RESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Citocromos c/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Piruvaldehído/metabolismo , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Biocatálisis , Western Blotting , Células Cultivadas , Citocromos c/genética , Transporte de Electrón , L-Lactato Deshidrogenasa/genética , Ácido Láctico/metabolismo , Espectrometría de Masas , Microscopía Confocal , Membranas Mitocondriales/metabolismo , Mutación , Oxidación-Reducción , Consumo de Oxígeno , Plantas Modificadas Genéticamente , Ácido Pirúvico/metabolismoRESUMEN
D-2-Hydroxyglutarate dehydrogenase (D-2HGDH) catalyzes the specific and efficient oxidation of D-2-hydroxyglutarate (D-2HG) to 2-oxoglutarate using FAD as a cofactor. In this work, we demonstrate that D-2HGDH localizes to plant mitochondria and that its expression increases gradually during developmental and dark-induced senescence in Arabidopsis thaliana, indicating an enhanced demand of respiration of alternative substrates through this enzymatic system under these conditions. Using loss-of-function mutants in D-2HGDH (d2hgdh1) and stable isotope dilution LC-MS/MS, we found that the D-isomer of 2HG accumulated in leaves of d2hgdh1 during both forms of carbon starvation. In addition to this, d2hgdh1 presented enhanced levels of most TCA cycle intermediates and free amino acids. In contrast to the deleterious effects caused by a deficiency in D-2HGDH in humans, d2hgdh1 and overexpressing lines of D-2HGDH showed normal developmental and senescence phenotypes, indicating a mild role of D-2HGDH in the tested conditions. Moreover, metabolic fingerprinting of leaves of plants grown in media supplemented with putative precursors indicated that D-2HG most probably originates during the catabolism of lysine. Finally, the L-isomer of 2HG was also detected in leaf extracts, indicating that both chiral forms of 2HG participate in plant metabolism.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Lisina/metabolismo , Oxidorreductasas de Alcohol/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ciclo del Ácido Cítrico/fisiología , Glutaratos/metabolismo , Lisina/genética , Consumo de Oxígeno/fisiologíaRESUMEN
D-Lactate negatively affects Arabidopsis thaliana seedling development in a concentration-dependent manner. At media D-lactate concentrations greater than 5-10mM the development of wild-type plants is arrested shortly after germination whereas plants overexpressing the endogenous D-lactate dehydrogenase (D-LDH) detoxify D-lactate to pyruvate and survive. When the transgenic plants are further transferred to normal growth conditions they develop indistinguishably from the wild type. Thus, D-LDH was successfully established as a new marker in A. thaliana allowing selecting transgenic plants shortly after germination. The selection on D-lactate containing media adds a new optional marker system, which is especially useful if the simultaneous selection of multiple constructs is desired.