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Institut für Pflanzengenetik
Logo Leibniz Universität Hannover
Institut für Pflanzengenetik
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Research Projects

1. Supramolecular structure of the respiratory chain in mitochondria

Prerequisite for oxidative phosphorylation (OXPHOS) in mitochondria are five protein complexes termed NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), cytochrome c reductase (complex III), cytochrome c oxidase (complex IV), and ATP synthase (complex V). Currently there is some controversy whether the OXPHOS complexes can freely diffuse within the inner mitochondrial membrane (liquid state model) or rather form defined supramolecular structures called “respiratory supercomplexes” or “respirasomes” (solid state model). Using very gentle biochemical procedures, several types of supercomplexes have been identified in higher plants and green alga: (i) a I+III2 supercomplex, (ii) III2+IV1-2 supercomplexes, (iii) I+III2+IV1-4 supercomplexes and (iv) dimeric ATP synthase [1][2][3]. The structures of several of these supercomplexes were resolved for the first time using electron microscopy in combination with single particle analysis [3][4][5]. ATP synthase monomers interact within the dimeric ATP synthase supercomplex in a way that bends the inner mitochondrial membrane. This bending is believed to be important for cristae formation of the inner mitochondrial membrane [5][6]. The function of respiratory supercomplexes is currently characterized in our laboratory.

2. Special functions of respiratory protein complexes in plant mitochondria

The respiratory chain of plant mitochondria has several special features: (i) respiratory electron transport is very much branched due to the presence of numerous so-called “alternative” oxidoreductases, (ii) the mitochondrial genomes of plants code for more subunits of respiratory protein complexes than those of heterotrophic eukaryotes which requires special assembly pathways for protein complexes and (iii) the respiratory protein complexes contain additional subunits that allow them to catalyse secondary or modified functions. A project was started to systematically characterize the subunits of OXPHOS complexes in plants, which is based on protein separations by two-dimensional Blue-native / SDS polyacrylamide gel electrophoresis and protein identifications by Edman degradation and mass spectrometry [1][7][8]. More than 20 plant specific subunits could be discovered. The functions of these extra-proteins were studied by physiological measurements and by reverse genetics. Complex III of plants includes the mitochondrial processing peptidase [9][10][11] and complex I includes an acyl carrier protein, carbonic anhydrases and a galactonolactone dehydrogenase [12][13][14]. The complex I-integrated carbonic anhydrases were recently found to be important for CO2 recycling between mitochondria and chloroplasts during photorespiration [15]. This mechanisms is currently further investigated in our laboratory.

3. Proteomic approach to investigate mitochondrial functions in plants

An Arabidopsis mitochondrial proteome project was started for a comprehensive investigation of mitochondrial functions in plants. Plant mitochondria comprise a large number of special functions, which partially are a consequence of photosynthesis. Besides the unique features of the respiratory chain they also have several additional metabolic functions. About 700 proteins are present in yeast mitochondria [16], but more than 2000 proteins are predicted to be present in mitochondria from Arabidopsis thaliana [17]. Starting material for the Arabidopsis mitochondrial proteome project are leaves or suspension cell cultures. Protein separations are based on two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) and protein identifications on mass spectrometry. In the course of the project, about 800 mitochondrial proteins were detected on 2D gels and more than 100 proteins were identified by mass spectrometry, allowing to predict new mitochondrial functions ([18][19], www.gartenbau.uni-hannover.de/genetik/AMPP). This project is currently continued in our laboratory. To reduce overlappings on our 2D gels, a three-dimensional gel electrophoresis procedure was developed, which is based on Blue-native PAGE, isoelectric focussing and SDS-PAGE [20]. This procedure allows to visualize additional plant mitochondrial proteins and also proved to be useful for the separation of isoforms of subunits forming part of protein complexes.

4. Signal transduction in plant mitochondria

Protein phosphorylation represents a universal mechanism in eukaryotes for the modulation of physiological processes in response to inner- or extracellular signals. Plant mitochondria have a large number of phosphorylated proteins, as revealed by in organello phosphorylation experiments and by protein characterizations using mass spectrometry in the course of the Arabidopsis mitochondrial proteome project [18]. Only a few of the phosphorylated proteins were identified so far, including a subunit of the pyruvate dehydrogenase and of the branched-chain alpha-oxoacid dehydrogenase complexes, two subunits of the F0F1-ATP synthase, a 18 kDa subunit of the NADH dehydrogenase complex and the Heat Stress Protein HSP22. The specific roles of most phosphorylations is unknown. A project was started to systematically identify phosphorylated proteins in plant mitochondria.

The characterization of protein kinases responsible for specific phosphorylations in plant mitochondria is addressed by comparative proteome analyses with selected Arabidopsis knockout mutants and corresponding wild-type plants. Detection of differentially phosphorylated proteins in mutant and wild-type plants allows to assign protein kinases to specific substrates. The long term aim of this project is the biochemical characterization of individual protein phosphorylations to elucidate their roles in the regulation of mitochondrial functions in plants.

5. Protein transport into plant mitochondria

Import of proteins into the mitochondrial compartment is a complex process which involves the coordinated action of a variety of different components including receptors, translocases, chaperones and proteases (reviewed in [21]). This so-called mitochondrial protein import apparatus of plant cells differs from the one of heterotrophic cells in many respects, which most likely is a consequence of the occurrence of plastids. Our current research focuses on the mitochondrial processing peptidase, which in plants forms part of complex III of the respiratory chain [9][10][11], and on the preprotein translocase of the outer mitochondrial membrane, which includes plant specific preprotein receptors [22][23][24].

6. Supramolecular structure of photosystems in higher plants

In parallel to the characterization of mitochondrial protein complexes we started to study the structure and function of the thylakoid membrane of higher plants by two-dimensional Blue-native / SDS-PAGE. This method was optimized for the analysis of chloroplast proteins and protein complexes [25][26]. Further improvement of native gel electrophoresis procedures gave new insights into the supramolecular structure of the protein complexes involved in photosynthesis in higher plants [27].

7. Proteomic approach to identify proteins involved in the stress and pathogen response of crop plants

Systematic analyses are carried out to identify proteins important for the response of plants towards abiotic stress factors or pathogen attack. These projects are carried out in the framework of co-operations at Hannover University: (i) Manganese toxicity in Cowpea (cooperation with Prof. Dr. W. Horst, Hannover University, [28][29]), (ii) Molecular interaction of Aphanomyces euteiches and Medicago trunculata (Cooperation with PD Dr. Franziska Krajinsky, Hannover University, [30][31]) and (iii) Proteomic analyses of somatic and zygotic embryos of Cyclamen persicum (Cooperation with PD Dr. T. Winkelmann, Hannover University, [32]). These co-operations are currently continued in our laboratory.

Literature cited

[1] Eubel, H., Jänsch, L. and Braun, H.P. (2003) New insights into the respiratory chain of plant mitochondria: supercomplexes and a unique composition of complex II. Plant Physiol. 133, 274-286.

[2] Eubel, H., Heinemeyer, J. and Braun, H.P. (2004) Identification and characterization of respirasomes in potato mitochondria. Plant Physiol. 134, 1450-1459.

[3] Dudkina, N.V., Heinemeyer, J., Sunderhaus, S., Boekema, E.J. and Braun, H.P. (2006a) Respiratory chain supercomplexes in the plant mitochondrial membrane. Trends in Plant Science 11, 232-240.

[4] Dudkina, N.V., Eubel, H., Keegstra, W., Boekema, E.J. and Braun, H.P. (2005a) Structure of a mitochondrial supercomplex formed by respiratory chain complexes I and III. Proc. Natl. Acad. Sci. USA 102, 3225-3229.

[5] Dudkina, N.V., Heinemeyer, H., Keegstra, W., Boekema, E.J. and Braun, H.P. (2005b)
Structure of dimeric ATP synthase from mitochondria: An angular association of monomers induces the strong curvature of the inner membrane. FEBS Lett. 579, 5769-5772.  

[6] Dudkina, N.V., Sunderhaus, S., Braun, H.P. and Boekema, E.J. (2006) Characterization of dimeric ATP synthase and cristae membrane ultrastructure from Saccharomyces and Polytomella mitochondria. FEBS Lett. 580, 3427-3432.

[7] Jänsch, L., Kruft, V., Schmitz, U.K. and Braun, H.P. (1996) New insights into the composition, molecular mass and stoichiometry of the protein complexes of plant mitochondria. Plant J. 9, 357-368.

[8] Millar, A.H., Eubel, H., Jänsch, L., Kruft, V., Heazlewood, L. and Braun, H.P. (2004) Mitochondrial cytochrome c oxidase and succinate dehydrogenase contain plant-specific subunits. Plant Mol. Biol. 56, 77-89.

[9] Braun, H.P., Emmermann, M., Kruft, V. and Schmitz, U.K. (1992) The general mitochondrial processing peptidase from potato is an integral part of cytochrome c reductase of the respiratory chain. EMBO J. 11, 3219-3227.

[10] Braun, H.P. and Schmitz, U.K. (1995) Are the "core" proteins of the mitochondrial bc1 complex evolutionary relics of a processing peptidase? Trends Biochem. Sci. 20, 171-175.

[11] Braun, H.P. and Schmitz, U.K. (1995) The bifunctional cytochrome c reductase / processing peptidase complex from plant mitochondria. J. Bioenerg. Biomembr. 27, 423-436.

[12] Perales, M., Parisi, G., Fornasari, M.S., Colaneri, A., Villarreal, F., Gonzalez-Schain, N., Gómez-Casati, D., Braun, H.P., Araya, A., Echave, J. and Zabaleta, E. (2004) Gamma carbonic anhydrase subunits physically interact within complex I of plant mitochondria. Plant Mol. Biol. 56, 947-957.

[13] Perales, M., Eubel, H. Heinemeyer, H., Colaneri, A., Zabaleta, E. and Braun, H.P. (2005) Disruption of a nuclear gene encoding a mitochondrial gamma carbonic anhydrase reduces complex I and supercomplex I+III2 levels and alters mitochondrial physiology in Arabidopsis. J. Mol. Biol. 350, 263-277.

[14] Sunderhaus, S., Dudkina, N., Jänsch, L., Klodmann, J., Heinemeyer, J., Perales, M.,  Zabaleta, E., Boekema, E. and Braun, H.P. (2006) Carbonic anhydrase subunits form a matrix-exposed domain attached to the membrane arm of mitochondrial complex I in plants. J. Biol. Chem. 281, 6482-6488.

[15] Braun, H.P. and Zabaleta, E. (2006) Carbonic anhydrase subunits of the mitochondrial NADH dehydrogenase complex (complex I) in plants. Physiologia Plantarum 129, 114-122.

[16] Sickmann, A., Reinders, J., Wagner, Y., Joppich, C., Zahedi, R., Meyer, H.E., Schönfisch, B., Perschil, I., Chacinska, A., Guiard, B., Rehling, P., Pfanner, N. and Meisinger, C. (2003) The proteome of Saccharomyces cerevisiae mitochondria. Proc. Natl. Acad. Sci. USA 100, 13207-13212.

[17] The Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796-815.

[18] Kruft, V., Eubel, H., Werhahn, W.H., Jänsch, L. and Braun, H.P. (2001) Proteomic approach to identify novel mitochondrial functions in Arabidopsis thaliana. Plant Physiology 127, 1694-1710.

[19] Millar, A.H., Heazlewood, L., Kristense, B.K., Braun, H.P. and Møller, IA (2005) The plant mitochondrial proteome. Trends in Plant Science 10, 36-43.

[20] Werhahn, W.H. and Braun, H.P. (2002) Biochemical dissection of the mitochondrial proteome of Arabidopsis thaliana by three-dimensional gel elelctrophoresis. Electrophoresis 23, 640-646.

[21] Braun, H.P. and Schmitz, U.K. (1999) The protein import apparatus of plant mitochondria. Planta 209, 267-274.

[22] Jänsch, L., Kruft, V., Schmitz, U.K. and Braun, H.P. (1998) Unique composition of the preprotein translocase of the outer mitochondrial membrane from plants. J. Biol. Chem. 273, 17251-17257.

[23] Werhahn, W.H., Niemeyer, A., Jänsch, L., Kruft, V., Schmitz, U.K. and Braun, H.P. (2001) Purification and characterization of the preprotein translocase of the outer mitochondrial membrane from Arabidopsis thaliana: identification of multiple forms of TOM20. Plant Physiol. 125, 943-954.

[24] Werhahn, W., Jänsch, L. and Braun, H.P. (2003) Identification of novel subunits of the TOM complex from Arabidopsis thaliana. Plant Physiol. Biochem. 41, 407-416.

[25] Kügler, M., Jänsch, L., Kruft, V., Schmitz, U.K. and Braun, H.P. (1997) Analysis of the chloroplast protein complexes by blue-native polyacrylamide gelelectrophoresis. Photosynthesis Research 53, 35-44.

[26] Kügler, M., Kruft, V., Schmitz, U.K. and Braun, H.P. (1998) Characterization of the PetM subunit of the b6f complex from higher plants. J. Plant Physiol. 153, 581-586.

[27] Heinemeyer, J., Eubel, H., Wehmhöner, D., Jänsch, L. and Braun, H.P. (2004) Proteomic approach to characterize the supramolecular organization of photosystems in higher plants. Phytochemistry 65, 1683-1692.

[28] Fecht-Christoffers, M.M., Braun, H.P., Lemaitre-Guillier, C., VanDorsselaer, A. and Horst, W.J. (2003) Effect of manganese toxicity on the proteome of the leaf apoplast in cowpea (Vigna unguiculata). Plant Physiol. 133, 1935-1946.

[29] Fecht-Christoffers, M.M., Führs, H. Braun, H.P. and Horst, W.J. (2006) The role of H2O2-producing and H2O2-consuming peroxidases in the leaf apoplast of Vigna unguiculata L. in manganese tolerance. Plant Physiol. 140, 1451-1463.

[30] Colditz, F., Nyamsuren, O., Niehaus, K., Eubel, H., Braun, H.P. and Krajinski, F. (2004) Proteomic approach: Identification of Medicago trunculata proteins differentially expressed after infection with the pathogenic oomycete Aphanomyces euteiches. Plant Mol. Biol. 55, 109-120.

[31] Colditz, F., Braun, H.P., Jacquet, C., Niehaus, K. and Krajinski, F. (2005) Proteomic profiling unravels insights into the molecular background underlying increased Apha-nomyces euteiches-tolerance of Medicago trunculata. Plant Mol. Biol. 59, 385-404.

[32] Winkelmann, T., Heintz, D., van Dorsselaer, A., Serek, M. and Braun, H.P. (2006) Proteomic analyses of somatic and zygotic embryos of Cyclamen persicum MILL. reveal new insights into seed and germination physiology. Planta 204, 508-519.

Cooperations:

  • PD Dr. S. Binder, Ulm University, Germany
  • Prof. Dr. G. Burger & Prof. Dr. F. Lang, Université de Montreal, Canada
  • Prof. Dr. E. Boekema, University of Groningen, The Netherlands
  • Dr. M. Focke, Karlsruhe University, Germany
  • Prof. Dr. M. Frentzen, Aachen University, Germany
  • Dr. M.L. Genova, Università di Bologna, Italy
  • Dr. D. Gomez-Casati, Inst. de Investigaciones Biotecnologicas, Chascomus, Argentinia
  • Prof. Dr. W. Horst, Hannover University, Germany
  • Dr. L. Jänsch, Gesellschaft for Biotechnol. Research, Braunschweig, Germany
  • Prof. Dr. H. Janska, Wroclaw University, Poland
  • PD Dr. Franziska Krajinski, Hannover University, Germany
  • Dr. V. Kruft, Applied Biosystems, Weiterstadt, Germany•      Dr. S. Kushnir, Gent University, Belgien
  • Prof. Dr. D. Leister, Munich University, GermanyProf.
  • Dr. A.H. Millar, Perth University, Australia
  • PD. Dr. H.-P. Mock, IPK Gatersleben, Germany
  • Prof. Dr. J. Papenbrock, Hannover University, Germany
  • Prof. Dr. H. Schägger, Frankfurt University, Germany
  • Prof. Dr. J. Whelan, Perth University, Australia
  • Prof Dr. Traud Winkelmann, Hannover University, Germany
  • Prof. Dr. E. Zabaleta, Universidad Nacional de Mar del Plata, Mar del Plata, Argentina.

Financial support

  • Deutsche Forschungsgemeinschaft (Br1829/7-3, Br1829/8-1, Br1829/10-1)
  • Deutscher Akademischer Austauschdienst (DAAD)
  • Volkswagenstiftung
  • Federation of European Biochemical Societies (FEBS)