, Volume 230, Issue 1, pp 135–147 | Cite as

Lipophilic components of the brown seaweed, Ascophyllum nodosum, enhance freezing tolerance in Arabidopsis thaliana

  • Prasanth Rayirath
  • Bernhard Benkel
  • D. Mark Hodges
  • Paula Allan-Wojtas
  • Shawna MacKinnon
  • Alan T. Critchley
  • Balakrishnan PrithivirajEmail author
Original Article


Extracts of the brown seaweed Ascophyllum nodosum enhance plant tolerance against environmental stresses such as drought, salinity, and frost. However, the molecular mechanisms underlying this improved stress tolerance and the nature of the bioactive compounds present in the seaweed extracts that elicits stress tolerance remain largely unknown. We investigated the effect of A. nodosum extracts and its organic sub-fractions on freezing tolerance of Arabidopsis thaliana. Ascophyllum nodosum extracts and its lipophilic fraction significantly increased tolerance to freezing temperatures in in vitro and in vivo assays. Untreated plants exhibited severe chlorosis, tissue damage, and failed to recover from freezing treatments while the extract-treated plants recovered from freezing temperature of −7.5°C in in vitro and −5.5°C in in vivo assays. Electrolyte leakage measurements revealed that the LT50 value was lowered by 3°C while cell viability staining demonstrated a 30–40% reduction in area of damaged tissue in extract treated plants as compared to water controls. Moreover, histological observations of leaf sections revealed that extracts have a significant effect on maintaining membrane integrity during freezing stress. Treated plants exhibited 70% less chlorophyll damage during freezing recovery as compared to the controls, and this correlated with reduced expression of the chlorphyllase genes AtCHL1 and AtCHL2. Further, the A. nodosum extract treatment modulated the expression of the cold response genes, COR15A, RD29A, and CBF3, resulting in enhanced tolerance to freezing temperatures. More than 2.6-fold increase in expression of RD29A, 1.8-fold increase of CBF3 and two-fold increase in the transcript level of COR15A was observed in plants treated with lipophilic fraction of A. nodosum at −2°C. Taken together, the results suggest that chemical components in A. nodosum extracts protect membrane integrity and affect the expression of stress response genes leading to freezing stress tolerance in A. thaliana.


Arabidopsis thaliana Ascophyllum nodosum Freezing tolerance Lipophilic compounds Seaweed extract Sustainable agriculture 



Nuclear magnetic resonance


Farmyard manure


Cold responsive


Ascophyllum nodosum extract




National Institutes of Health


Pectin methyl esterase


CRT/DRE binding factor



BP’s lab is supported by the grants from Atlantic Canada Opportunities Agency (ACOA), Natural Sciences and Engineering Research Council of Canada (NSERC), Nova Scotia Department of Agriculture & Marketing (NSDAF) and Acadian Seaplants Limited. PR is grateful to Kalyani Prithiviraj, Nova Scotia Agricultural College for her help with real time PCR experiments.

Supplementary material

425_2009_920_MOESM1_ESM.tif (6.2 mb)
Supplementary Figure 1. Pectin methyl esterase activity in plants treated with ANE (1.0 g L-1) orethyl acetate sub-fractions (1.0 g L-1) and untreated controls. (TIFF 6357 kb)
425_2009_920_MOESM2_ESM.doc (38 kb)
Supplementary material 2 (DOC 37 kb)
425_2009_920_MOESM3_ESM.doc (47 kb)
Supplementary material 3 (DOC 47 kb)


  1. Arnon DI (1949) Copper enzymes in isolated chloroplasts; polyphenoloxidse in Beta vulgaris. Plant Physiol 24:1–15PubMedCrossRefGoogle Scholar
  2. Ashrafi K, Chang FY, Watts JL, Fraser AG, Kamath RS, Ahringer J, Ruvkun G (2003) Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421:268–272PubMedCrossRefGoogle Scholar
  3. Blunden G (1991) Agricultural uses of seaweeds and seaweed extracts. In: Guiry MD, Blunden G (eds) Seaweed resources in Europe: uses and potential. Wiley, Chichester, pp 65–81Google Scholar
  4. Bray EA, Bailey-Serres J, Weretilnyk E (2000) Responses to abiotic stresses. In: Gruissem W, Buchannan B, Jones R (eds) Biochemistry and molecular biology of plants. American Society of Plant Biologists, Rockville, pp 1158–1249Google Scholar
  5. Burchett S, Fuller MP, Jellings AJ (1998) Application of seaweed extract improves winter hardiness of winter barley cv Igri. Abstracts—plant cell topics. York MeetingGoogle Scholar
  6. Crouch I, Van Staden J (1993) Evidence for the presence of plant growth regulators in commercial seaweed products. Plant Growth Regul 13:21–29Google Scholar
  7. Downie B, Dirk LMA, Hadfield KA, Wilkins TA, Bennett AB, Bradford KJ (1998) A gel diffusion assay for quantification of pectin methylesterase activity. Anal Biochem 264:149–157PubMedCrossRefGoogle Scholar
  8. Featonby-Smith BC, van-Staden J (1983) The effect of seaweed concentrate on the growth of tomatoes in nematode infested soil. Scient Hort 20:137–146CrossRefGoogle Scholar
  9. Gilmour SJ, Hajela RK, Thomashow MF (1988) Cold acclimation in Arabidopsis thaliana. Plant Physiol 87:745–750PubMedCrossRefGoogle Scholar
  10. Guy CL (2003) Freezing tolerance of plants: current understanding and selected emerging concepts. Can J Bot 81:1216–1223CrossRefGoogle Scholar
  11. Horváth I, Van Hasselt PR (1985) Inhibition of chilling-induced photooxidative damage to leaves of Cucumis sativus L. by treatment with amino alcohols. Planta 164:83–88CrossRefGoogle Scholar
  12. Huner NPA, Oquist G, Hurry VM, Krol M, Falk S, Griffith M (1993) Photosynthesis, photoinhibition and low temperature acclimation tolerant plants in cold. Photosynth Res 37:19–39CrossRefGoogle Scholar
  13. Ilker R, Warring AJ, Lyons JM, Breidenbach RW (1976) The cytological responses of tomato seedling cotyledons to chilling and the influence of membrane modifications upon these responses. Protoplasma 90:229–252CrossRefGoogle Scholar
  14. Jones PG, Inouye M (1994) The cold shock response: a hot topic. Mol Microbiol 11:811–818PubMedCrossRefGoogle Scholar
  15. Kamal-Eldin A, Määttä K, Toivo J, Lampi A-M, Piironen V (1998) Acid-catalyzed isomerization of fucosterol and Δ5-avenasterol. Lipids 33:1073–1077PubMedCrossRefGoogle Scholar
  16. Kariola T, Brader G, Li J, Palva ET (2005) Chlorophyllase 1, a damage control enzyme, affects the balance between defense pathways in plants. Plant Cell 17:282–294PubMedCrossRefGoogle Scholar
  17. Karpinski S, Gabrys H, Mateo A, Karpinska B, Mullineaux PM (2003) Light perception in plant disease defense signalling. Curr Opin Plant Biol 6:390–396PubMedCrossRefGoogle Scholar
  18. Lin C, Thomashow MF (1992) A cold-regulated Arabidopsis gene encodes a polypeptide having potent cryoprotective activity. Biochem Biophys Res Commun 183:1103–1108PubMedCrossRefGoogle Scholar
  19. McConn M, Hugly S, Browse J, Somerville C (1994) A mutation at the fad8 locus of Arabidopsis identifies a second chloroplast ω-3 desaturase. Plant Physiol 106:1609–1614PubMedGoogle Scholar
  20. Metting B, Zimmerman WJ, Crouch T, van-Staden J (1990) Agronomic uses of seaweed and microalgae. In: Akatsuka I (ed) Introduction of applied phycology. SPB Academic, Hague, pp 589–627Google Scholar
  21. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  22. Nabati DA (1991) Response of two grass species to plant growth regulators, fertilizer N, chelated Fe, salinity and water stress. Ph.D. dissertation. Virginia Polytechnic Institute and State University, BlacksburgGoogle Scholar
  23. Nabati DA, Schmidt RE, Parrish DJ (1994) Alleviation of salinity stress in Kentucky bluegrass by plant growth regulators and iron. Crop Sci 34:198–202CrossRefGoogle Scholar
  24. Nakayama K, Okawa K, Kakizaki T, Honma T, Itoh H, Inaba T (2007) Arabidopsis COR15A is a chloroplast stromal protein that has cryoprotective activity and forms oligomers. Plant Physiol 144:513–523PubMedCrossRefGoogle Scholar
  25. Nishida I, Murata N (1996) Chilling sensitivity in plants and Cyanobacteria: the crucial contribution of membrane lipid. Annu Rev Plant Physiol Plant Mol Biol 47:541–568PubMedCrossRefGoogle Scholar
  26. Rate DN, Cuenca JV, Bowman GR, Guttman DS, Greenberg JT (1999) The gain-of-function Arabidopsis acd6 mutant reveals novel regulation and function of the salicylic acid signaling pathway in controlling cell death, defenses, and cell growth. Plant Cell 11:1695–1708PubMedCrossRefGoogle Scholar
  27. Sharma P, Sharma N, Deswal R (2005) The molecular biology of the low-temperature response in plants. Bioessays 27:1048–1059PubMedCrossRefGoogle Scholar
  28. Spurlock BO, Skinner MS, Kattine AA (1966) A simple rapid method for staining epoxy- embedded specimens for light microscopy with the polychromatic stain paragon-1301. Am J Clin Path 46:252PubMedGoogle Scholar
  29. Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31PubMedCrossRefGoogle Scholar
  30. Steponkus PL, Uemura M, Joseph RA, Gilmour SJ, Thomashow MF (1998) Mode of action of the COR15A gene on the freezing tolerance of Arabidopsis thaliana. Proc Natl Acad Sci USA 95:14570–14575PubMedCrossRefGoogle Scholar
  31. Stockinger EJ, Gilmour SJ, Thomashow MF (1997) Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcription activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci USA 94:1035–1040PubMedCrossRefGoogle Scholar
  32. Temple WD, Bomke AA (1989) Effects of Kelp (Macrocystis integrifolia and Eklonia maxima) foliar applications on bean crop growth. Plant Soil 117:85–92CrossRefGoogle Scholar
  33. Thomashow MF (1994) Arabidopsis thaliana as a model for studying mechanisms of plant cold tolerance. In: Meyerowitz E, Somerville C (eds) Arabidopsis. Cold Spring Harbor Laboratory Press, New York, pp 807–834Google Scholar
  34. Thomashow MF (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol Plant Mol Biol 50:571–599PubMedCrossRefGoogle Scholar
  35. Upchurch RG (2008) Fatty acid unsaturation, mobilization, and regulation in the response of plants to stress. Biotechnol Lett 30:967–977PubMedCrossRefGoogle Scholar
  36. Wilson S (2001) Frost management in cool climate vineyards. In: University of Tasmania Research Report—UT 99/1. Grape and Wine Research and Development CorporationGoogle Scholar
  37. Xin Z, Browse B (1998) Eskimo1 mutants of Arabidopsis are constitutively freezing-tolerant. Proc Natl Acad USA 95:7799–7804CrossRefGoogle Scholar
  38. Yamaguchi-Shinozaki K, Shinozaki K (1993) Arabidopsis DNA encoding two desiccation-responsive rd29 genes. Plant Physiol 101:1119–1120PubMedCrossRefGoogle Scholar
  39. Yan J (1993) Influence of plant growth regulators on turfgrass polar lipid composition, tolerance to drought and salinity stresses, and nutrient efficiency. Ph.D. dissertation. Virginia Polytechnic Institute and State University, BlacksburgGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Prasanth Rayirath
    • 1
  • Bernhard Benkel
    • 1
  • D. Mark Hodges
    • 2
  • Paula Allan-Wojtas
    • 2
  • Shawna MacKinnon
    • 3
  • Alan T. Critchley
    • 4
  • Balakrishnan Prithiviraj
    • 1
    Email author
  1. 1.Department of Plant and Animal SciencesNova Scotia Agricultural CollegeTruroCanada
  2. 2.Atlantic Food and Horticulture Research CentreAgriculture and Agri-Food CanadaKentvilleCanada
  3. 3.Institute for Marine BiosciencesNational Research Council of CanadaHalifaxCanada
  4. 4.Acadian Seaplants LimitedDartmouthCanada

Personalised recommendations