płażek A. & Rapacz M. 2000. The intensity of respiration and heat emission from seedlings of Festuca pratensis (Hud.) and Hordeum, vulgare L. during pathogenesis caused by Bipolaris sorokiniana (Sacc.) Shoem. Acta Physiol. Plant. 22: 25–30.CrossRefGoogle Scholar
  43. Płażek A. & Żur I. 2003. Cold-induced plant resistance to necrotrophic pathogens and antioxidant enzyme activities and cell membrane permeability. Plant Sci. 164: 1019–1028.CrossRefGoogle Scholar
  44. Rapacz M. 1998. The after-effects of temperature and irradiance during early growth of winter oilseed rape (Brassica napus L. var. oleifero, cv. Gárczański) seedlings on the progress of their cold acclimation. Acta Physiol. Plant. 20: 73–78.CrossRefGoogle Scholar
  45. Rapacz M. 2002. Cold-deacclimation of oilseed rape (Brassica napus var. oleifero) in response to fluctuating temperatures and photoperiod. Ann. Bot. 89: 543–549.PubMedPubMedCentralCrossRefGoogle Scholar
  46. Reyes E. & Jennings P.H. 1997. Effects of chilling on respiration and induction of cyanide-resistant respiration in seedling roots of cucumber. J. Amer. Soc. Hort. Sci. 122: 190–194.CrossRefGoogle Scholar
  47. Schopfer P. 1996. Hydrogen peroxide-mediated cell-wall stiffening in vitro in maize coleoptiles. Planta 199: 43–49.CrossRefGoogle Scholar
  48. Schweiger J., Lang M. & Lichtenthaler H.K. 1996. Differences in fluorescence excitation spectra of leaves between stressed and non-stressed plants. J. Plant Physiol. 148: 536–547.CrossRefGoogle Scholar
  49. Singleton V.S. & Rossi J.A., Jr. 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagent. Amer. J. Enol. Viticult. 16: 144–157.Google Scholar
  50. Solecka D., Boudet A.M. & Kacperska A. 1999. Phenylpropanoid and anthocyanin changes in low-temperature treated winter oilseed rape leaves. Plant Physiol. Biochem. 37: 491–496.CrossRefGoogle Scholar
  51. Solecka D. & Kacperska A. 2003. Phenylpropanoid deficiency affects the course of plant acclimation to cold. Physiol. Plant. 119: 253–262.CrossRefGoogle Scholar
  52. Stefanowska M., Kuraś M. & Kacperska A. 2002. Low temperature-induced modifications in cell ultrastructure and localization of phenolics in winter oilseed rape (Brassica napus L. var. oleifero, L.) leaves. Ann. Bot. 90: 637–645.PubMedPubMedCentralCrossRefGoogle Scholar
  53. Suzuki N. & Mittler R. 2006. Reactive oxygen species and temperature stresses: A delicate balance between signaling and destruction. Physiol. Plant. 126: 45–51.CrossRefGoogle Scholar
  54. Trillas M.I. & Azcon-Bieto J. 1995. Short- and long-term effects of Fusarium, oxysporum, elicitors on respiration of carnation callus. Plant Physiol. Biochem. 33: 47–53.Google Scholar
  55. Tronsmo A.M. 1984. Resistance to the rust fungus Puccinia poae-nemoralis in Poa pratensis induced by low-temperature hardening. Can. J. Bot. 62: 2891–2892.CrossRefGoogle Scholar
  56. Truesdale G.A. & Downing A.L. 1954. Solubility of oxygen in water. Nature 173: 1236.Google Scholar
  57. Wakabayashi K., Hoson T. & Kamisaka S. 1997. Osmotic stress suppresses cell wall stiffening and the increase in cell wallbound ferulic and diferulic acids in wheat coleoptiles. Plant Physiol. 113: 967–973.PubMedPubMedCentralCrossRefGoogle Scholar
  58. West J.S., Kharbanda P.D., Barbetti M.J. & Fitt B.D.L. 2001. Epidemiology and management of Leptosphaeria maculans (phoma stem canker) on oilseed rape in Australia, Canada and Europe. Plant Pathol. 50: 10–27.CrossRefGoogle Scholar

Copyright information

© Slovak Academy of Sciences 2015

Authors and Affiliations

  • Katarzyna Hura
    • 1
    Email author
  • Tomasz Hura
    • 2
  • Marcin Rapacz
    • 1
  • Agnieszka Płażek
    • 1
  1. 1.Department of Plant Physiology, Faculty of Agriculture and EconomicsAgricultural UniversityKrakówPoland
  2. 2.The Franciszek Górski Institute of Plant PhysiologyPolish Academy of SciencesKrakówPoland

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