title: d-Lactate Dehydrogenase Links Methylglyoxal Degradation and Electron Transport through Cytochrome c.
abstract: Glycolysis generates methylglyoxal (MGO) as an unavoidable, cytotoxic by-product in plant cells. MGO scavenging is performed by the glyoxalase system, which produces d-lactate as an end product. d-Lactate dehydrogenase (d-LDH) is encoded by a single gene in Arabidopsis (Arabidopsis thaliana; At5g06580). It catalyzes in vitro the oxidation of d-lactate to pyruvate using flavin adenine dinucleotide as a cofactor; knowledge of its function in the context of the plant cell remains sketchy. Blue native-polyacrylamide gel electrophoresis of mitochondrial extracts combined with in gel activity assays using different substrates and tandem mass spectrometry allowed us to definitely show that d-LDH acts specifically on d-lactate, is active as a dimer, and does not associate with respiratory supercomplexes of the inner mitochondrial membrane. The combined use of cytochrome c (CYTc) loss-of-function mutants and respiratory complex III inhibitors showed that CYTc acts as the in vivo electron acceptor of d-LDH. CYTc loss-of-function mutants, as well as the d-LDH mutants, were more sensitive to d-lactate and MGO, indicating that they function in the same pathway. In addition, overexpression of d-LDH and CYTc increased tolerance to d-lactate and MGO Together with fine-localization of d-LDH, the functional interaction with CYTc in vivo strongly suggests that d-lactate oxidation takes place in the mitochondrial intermembrane space, delivering electrons to the respiratory chain through CYTc These results provide a comprehensive picture of the organization and function of d-LDH in the plant cell and exemplify how the plant mitochondrial respiratory chain can act as a multifunctional electron sink for reductant from cytosolic pathways.
authors: Instituto de Agrobiotecnología del Litoral, Consejo Nacional de Investigaciones Científicas y Técnicas, Cátedra de Biología Celular y Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina (E.W., L.G., D.H.G.);Institute of Developmental and Molecular Biology of Plants, Plant Molecular Physiology and Biotechnology Group, Heinrich-Heine-Universität, and Cluster of Excellence on Plant Sciences, 40225 Duesseldorf, Germany (J.S., J.W., V.G.M.);Plant Energy Biology Laboratory, Institute of Crop Science and Resource Conservation, Rheinische Friedrich-Wilhelms-Universität Bonn, 53113 Bonn, Germany (P.F., S.W., M.S.); andPflanzengenetik, Abteilung Pflanzenproteomik, Leibniz Universität Hannover, 30419 Hannover, Germany (P.S., H.-P.B.).
authors: Elina
authors: 0000-0003-4025-573X
authors: E
authors: Welchen
authors: Instituto de Agrobiotecnología del Litoral, Consejo Nacional de Investigaciones Científicas y Técnicas, Cátedra de Biología Celular y Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina (E.W., L.G., D.H.G.);Institute of Developmental and Molecular Biology of Plants, Plant Molecular Physiology and Biotechnology Group, Heinrich-Heine-Universität, and Cluster of Excellence on Plant Sciences, 40225 Duesseldorf, Germany (J.S., J.W., V.G.M.);Plant Energy Biology Laboratory, Institute of Crop Science and Resource Conservation, Rheinische Friedrich-Wilhelms-Universität Bonn, 53113 Bonn, Germany (P.F., S.W., M.S.); andPflanzengenetik, Abteilung Pflanzenproteomik, Leibniz Universität Hannover, 30419 Hannover, Germany (P.S., H.-P.B.).
authors: Jessica
authors: J
authors: Schmitz
authors: Instituto de Agrobiotecnología del Litoral, Consejo Nacional de Investigaciones Científicas y Técnicas, Cátedra de Biología Celular y Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina (E.W., L.G., D.H.G.);Institute of Developmental and Molecular Biology of Plants, Plant Molecular Physiology and Biotechnology Group, Heinrich-Heine-Universität, and Cluster of Excellence on Plant Sciences, 40225 Duesseldorf, Germany (J.S., J.W., V.G.M.);Plant Energy Biology Laboratory, Institute of Crop Science and Resource Conservation, Rheinische Friedrich-Wilhelms-Universität Bonn, 53113 Bonn, Germany (P.F., S.W., M.S.); andPflanzengenetik, Abteilung Pflanzenproteomik, Leibniz Universität Hannover, 30419 Hannover, Germany (P.S., H.-P.B.).
authors: Philippe
authors: 0000-0001-6379-853X
authors: P
authors: Fuchs
authors: Instituto de Agrobiotecnología del Litoral, Consejo Nacional de Investigaciones Científicas y Técnicas, Cátedra de Biología Celular y Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina (E.W., L.G., D.H.G.);Institute of Developmental and Molecular Biology of Plants, Plant Molecular Physiology and Biotechnology Group, Heinrich-Heine-Universität, and Cluster of Excellence on Plant Sciences, 40225 Duesseldorf, Germany (J.S., J.W., V.G.M.);Plant Energy Biology Laboratory, Institute of Crop Science and Resource Conservation, Rheinische Friedrich-Wilhelms-Universität Bonn, 53113 Bonn, Germany (P.F., S.W., M.S.); andPflanzengenetik, Abteilung Pflanzenproteomik, Leibniz Universität Hannover, 30419 Hannover, Germany (P.S., H.-P.B.).
authors: Lucila
authors: 0000-0002-5109-2043
authors: L
authors: García
authors: Instituto de Agrobiotecnología del Litoral, Consejo Nacional de Investigaciones Científicas y Técnicas, Cátedra de Biología Celular y Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina (E.W., L.G., D.H.G.);Institute of Developmental and Molecular Biology of Plants, Plant Molecular Physiology and Biotechnology Group, Heinrich-Heine-Universität, and Cluster of Excellence on Plant Sciences, 40225 Duesseldorf, Germany (J.S., J.W., V.G.M.);Plant Energy Biology Laboratory, Institute of Crop Science and Resource Conservation, Rheinische Friedrich-Wilhelms-Universität Bonn, 53113 Bonn, Germany (P.F., S.W., M.S.); andPflanzengenetik, Abteilung Pflanzenproteomik, Leibniz Universität Hannover, 30419 Hannover, Germany (P.S., H.-P.B.).
authors: Stephan
authors: 0000-0001-5369-7911
authors: S
authors: Wagner
authors: Instituto de Agrobiotecnología del Litoral, Consejo Nacional de Investigaciones Científicas y Técnicas, Cátedra de Biología Celular y Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina (E.W., L.G., D.H.G.);Institute of Developmental and Molecular Biology of Plants, Plant Molecular Physiology and Biotechnology Group, Heinrich-Heine-Universität, and Cluster of Excellence on Plant Sciences, 40225 Duesseldorf, Germany (J.S., J.W., V.G.M.);Plant Energy Biology Laboratory, Institute of Crop Science and Resource Conservation, Rheinische Friedrich-Wilhelms-Universität Bonn, 53113 Bonn, Germany (P.F., S.W., M.S.); andPflanzengenetik, Abteilung Pflanzenproteomik, Leibniz Universität Hannover, 30419 Hannover, Germany (P.S., H.-P.B.).
authors: Judith
authors: J
authors: Wienstroer
authors: Instituto de Agrobiotecnología del Litoral, Consejo Nacional de Investigaciones Científicas y Técnicas, Cátedra de Biología Celular y Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina (E.W., L.G., D.H.G.);Institute of Developmental and Molecular Biology of Plants, Plant Molecular Physiology and Biotechnology Group, Heinrich-Heine-Universität, and Cluster of Excellence on Plant Sciences, 40225 Duesseldorf, Germany (J.S., J.W., V.G.M.);Plant Energy Biology Laboratory, Institute of Crop Science and Resource Conservation, Rheinische Friedrich-Wilhelms-Universität Bonn, 53113 Bonn, Germany (P.F., S.W., M.S.); andPflanzengenetik, Abteilung Pflanzenproteomik, Leibniz Universität Hannover, 30419 Hannover, Germany (P.S., H.-P.B.).
authors: Peter
authors: P
authors: Schertl
authors: Instituto de Agrobiotecnología del Litoral, Consejo Nacional de Investigaciones Científicas y Técnicas, Cátedra de Biología Celular y Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina (E.W., L.G., D.H.G.);Institute of Developmental and Molecular Biology of Plants, Plant Molecular Physiology and Biotechnology Group, Heinrich-Heine-Universität, and Cluster of Excellence on Plant Sciences, 40225 Duesseldorf, Germany (J.S., J.W., V.G.M.);Plant Energy Biology Laboratory, Institute of Crop Science and Resource Conservation, Rheinische Friedrich-Wilhelms-Universität Bonn, 53113 Bonn, Germany (P.F., S.W., M.S.); andPflanzengenetik, Abteilung Pflanzenproteomik, Leibniz Universität Hannover, 30419 Hannover, Germany (P.S., H.-P.B.).
authors: Hans-Peter
authors: 0000-0002-4459-9727
authors: HP
authors: Braun
authors: Instituto de Agrobiotecnología del Litoral, Consejo Nacional de Investigaciones Científicas y Técnicas, Cátedra de Biología Celular y Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina (E.W., L.G., D.H.G.);Institute of Developmental and Molecular Biology of Plants, Plant Molecular Physiology and Biotechnology Group, Heinrich-Heine-Universität, and Cluster of Excellence on Plant Sciences, 40225 Duesseldorf, Germany (J.S., J.W., V.G.M.);Plant Energy Biology Laboratory, Institute of Crop Science and Resource Conservation, Rheinische Friedrich-Wilhelms-Universität Bonn, 53113 Bonn, Germany (P.F., S.W., M.S.); andPflanzengenetik, Abteilung Pflanzenproteomik, Leibniz Universität Hannover, 30419 Hannover, Germany (P.S., H.-P.B.).
authors: Markus
authors: 0000-0003-0796-8308
authors: M
authors: Schwarzländer
authors: Instituto de Agrobiotecnología del Litoral, Consejo Nacional de Investigaciones Científicas y Técnicas, Cátedra de Biología Celular y Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina (E.W., L.G., D.H.G.);Institute of Developmental and Molecular Biology of Plants, Plant Molecular Physiology and Biotechnology Group, Heinrich-Heine-Universität, and Cluster of Excellence on Plant Sciences, 40225 Duesseldorf, Germany (J.S., J.W., V.G.M.);Plant Energy Biology Laboratory, Institute of Crop Science and Resource Conservation, Rheinische Friedrich-Wilhelms-Universität Bonn, 53113 Bonn, Germany (P.F., S.W., M.S.); andPflanzengenetik, Abteilung Pflanzenproteomik, Leibniz Universität Hannover, 30419 Hannover, Germany (P.S., H.-P.B.).
authors: Daniel H
authors: DH
authors: Gonzalez
authors: Instituto de Agrobiotecnología del Litoral, Consejo Nacional de Investigaciones Científicas y Técnicas, Cátedra de Biología Celular y Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina (E.W., L.G., D.H.G.);Institute of Developmental and Molecular Biology of Plants, Plant Molecular Physiology and Biotechnology Group, Heinrich-Heine-Universität, and Cluster of Excellence on Plant Sciences, 40225 Duesseldorf, Germany (J.S., J.W., V.G.M.);Plant Energy Biology Laboratory, Institute of Crop Science and Resource Conservation, Rheinische Friedrich-Wilhelms-Universität Bonn, 53113 Bonn, Germany (P.F., S.W., M.S.); andPflanzengenetik, Abteilung Pflanzenproteomik, Leibniz Universität Hannover, 30419 Hannover, Germany (P.S., H.-P.B.) veronica.maurino@uni-duesseldorf.de.
authors: Veronica G
authors: 0000-0002-3124-1451
authors: VG
authors: Maurino
mesh_terms: D017360:Arabidopsis / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D015153:Blotting, Western; D002478:Cells, Cultured; D045304:Cytochromes c / Q000235:genetics / Q000378:metabolism*; D004579:Electron Transport; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D019344:Lactic Acid / Q000378:metabolism; D013058:Mass Spectrometry; D018613:Microscopy, Confocal; D051336:Mitochondrial Membranes / Q000378:metabolism; D009154:Mutation; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D030821:Plants, Genetically Modified; D011765:Pyruvaldehyde / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D019344:Lactic Acid; D011765:Pyruvaldehyde; D019289:Pyruvic Acid; D045304:Cytochromes c; D007770:L-Lactate Dehydrogenase