Abstract
Adenylate levels in chloroplasts, mitochondria and the cytosol of oat mesophyll protoplasts were determined under light and dark conditions, in the absence and presence of plasmalemma-permeable inhibitors of electron transfer and uncouplers of phosphorylation. This was achieved using a microgradient technique which allowed an integrated homogenization and fractionation of protoplasts within 60 s (Hampp et al. 1982, Plant Physiol. 69, 448–455), under conditions which quench bulk activities of metabolic interconversion in less than 2 s. In illuminated controls, ATP/ADP ratios were found to be 2.1 in chloroplasts, about unity in mitochondria, and 11 in the cytosol; whereas, in the dark, this ratio only showed a large drop in chloroplasts (0.4). None of the compounds used [carbonylcyanide m-chlorophenylhydrazone (CCCP), carbonylcyanide p-trifluoromethoxy-phenylhydrazone (FCCP), antimycin A, dibromothymoquinone (DBMIB), dichlorophenyldi-methylurea (DCMU), or salicylhydroxamic acid (SHAM)] affected the stroma adenylate ratio in the dark. Under illumination, however, the ATP/ADP ratios were partly reduced in the presence of antimycin (inhibitor of cyclic photophosphorylation) and of DCMU (inhibitor of linear electron flow), while in the presence of DBMIB, DCMU+ antimycin (inhibition of both cyclic and linear electron flow), and CCCP (uncoupling) the ratio obtained was the same as that occurring in the dark. In contrast, mitochondrial adenylate levels did not exhibit large variations under the various treatments. The cytosolic ATP/ADP ratio, however, showed dramatic changes: in darkened protoplasts, cytosolic values dropped to 0.2 and 0.1 in the presence of uncouplers and antimycin, respectively, while SHAM did not induce any significant alteration. In the light, a similar pronounced decrease in ATP levels was observed only after the application of uncouplers or inhibitors of both mitochondrial and photosynthetic electron transport, whereas selective inhibition of the latter was largely ineffective in reducing the cytosolic ATP/ADP ratio. Thus, the results show that the antimycin-sensitive electron transport is, potentially, equally active in light and darkness. In addition, they indicate that antimycin-insensitive electron transport in mitochondria (alternative pathway) does not significantly contribute to the cytosolic energy state.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- CCCP:
-
carbonylcyanide m-chlorophenylhydrazone
- DBMIB:
-
dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropy-p-benzoquinone)
- DCMU:
-
dichlorophenyldimethylurea
- FCCP:
-
carbonylcyanide-p-trifluoromethoxy-phenylhydrazone
- SHAM:
-
sancylhydroxamic acid
References
Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1–15
Arnon, D.I. (1969) Role of ferredoxin in photosynthesis. Naturwissenschaften 56, 295–305
Berden, J.A., Slater, E.C. (1972) The allosteric binding of antimycin to cytochrome b in the mitochondrial membrane. Biochim. Biophys. Acta 256, 199–215
Crowther, D., Hind, G. (1980) Partial characterisation of cyclic electron transport in intact chloroplasts. Arch. Biochem. Biophys. 204, 568–577
Day, D.A., Arron, G.P., Laties, G.G. (1980) Nature and control of respiratory pathways in plants: the interaction of cyanide-resistant respiration with the cyanide-sensitive pathway. In: The biochemistry of plants, vol. 2, pp. 198–241, Davies, D.D., ed. Academic Press, New York London
Delieu, T., Walker, D.A. (1972) An improved cathode for the measurement of photosynthetic oxygen evolution by isolated chloroplasts. New Phytol. 71, 201–225
Graham, D. (1980) Effects of light on “dark respiration”. In: The biochemistry of plants, vol. II, pp. 525–579, Davies, D.D., ed. Academic Press, New York London
Graham, D., Chapman, E.A. (1979) Interactions between photosynthesis and respiration in higher plants. In: Encyclopedia of plant physiology, N.S., vol. 6: Photosynthesis II, pp. 150–162, M. Gibbs, E. Latzko, eds. Springer, Berlin Heidelberg New York
Hampp, R. (1980) Rapid separation of the plastid, mitochondrial and cytoplasmic fractions from intact leaf protoplasts of Avena. Planta 150, 291–298
Hampp, R., Goller, M., Ziegler, H. (1982) Adenylate levels, energy charge and phosphorylation potential during dark-light and light-dark transition in chloroplasts, mitochondria and cytosol of mesophyll protoplasts from Avena sativa L. Plant Physiol. 69, 448–455
Hampp, R., Wellburn, A.R. (1980) Translocation and phosphorylation of adenine nucleotides by mitochondria and plastids during greening. Z. Pflanzenphysiol. 98, 289–303
Hampp, R., Ziegler, H. (1980) On the use of Avena protoplasts to study chloroplast development. Planta 147, 485–494
Heber, U. (1974) Metabolite exchange between chloroplasts and cytoplasm. Annu. Rev. Plant Physiol. 25, 393–421
Heber, U. (1975) Energy transfer within leaf cells. In: Proc. 3rd Int. Congr. Photosynthesis, vol. II, pp. 1335–1348, Avron, M., ed. Elsevier, Amsterdam
Heber, U., Heldt, H.W. (1981) The chloroplast envelope: structure, function, and role in leaf metabolism. Annu. Rev. Plant Physiol. 32, 139–168
Heber, U., Santarius, K.A. (1970) Direct and indirect transfer of ATP and ADP across the chloroplast envelope. Z. Naturforsch. 25b, 718–728
Heldt, H.W. (1976) Metabolite carriers of chloroplasts. In: Encyclopedia of plant physiology, N.S., vol. 3: Transport in plants III, pp. 137–143, Stocking, C.R., Heber, U., eds. Springer, Berlin Heidelberg New York
Heldt, H.W., Klingenberg, M., Milovancev, M. (1972) Differences between the ATP/ADP ratios in the mitochondrial matrix and in the extramitochondrial space. Eur. J. Biochem. 30, 434–440
Heytler, P.G. (1963) Uncoupling of oxidative phosphorylation by carbonyl cyanide phenylhydrazons. I. Some characteristies of m-ClCCP action on mitochondria and chloroplasts. Biochemistry 2, 357–361
Hopfer, U., Lehninger, A.L., Thompson, T.E. (1968) Protonic conductance across phospholipid bilayer membranes induced by uncoupling agents for oxidative phosphorylation. Proc. Natl. Acad. Sci. USA 59, 484–490
Izawa, S. (1977) Inhibitors of electron transport. In: Encyclopedia of plant physiology, N.S., vol. 5: Photosynthesis I, pp. 266–282, Trebst, A., Avron, M., eds. Springer, Berlin Heidelberg New York
Kaiser, W., Urbach, W. (1976) Rates and properties of endogenous cyclic photophosphorylation of isolated intact chloroplasts measured by CO2 fixation in the presence of dihydroxyacetone phosphate. Biochim. Biophys. Acta 423, 91–102
Krause, G.H., Heber, U. (1976) Energetics of intact chloroplasts. In: The intact chloroplast, pp. 171–214, Barber, J., ed. Elsevier, Amsterdam
Kuylenstierna, B., Nicholls, D.G., Hovmoller, S., Ernster, L. (1970) Effect of trypsin on mitochondrial and microsomal enzymes. Eur. J. Biochem. 12, 419–426
Loschen, G., Azzi, A. (1974) Dibromothymoquinone: a new inhibitor of mitochondrial electron transport at the level of ubiquinone. FEBS Lett. 41, 115–117
Meeuse, B.J.D. (1975) Thermogenic respiration in aroids. Annu. Rev. Plant Physiol. 26, 117–126
Mills, J.D., Slovacek, R.E., Hind, G. (1978) Cyclic electron transport in isolated intact chloroplasts. Further studies with antimycin. Biochim. Biophys. Acta 504, 298–309
Santarius, K.A., Heber, U. (1965) Changes in the intracellular levels of ATP, ADP, AMP and Pi and regulatory function of the adenylate system in leaf cells during photosynthesis. Biochim. Biophys. Acta 102, 39–54
Schonbaum, G.S., Bonner, W.D., Storey, B.T., Bahr, J.T. (1971) Specific inhibition of the cyanide-insensitive respiratory pathway in plant mitochondria by hydroxamic acids. Plant Physiol. 47, 124–128
Schwenke, U.-D., Soboll, S., Seitz, H.J., Sies, H. (1981) Mitochondrial and cytosolic ATP/ADP ratios in rat livers in vivo. Biochem. J. 200, 405–408
Shahak, Y., Crowther, D., Hind, G. (1980) Endogenous cyclic electron transport in broken chloroplasts. FEBS Lett. 114, 73–78
Siedow, J.N., Huber, S.C., Moreland, D.E. (1979) Effects of dibromothymoquinone on mung bean mitochondrial electron transfer and membrane fluidity. Biochim. Biophys. Acta 547, 282–295
Slater, E.C. (1973) The mechanism of action of the respiratory inhibitor, antimycin. Biochim. Biophys. Acta 301, 129–154
Slovacek, R.E., Crowther, D., Hind, G. (1979) Cytochrome function in the cyclic electron transport pathway of chloroplasts. Biochim. Biophys. Acta 547, 138–148
Soboll, S., Scholz, R., Heldt, H.W. (1978) Subcellular metabolite concentrations. Dependence of mitochondrial and cytosolic ATP systems on the metabolic state of perfused rat liver. Eur. J. Biochem. 87, 377–390
Tagawa, K., Tsujimoto, H., Arnon, D.I. (1963) Role of chloroplast ferredoxin in the energy conversion process of photosynthesis. Proc. Natl. Acad. Sci. USA 49, 567
Trebst, A., Harth, E., Draber, W. (1970) On a new inhibitor of photosynthetic electron transport in isolated chloroplasts. Z. Naturforsch. 25b, 1157–1159
Walker, D.A. (1976) CO2 fixation by intact chloroplasts: photosynthetic induction and its relation to transport phenomena and control mechanisms. In: The intact chloroplasts, pp. 235–278, Barber, J., ed. Elsevier, Amsterdam
Wessels, J.S.C., Van der Veen, R. (1956) The action of some derivatives of phenylurethan and of 3-phenyl-1,1-dimethylurea on the Hill reaction. Biochim. Biophys. Acta 19, 548–549
Wirtz, W., Stitt, M., Heldt, H.W. (1980) Enzymic determination of metabolites in the subcellular compartments of spinach protoplasts. Plant Physiol. 66, 187–193
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Goller, M., Hampp, R. & Ziegler, H. Regulation of the cytosolic adenylate ratio as determined by rapid fractionation of mesophyll protoplasts of oat. Planta 156, 255–263 (1982). https://doi.org/10.1007/BF00393733
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00393733