RESUMO
KEY MESSAGE: The functional absence of the electron-transfer flavoprotein: ubiquinone oxidoreductase (ETFQO) directly impacts electrons donation to the mitochondrial electron transport chain under carbohydrate-limiting conditions without major impacts on the respiration of cell cultures. Alternative substrates (e.g., amino acids) can directly feed electrons into the mitochondrial electron transport chain (mETC) via the electron transfer flavoprotein/electron-transfer flavoprotein: ubiquinone oxidoreductase (ETF/ETFQO) complex, which supports plant respiration during stress situations. By using a cell culture system, here we investigated the responses of Arabidopsis thaliana mutants deficient in the expression of ETFQO (etfqo-1) following carbon limitation and supplied with amino acids. Our results demonstrate that isovaleryl-CoA dehydrogenase (IVDH) activity was induced during carbon limitation only in wild-type and that these changes occurred concomit with enhanced protein content. By contrast, neither the activity nor the total amount of IVDH was altered in etfqo-1 mutants. We also demonstrate that the activities of mitochondrial complexes in etfqo-1 mutants, display a similar pattern as in wild-type cells. Our findings suggest that the defect of ETFQO protein culminates with an impaired functioning of the IVDH, since no induction of IVDH activity was observed. However, the functional absence of the ETFQO seems not to cause major impacts on plant respiration under carbon limiting conditions, most likely due to other alternative electron entry pathways.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Flavoproteínas Transferidoras de Elétrons , Aminoácidos de Cadeia Ramificada/farmacologia , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Metabolismo dos Carboidratos , Técnicas de Cultura de Células , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Flavoproteínas Transferidoras de Elétrons/genética , Flavoproteínas Transferidoras de Elétrons/metabolismo , Regulação da Expressão Gênica de Plantas , Isovaleril-CoA Desidrogenase/genética , Isovaleril-CoA Desidrogenase/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , MutaçãoRESUMO
Plant responses to abiotic stress include various modifications in amino acid metabolism. By using a hydroponic culture system, we systematically investigate modification in amino acid profiles and the proteome of Arabidopsis thaliana leaves during initial recovery from low water potential or high salinity. Both treatments elicited oxidative stress leading to a biphasic stress response during recovery. Degradation of highly abundant proteins such as subunits of photosystems and ribosomes contributed to an accumulation of free amino acids. Catabolic pathways for several low abundant amino acids were induced indicating their usage as an alternative respiratory substrate to compensate for the decreased photosynthesis. Our results demonstrate that rapid detoxification of potentially detrimental amino acids such as Lys is a priority during the initial stress recovery period. The content of Pro, which acts as a compatible osmolyte during stress, was adjusted by balancing its synthesis and catabolism both of which were induced both during and after stress treatments. The production of amino acid derived secondary metabolites was up-regulated specifically during the recovery period, and our dataset also indicates increased synthesis rates of the precursor amino acids. Overall, our results support a tight relationship between amino acid metabolism and stress responses.
Assuntos
Aminoácidos/metabolismo , Arabidopsis/fisiologia , Estresse Fisiológico , Proteínas de Arabidopsis/metabolismo , Desidratação , Lisina/metabolismo , Estresse Oxidativo , Folhas de Planta/metabolismo , Prolina/metabolismo , Proteoma/metabolismo , Estresse SalinoRESUMO
Plant respiration mostly depends on the activity of glycolysis and the oxidation of organic acids in the tricarboxylic acid cycle to synthesize ATP. However, during stress situations plant cells also use amino acids as alternative substrates to donate electrons through the electron-transfer flavoprotein (ETF)/ETF:ubiquinone oxidoreductase (ETF/ETFQO) complex to the mitochondrial electron transport chain (mETC). Given this, we investigated changes of the oxidative phosphorylation (OXPHOS) system in Arabidopsis thaliana cell culture under carbohydrate starvation supplied with a range of amino acids. Induction of isovaleryl-CoA dehydrogenase (IVDH) activity was observed under carbohydrate starvation which was associated with increased amounts of IVDH protein detected by immunoblotting. Furthermore, activities of the protein complexes of the mETC were reduced under carbohydrate starvation. We also observed that OXPHOS system activity behavior is differently affected by different amino acids and that proteins associated with amino acids catabolism are upregulated in cells following carbohydrate starvation. Collectively, our results support the contention that ETF/ETFQO is an essential pathway to donate electrons to the mETC and that amino acids are alternative substrates to maintain respiration under carbohydrate starvation.
Assuntos
Aminoácidos/metabolismo , Arabidopsis/metabolismo , Flavoproteínas Transferidoras de Elétrons/metabolismo , Mitocôndrias/metabolismo , Oxirredução , Fosforilação OxidativaRESUMO
The NADH-ubiquinone oxidoreductase complex (complex I) (EC 1.6.5.3) is the main entrance site of electrons into the respiratory chain. In a variety of eukaryotic organisms, except animals and fungi (Opisthokonta), it contains an extra domain comprising trimers of putative γ-carbonic anhydrases, named the CA domain, which has been proposed to be essential for assembly of complex I. However, its physiological role in plants is not fully understood. Here, we report that Arabidopsis mutants defective in two CA subunits show an altered photorespiratory phenotype. Mutants grown in ambient air show growth retardation compared to wild-type plants, a feature that is reversed by cultivating plants in a high-CO2 atmosphere. Moreover, under photorespiratory conditions, carbon assimilation is diminished and glycine accumulates, suggesting an imbalance with respect to photorespiration. Additionally, transcript levels of specific CA subunits are reduced in plants grown under non-photorespiratory conditions. Taken together, these results suggest that the CA domain of plant complex I contributes to sustaining efficient photosynthesis under ambient (photorespiratory) conditions.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Anidrases Carbônicas/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Proteínas de Arabidopsis/genética , Dióxido de Carbono/metabolismo , Anidrases Carbônicas/genética , Complexo I de Transporte de Elétrons/genética , Regulação da Expressão Gênica de Plantas , Glicina/metabolismo , Mutação , Oxigênio/metabolismo , Fotossíntese/genética , Folhas de Planta/genética , Folhas de Planta/fisiologia , Estrutura Terciária de Proteína , Espécies Reativas de Oxigênio/metabolismoRESUMO
We studied the role of cytochrome c (CYTc), which mediates electron transfer between Complexes III and IV, in cellular events related with mitochondrial respiration, plant development and redox homeostasis. We analyzed single and double homozygous mutants in both CYTc-encoding genes from Arabidopsis: CYTC-1 and CYTC-2. While individual mutants were similar to wild-type, knock-out of both genes produced an arrest of embryo development, showing that CYTc function is essential at early stages of plant development. Mutants in which CYTc levels were extremely reduced respective to wild-type had smaller rosettes with a pronounced decrease in parenchymatic cell size and an overall delay in development. Mitochondria from these mutants had lower respiration rates and a relative increase in alternative respiration. Furthermore, the decrease in CYTc severely affected the activity and the amount of Complex IV, without affecting Complexes I and III. Reactive oxygen species levels were reduced in these mutants, which showed induction of genes encoding antioxidant enzymes. Ascorbic acid levels were not affected, suggesting that a small amount of CYTc is enough to support its normal synthesis. We postulate that, in addition to its role as an electron carrier between Complexes III and IV, CYTc influences Complex IV levels in plants, probably reflecting a role of this protein in Complex IV stability. This double function of CYTc most likely explains why it is essential for plant survival.
Assuntos
Arabidopsis/enzimologia , Citocromos c/deficiência , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Antioxidantes/metabolismo , Arabidopsis/citologia , Arabidopsis/embriologia , Arabidopsis/genética , Ácido Ascórbico/metabolismo , Respiração Celular , Citocromos c/genética , Eletroforese em Gel Bidimensional , Estabilidade Enzimática , Genes de Plantas/genética , Homozigoto , Mutação/genética , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Sementes/metabolismo , Estresse FisiológicoRESUMO
Many photosynthetic organisms have developed inorganic carbon (Ci) concentrating mechanisms (CCMs) that increase the CO2 concentration within the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). Several CCMs, such as four carbon (C4) and crassulacean acid metabolism (CAM), bicarbonate accumulation systems and capsular structures around RubisCO have been described in great detail. These systems are believed to have evolved several times as mechanisms that acclimate organisms to unfavourable growth conditions. Based on recent experimental evidence we propose the occurrence of another more general CCM system present in all plants. This basal CCM (bCCM) is supposed to be composed of mitochondrial carbonic anhydrases (a ß-type carbonic anhydrase and the γ-type carbonic anhydrase domain of the mitochondrial NADH dehydrogenase complex) and probably further unknown components. The bCCM is proposed to reduce leakage of CO2 from plant cells and allow efficient recycling of mitochondrial CO2 for carbon fixation in chloroplasts.
Assuntos
Dióxido de Carbono/metabolismo , Carbono/metabolismo , Anidrases Carbônicas/metabolismo , Cloroplastos/metabolismo , Mitocôndrias/metabolismo , Fotossíntese/fisiologia , Plantas/metabolismo , Modelos Biológicos , NADH Desidrogenase/metabolismo , Células Vegetais/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismoRESUMO
Plant mitochondria include gamma-type carbonic anhydrases (gammaCAs) of unknown function. In Arabidopsis, the gammaCAs form a gene family of five members which all are attached to the NADH dehydrogenase complex (complex I) of the respiratory chain. Here we report a functional analysis of gamma carbonic anhydrase 2 (CA2). The gene encoding CA2 is constitutively expressed in all plant organs investigated but it is ten fold induced in flowers, particularly in tapetal tissue. Ectopic expression of CA2 in Arabidopsis causes male sterility in transgenic plants. In normal anther development, secondary thickenings of the endothecial cell wall cause anthers to open upon dehydration. Histological analyses revealed that abnormal secondary thickening prevents anther opening in 35S::CA2 transgenic plants. CA2 abundance in transgenic plants is increased 2-3 fold compared to wild-type plants as revealed by Western blotting analyses. Moreover, abundance of other members of the CA family, termed CA3 and CAL2, is increased in transgenic plants. Oxygen uptake measurements revealed that respiration in transgenic plants is mainly based on NADH reduction by the alternative NADH dehydrogenases present in plant mitochondria. Furthermore, the formation of reactive oxygen species (ROS) is very low in transgenic plants. We propose that reduction in ROS inhibits H(2)O(2) dependent lignin polymerization in CA2 over-expressing plants, thereby causing male sterility.
Assuntos
Proteínas de Arabidopsis/genética , Anidrases Carbônicas/genética , Flores/genética , Proteínas Mitocondriais/genética , Infertilidade das Plantas/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Ácido Ascórbico/metabolismo , Western Blotting , Anidrases Carbônicas/metabolismo , Eletroforese em Gel de Poliacrilamida , Flores/crescimento & desenvolvimento , Flores/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genótipo , Peróxido de Hidrogênio/metabolismo , Hibridização In Situ , Lignina/metabolismo , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Consumo de Oxigênio , Fenótipo , Plantas Geneticamente Modificadas , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We report the identification by two hybrid screens of two novel similar proteins, called Arabidopsis thaliana gamma carbonic anhydrase like1 and 2 (AtgammaCAL1 and AtgammaCAL2), that interact specifically with putative Arabidopsis thaliana gamma Carbonic Anhydrase (AtgammaCA) proteins in plant mitochondria. The interaction region that was located in the N-terminal 150 amino acids of mature AtgammaCA and AtgammaCA like proteins represents a new interaction domain. In vitro experiments indicate that these proteins are imported into mitochondria and are associated with mitochondrial complex I as AtgammaCAs. All plant species analyzed contain both AtgammaCA and AtgammaCAL sequences indicating that these genes were conserved throughout plant evolution. Structural modeling of AtgammaCAL sequences show a deviation of functionally important active site residues with respect to gammaCAs but could form active interfaces in the interaction with AtgammaCAs. We postulate a CA complex tightly associated to plant mitochondrial complex.