Abstract
Mitochondria were purified from tomato leaves by a differential centrifugation method and a Percoll density-gradient method. A comparative study on the ascorbate-glutathione cycle activity revealed that there was no difference in the specific activity of the antioxidative enzymes and ascorbate and glutathione concentrations between mitochondria prepared by these two methods. However, the contents of dehydroascorbic acid and oxidised glutathione in Percoll-separated organelles were 260 and 210 %, respectively, of those found in mitochondria purified by the differential centrifugation method.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bartoli, C.G., Pastori, G.M., Foyer, C.H.: Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV.-Plant Physiol. 123: 335-343, 2000.
Bergman, A., Gardeström, P., Ericson, I.: Method to obtain a chlorophyll-free preparation of intact mitochondria from spinach leaves.-Plant Physiol. 66: 442-445, 1980.
Blokhina, O., Virolainen, E., Fagerstedt, K.V.: Antioxidants, oxidative damage and oxygen deprivation stress: a review.-Ann. Bot. 91: 179-194, 2003.
Bradford, M.M.: A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.-Anal. Biochem. 72: 248-254, 1976.
Burgess, N., Beakes, G.W., Thomas, D.R.: Separation of mitochondria from microbodies of Pisum sativum L. cv. Alaska cotyledones.-Planta 166:151-155, 1985.
Corpas, F.J., Barroso, J.B., Sandalio, L.M., Distefano, S., Palma, J.M., Lupiáñez, J.A.: A dehydrogenase-mediated recycling system of NADPH in plant peroxisomes.-Biochem. J. 330: 777-784, 1998.
Douce, R., Christensen, E.L., Bonner, W.D., Jr.: Preparation of intact plant mitochondria.-Biochim. biophys. Acta 275: 148-160, 1972.
Drążkiewicz, M., Skórzyóska-Polit, E. Krupa, Z.: Response of ascorbate-glutathione cycle to excess copper in Arabidopsis thaliana (L.).-Plant Sci. 164: 195-202, 2003.
Hernández, J.A., Corpas, F.J., Gómez, M., Del Río, L.A.: Salt-induced oxidative stress mediated by activated oxygen species in pea leaf mitochondria.-Plant Physiol. 89: 103-110, 1993.
Jiménez, A., Hernández, J.A., Del Río, L.A., Sevilla, F.: Evidence for the presence of the ascorbate-glutathione cycle in mitochondria and peroxisomes of pea leaves.-Plant Physiol. 114: 275-284, 1997.
Jones, A.: Does the plant mitochondrion integrate cellular stress and regulate programmed cell death?-Trends Plant Sci. 5: 225-230, 2000.
Karpinski, S., Escobar, C., Karpinska, B., Creissen, G., Mullineaux, P.M.: Photosynthetic electron transport regulates the expression of cytosolic ascorbate peroxidase genes in Arabidopsis during excess light stress.-Plant Cell 9: 627-640, 1997.
Kuźniak, E., Skłodowska, M.: The effect of Botrytis cinerea infection on ascorbate-glutathione cycle in tomato leaves.-Plant Sci. 148: 69-76, 1999.
Kuźniak, E., Skłodowska, M.: Ascorbate, glutathione and related enzymes in chloroplasts of tomato leaves infected by Botrytis cinerea.-Plant Sci. 160: 723-731, 2001.
Liang, Z., Yu, C., Huang, A.H.C.: Isolation of spinach leaf peroxisomes in 0.25 molar sucrose solution by Percoll density gradient centrifugation.-Plant Physiol. 70: 1210-1212, 1982.
Mittova, V., Volokita, M., Tal, M.: Activities of SOD and the ascorbate-glutathione cycle enzymes in subcellular compartments in leaves and roots of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii.-Physiol. Plant. 110: 42-51, 2000.
Møller, I.M.: Plant mitochondria and oxidative stress: Electron transport, NADPH turnover, and metabolism of reactive oxygen species.-Annu. Rev. Plant Physiol. Plant mol. Biol. 52: 561-591, 2001.
Nishimura, M., Douce, R., Akazawa, T.: Isolation and characterization of metabolically competent mitochondria from spinach leaf protoplasts.-Plant Physiol. 69: 916-920, 1982.
Peckmann, K., Herpich, W.B.: Effects of short-term drought and rewatering on the activity of mitochondrial enzymes and the oxidative capacity of leaf mitochondria from a CAM plant, Aptenia cordifolia.-J. Plant Physiol. 152: 518-524, 1998.
Petit, P.X., Edman, K.A., Gardeström, P., Ericson, I.: Some properties of mitochondria, mitoplasts and submitochondrial particles of different polarities from plant tissues.-Biochim. biophys. Acta 890: 377-386, 1987.
Porra, J.R., Thompson, W.A., Kiedermann, P.E.: Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy.-Biochim. biophys. Acta 975: 384-394, 1989.
Rasmusson, A.G., Møller, I.M.: NADP-utilizing enzymes in the matrix of plant mitochondria.-Plant Physiol. 94: 1012-1018, 1990.
Sandalio, L.M., Palma, J.M., Del Río, L.A.: Localization of manganese superoxide dismutase in peroxisomes isolated from Pisum sativum L.-Plant Sci. 51: 1-8, 1987.
Schwitzguebel, J.-P., Siegenthaler, P.-A.: Purification of peroxisomes and mitochondria from spinach leaf by Percoll gradient centrifugatuion.-Plant Physiol. 75: 670-674, 1984.
Struglics, A., Fredlund, K.M., Rasmusson, A.G., Møller, I.M.: The presence of a short redox chain in the membrane of intact potato tuber peroxisomes and the association of malate dehydrogenase with peroxisomal membrane.-Physiol. Plant. 88: 19-28, 1993.
Tiwari, B.S., Belenghi, B., Levine, A.: Oxidative stress increased respiration and generation of reactive oxygen species, resulting in ADP depletion, opening mitochondrial permeability transition, and programmed cell death.-Plant Physiol. 128: 1271-1281, 2002.
Veljovic-Jovanovic, S.D., Pignocchi, C., Noctor, G., Foyer, C.H.: Low ascorbic acid in the vtc-1 mutant of Arabidopsis is associated with decreased growth and intracellular redistribution of the antioxidant system.-Plant Physiol. 127: 426-435, 2001.
Virolainen, E., Blokhina, O., Fagerstedt, K.: Ca 2+-induced high amplitude swelling and cytochrome c release from wheat (Triticum aestivum L.) mitochondria under anoxic stress.-Ann. Bot. 90: 509-516, 2002.
Warrilow, A.G.S., Hawkesford, M.J.: Separation, subcellular location and influence of sulphur nutrition on isoforms of cysteine synthase in spinach.-J. exp. Bot. 49: 1625-1636, 1998.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kuźniak, E., Skłodowska, M. Comparison of Two Methods for Preparing Mitochondria from Tomato Leaves to Study the Ascorbate-Glutathione Cycle Activity. Biologia Plantarum 48, 537–542 (2004). https://doi.org/10.1023/B:BIOP.0000047149.57946.a5
Issue Date:
DOI: https://doi.org/10.1023/B:BIOP.0000047149.57946.a5