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. 1982 Feb;69(2):448–455. doi: 10.1104/pp.69.2.448

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. 1

Rüdiger Hampp 1, Marion Goller 1, Hubert Ziegler 1
PMCID: PMC426228  PMID: 16662227

Abstract

The compartmentation of cellular energy relations during dark-light and light-dark transitions was studied by means of a newly developed technique to fractionate oat (Avena sativa L., var. Arnold) mesophyll protoplasts. Using an improved microgradient system with hydrophobic and hydrophilic layers of increasing density, a pure plastid pellet (up to 90% of total chloroplasts) could be separated from an interphase of only slightly contaminated mitochondria (70 to 80% of total mitochondria), and a cytoplasmic supernatant could be obtained within 60 seconds. Appropriate controls indicate that, under the conditions employed, metabolic interconversions of adenylates can be kept to a minimum and, thus, be determined and corrected for. Cross contamination of the fractions, as well as liberation of organelles to the supernatant, was assessed by specific markers, and the metabolite levels recorded were corrected accordingly. Using this technique, we found that, during dark-light transition, the chloroplastic and cytosolic ATP exhibits a rapid increase, while the mitochondrial ATP level decreases. In all compartments, ADP levels mirror alterations of the ATP pool in the opposite way, at least to some extent. To compensate fully for the rise in ATP, chloroplastic and mitochondrial AMP levels change accordingly, indicating that, due to the more or less unchanged level of total adenylates, there is no net flux of adenylates between the compartments. In contrast to the organelles, no AMP could be detected within the cytosol. When the light is turned off, a decrease of ATP coincides between chloroplast stroma and the cytosol for only about 30 seconds. Under prolonged dark treatment, cytosolic ATP rises again, while stroma ATP levels exhibit a further decrease. After about 60 seconds of darkness, the cytosolic ATP level is back to its initial value. This obviously is due to the immediate rise in mitochondrial ATP upon darkening, which cumulates after about 60 seconds; then, caused by an ATP/ADP exchange with the cytosol, it levels off again at the state before changing the conditions, as soon as the cytosolic ATP is also back to its original level. All of these events are closely mirrored by the change in the ATP/ADP ratio and the energy charge within the compartments. While the values for chloroplasts exhibit considerable differences between dark and light, those calculated for mitochondria and the cytosol exhibit only transient changes. These are limited to about 60 seconds of undershoot or overshoot, with respect to the cytosol, and then return to nearly the levels observed before changing the conditions. Adenylate kinase was found to be exclusively associated with chloroplasts (90% of total activity level) and mitochondria. Isotonic liberation of vacuoles did not point toward a significant association of adenylates with this compartment.

The results are discussed with respect to an effective collaboration between photosynthetic and oxidative phosphorylation in order to keep the cytosolic energy state at a constant, preset value.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arnon D. I. COPPER ENZYMES IN ISOLATED CHLOROPLASTS. POLYPHENOLOXIDASE IN BETA VULGARIS. Plant Physiol. 1949 Jan;24(1):1–15. doi: 10.1104/pp.24.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Atkinson D. E. The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry. 1968 Nov;7(11):4030–4034. doi: 10.1021/bi00851a033. [DOI] [PubMed] [Google Scholar]
  3. Bomsel J. L., Pradet A. Study of adenosine 5'-mono-,di- and triphosphates in plant tissues. IV. Regulation of the level of nucleotides, in vivo, by adenylate kinase: theoretical and experimental study. Biochim Biophys Acta. 1968 Aug 20;162(2):230–242. doi: 10.1016/0005-2728(68)90105-9. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Davis E. J., Davis-van Thienen W. I. Control of mitochondrial metabolism by the ATP/ADP ratio. Biochem Biophys Res Commun. 1978 Aug 29;83(4):1260–1266. doi: 10.1016/0006-291x(78)91357-8. [DOI] [PubMed] [Google Scholar]
  6. Dürr M., Boller T., Wiemken A. Polybase induced lysis of yeast spheroplasts. A new gentle method for preparation of vacuoles. Arch Microbiol. 1975 Nov 7;105(3):319–327. doi: 10.1007/BF00447152. [DOI] [PubMed] [Google Scholar]
  7. Giersch C., Heber U., Kobayashi Y., Inoue Y., Shibata K., Heldt H. W. Energy charge, phosphorylation potential and proton motive force in chloroplasts. Biochim Biophys Acta. 1980 Mar 7;590(1):59–73. doi: 10.1016/0005-2728(80)90146-2. [DOI] [PubMed] [Google Scholar]
  8. Harris D. A., Slater E. D. Tightly bound nucleotides of the energy-transducing ATPase of chloroplasts and their role in photophosphorylation. Biochim Biophys Acta. 1975 May 15;387(2):335–348. doi: 10.1016/0005-2728(75)90114-0. [DOI] [PubMed] [Google Scholar]
  9. Heber U., Santarius K. A. Direct and indirect transfer of ATP and ADP across the chloroplast envelope. Z Naturforsch B. 1970 Jul;25(7):718–728. doi: 10.1515/znb-1970-0714. [DOI] [PubMed] [Google Scholar]
  10. Heldt H. W., Klingenberg M., Milovancev M. Differences between the ATP-ADP ratios in the mitochondrial matrix and in the extramitochondrial space. Eur J Biochem. 1972 Nov 7;30(3):434–440. doi: 10.1111/j.1432-1033.1972.tb02115.x. [DOI] [PubMed] [Google Scholar]
  11. Heldt H. W., Sauer F. The inner membrane of the chloroplast envelope as the site of specific metabolite transport. Biochim Biophys Acta. 1971 Apr 6;234(1):83–91. doi: 10.1016/0005-2728(71)90133-2. [DOI] [PubMed] [Google Scholar]
  12. Inoue Y., Kobayashi Y., Shibata K., Heber U. Synthesis and hydrolysis of ATP by intact chloroplasts under flash illumination and in darkness. Biochim Biophys Acta. 1978 Oct 11;504(1):142–152. doi: 10.1016/0005-2728(78)90013-0. [DOI] [PubMed] [Google Scholar]
  13. Kringstad R., Kenyon W. H., Black C. C. The rapid isolation of vacuoles from leaves of crassulacean Acid metabolism plants. Plant Physiol. 1980 Sep;66(3):379–382. doi: 10.1104/pp.66.3.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Moore A. L., Bonner W. D., Jr A comparison of the phosphorylation potential and electrochemical proton gradient in mung bean mitochondria and phosphorylating sub-mitochondrial particles. Biochim Biophys Acta. 1981 Jan 14;634(1):117–128. doi: 10.1016/0005-2728(81)90132-8. [DOI] [PubMed] [Google Scholar]
  15. Santarius K. A., Heber U. Changes in the intracellular levels of ATP, ADP, AMP and P1 and regulatory function of the adenylate system in leaf cells during photosynthesis. Biochim Biophys Acta. 1965 May 25;102(1):39–54. doi: 10.1016/0926-6585(65)90201-3. [DOI] [PubMed] [Google Scholar]
  16. Schmidt R., Poole R. J. Isolation of protoplasts and vacuoles from storage tissue of red beet. Plant Physiol. 1980 Jul;66(1):25–28. doi: 10.1104/pp.66.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Wirtz W., Stitt M., Heldt H. W. Enzymic determination of metabolites in the subcellular compartments of spinach protoplasts. Plant Physiol. 1980 Jul;66(1):187–193. doi: 10.1104/pp.66.1.187. [DOI] [PMC free article] [PubMed] [Google Scholar]

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