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Biologia Plantarum

, 38:229 | Cite as

Enzymatic determination of ascorbic acid in leaf cell walls using acidic buffer during infiltration

  • H. Moldau
  • I. Bichele
  • H. Kollist
  • E. Padu
Original Paper

Abstract

A modification of the procedure of extraction of cell wall solution for enzymatic determination of ascorbic acid and its reduction level in the apoplast of leaf cells is proposed. The modification consists in infiltration of leaves with citric acid/sodium phosphate buffer, pH 3, instead of customarily used neutral solutions. In acidic media autooxidation of ascorbic acid is effectively suppressed, so that infiltration could be performed at laboratory temperatures. Using polyacrylamide gel electrophoresis and infiltration solutions of pH down to 1.5 it is shown, that at pH 3 the extracted fluid is not contaminated with intracellular substances if appropriate vacuum and centrifugation forces are used. The modification is shown to be more effective for leaves ofPhaseolus than for those ofSpinacia. In cell walls of mature leaves of these species the concentration of ascorbic acid was found to be around 1 mM, with reduction level up to 0.90. The role of ascorbic acid in cell walls as ozone scavenger is discussed

Additional key words

ascorbate oxidase ozone susceptibility Phaseolus vulgaris Spinacia oleracea 

Abbreviations

AA

ascorbic acid

AAO

ascorbate oxidase

AM

acidic method

DHA

dehydroascorbate

DTT

dithiothreitol

EDTA

ethylenediaminetetraacetic acid

IWF

intercellular washing fluid

NM

neutral method

PAGE

polyacrylamide gel electrophoresis

PPFD

photosynthetic photon flux density

RL

reduction level of AA

SU

solute uptake by infiltration

References

  1. Arrigoni, O., De Gara, L., Tommasi, F., Liso, R.: Changes in the ascorbate system during seed development ofVicia faba L. Plant. Physiol.99: 235–238, 1992.PubMedGoogle Scholar
  2. Castillo, F.J., Greppin, H.: Extracellular ascorbic acid and enzyme activities related to ascorbic acid metabolism inSedum album L. leaves after ozone exposure. Environ, exp. Bot.28: 231–238, 1988.CrossRefGoogle Scholar
  3. Castillo, F.J., Miller, P.R., Greppin, H.: Extracellular biochemical markers of photochemical oxidant air pollution damage to Norway spruce. Experientia43: 111–115, 1987.CrossRefGoogle Scholar
  4. Chameides, W.L.: The chemistry of ozone deposition to plant leaves: role of ascorbic acid. Environ. Sci. Technol.23: 595–600, 1989.CrossRefGoogle Scholar
  5. Chevrier, N., Sarhan, F.: Effect of ozone on energy metabolism and its relation to carbon dioxide fixation inEuglena gradlis. J. Plant Physiol.140: 521–526, 1992.Google Scholar
  6. Eckey-Kaltenbach, H., Heller, W., Sonnenbichler, J., Zetl, I., Schafer, W., Ernst, D., Sandermann, H.: Oxidative stress and plant secondary metabolism: 6-o-malonylapiin in parsley. Phytochemistry34: 687–691, 1993.CrossRefGoogle Scholar
  7. Giamalva, D., Church, D.F., Pryor W.A.: A comparison of the rates of ozonation of biological antioxidants and oleate and linoleate esters. Biochem. biophys. Res. Commun.133: 773–779, 1985.PubMedCrossRefGoogle Scholar
  8. Jaaska, V., Jaaska, V.: Isoenzyme differentiation between Asian beansVigna radiata andV. mungo. Biochem. Physiol. Pflanz.185: 41–53, 1989.Google Scholar
  9. Heath, R.L.: Biochemical mechanisms of pollutant stress. In: Heck, W.W., Taylor, O.C., Tingey, O.C. (ed.): Assessmen t of Crop Loss from Air Pollutants. Pp. 259–286. Elsevier Applied Science, London 1988.Google Scholar
  10. Kimoto, E., Tanaka, H., Ohmoto, T., Choami, M.: Analysis of the transformation products of dehydro-L-ascorbic acid by ion-pairing high-performance liquid chromatography. Anal. Biochem.214: 38–44, 1993.PubMedCrossRefGoogle Scholar
  11. Luwe, M.: Zur Entgiftung von Ozon im Blatt durch Antioxidanten unter besonderer Berücksichtigung des Apoplasten. Dissertation Thesis. University of Würzburg, Würzburg 1994.Google Scholar
  12. Luwe, M.W.F., Takahama, U., Heber, U.: Role of ascorbate in detoxifying ozone in the apoplast of spinach (Spinacia oleracea L.) leaves. Plant Physiol.101: 969–976, 1993.PubMedGoogle Scholar
  13. Okamura, M.: An improved method for determination of L-ascorbic acid and L-dehydroascorbic acid in blood plasma. Clin. chim. Acta103: 259–268, 1980.PubMedCrossRefGoogle Scholar
  14. Omasa, K., Abo, F., Natori, T., Totsuka, T.: Analysis of air pollutant sorption by plants. (3) Sorption under fumigation with NO2, O3 or NO2+O3. Res. Rep. nat. Inst. environ. Stud.11: 195–224, 1980.Google Scholar
  15. Polle, A., Rennenberg, H., Chakrabarti, K.: Extracellular and intracellular scavenging of toxic oxygen species in needles of Norway spruce. Physiol. Plant.94: 312–319, 1990.Google Scholar
  16. Sereikaite, J., Iljaseviciene, D., Dienys, G., Danilcenko, H., Gavrilova, V.: Ascorbate oxidase. Specificity and analytical application. Appl. Biochem. Biotechnol.43: 153–160, 1993.Google Scholar
  17. Speer, M., Kaiser, W.M.: Ion relations of symplastic and apoplastic space in leaves fromSpinacia andPisum under salinity. Plant Physiol.97: 990–997, 1991.PubMedCrossRefGoogle Scholar
  18. Takahama, U.: Redox state of ascorbic acid in the apoplast of stems ofKalanchoe daigremontiana. Physiol. Plant.89: 791–798, 1993.CrossRefGoogle Scholar
  19. Takahama, U., Oniki, T.: Regulation of peroxidase-dependent oxidation of phenolics in the apoplast of spinach leaves by ascorbate. Plant Cell Physiol.33: 379–387, 1992.Google Scholar
  20. Turner, N.C.: Use of the pressure chamber in membrane damage studies. J. exp. Bot.27: 1085–1092, 1976.CrossRefGoogle Scholar
  21. Wendel, J.F., Weeden, N.F.: Visualization and interpretation of plant isozymes. In: Soltis, D.E., Soltis, P.S. (ed): Isozymes in Plant Biology. Pp. 5–45. Dioscorides Press, Portland 1989.Google Scholar

Copyright information

© Institute of Experimental Botany, ASCR 1996

Authors and Affiliations

  • H. Moldau
    • 1
  • I. Bichele
    • 1
  • H. Kollist
    • 1
  • E. Padu
    • 1
  1. 1.Institute of Molecular and Cell BiologyUniversity of TartuTartu

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