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URI permanente para esta coleçãohttps://locus.ufv.br/handle/123456789/11846
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Item Sucrose breakdown within guard cells provides substrates for glycolysis and glutamine biosynthesis during light- induced stomatal opening(The Plant Journal, 2018-05) Medeiros, David B.; Souza, Leonardo Perez; Antunes, Werner C.; Araújo, Wagner L.; Daloso, Danilo M.; Fernie, Alisdair R.Sucrose has long been thought to play an osmolytic role in stomatal opening. However, recent evidence supports the idea that the role of sucrose in this process is primarily energetic. Here we used a combination of stomatal aperture assays and kinetic [U- 13 C]-sucrose isotope labelling experiments to confirm that sucrose is degraded during light-induced stomatal opening and to define the fate of the C released from sucrose breakdown. We additionally show that addition of sucrose to the medium did not enhance light- induced stomatal opening. The isotope experiment showed a consistent 13 C enrichment in fructose and glu- cose, indicating that during light-induced stomatal opening sucrose is indeed degraded. We also observed a clear 13 C enrichment in glutamate and glutamine (Gln), suggesting a concerted activation of sucrose degra- dation, glycolysis and the tricarboxylic acid cycle. This is in contrast to the situation for Gln biosynthesis in leaves under light, which has been demonstrated to rely on previously stored C. Our results thus collectively allow us to redraw current models concerning the influence of sucrose during light-induced stomatal open- ing, in which, instead of being accumulated, sucrose is degraded providing C skeletons for Gln biosynthesis.Item On the role of the mitochondrial 2-oxoglutarate dehydrogenase complex in amino acid metabolism(Amino Acids, 2013-02) Araújo, Wagner L.; Steinhauser, Dirk; Krall, Leonard; Fernie, Alisdair R.; Trofimova, Lidia; Mkrtchyan, Garik; Graf, Anastasia; Bunik, Victoria I.Mitochondria are tightly linked to cellular nutrient sensing, and provide not only energy, but also intermediates for the de novo synthesis of cellular compounds including amino acids. Mitochondrial metabolic enzymes as generators and/or targets of signals are therefore important players in the distribution of intermediates between catabolic and anabolic pathways. The highly regulated 2-oxoglutarate dehydrogenase complex (OGDHC) participates in glucose oxidation via the tricarboxylic acid cycle. It occupies an amphibolic branch point in the cycle, where the energy-producing reaction of the 2-oxoglutarate degradation competes with glutamate (Glu) synthesis via nitrogen incorporation into 2-oxoglutarate. To characterize the specific impact of the OGDHC inhibition on amino acid metabolism in both plant and animal mitochondria, a synthetic analog of 2-oxoglutarate, namely succinyl phosphonate (SP), was applied to living systems from different kingdoms, both in situ and in vivo. Using a high-throughput mass spectrometry-based approach, we showed that organisms possessing OGDHC respond to SP by significantly changing their amino acid pools. By contrast, cyanobacteria which lack OGDHC do not show perturbations in amino acids following SP treatment. Increases in Glu, 4-aminobutyrate and alanine represent the most universal change accompanying the 2-oxoglutarate accumulation upon OGDHC inhibition. Other amino acids were affected in a species-specific manner, suggesting specific metabolic rearrangements and substrate availability mediating secondary changes. Strong perturbation in the relative abundance of amino acids due to the OGDHC inhibition was accompanied by decreased protein content. Our results provide specific evidence of a considerable role of OGDHC in amino acid metabolism.Item Metabolic control and regulation of the tricarboxylic acid cycle in photosynthetic and heterotrophic plant tissues(Plant, Cell and Environment, 2011-04-08) Nunes-Nesi, Adriano; Araújo, Wagner L.; Nikoloski, Zoran; Sweetlove, Lee J.; Fernie, Alisdair R.The tricarboxylic acid (TCA) cycle is a crucial component of respiratory metabolism in both photosynthetic and heterotrophic plant organs. All of the major genes of the tomato TCA cycle have been cloned recently, allowing the generation of a suite of transgenic plants in which the majority of the enzymes in the pathway are progres- sively decreased. Investigations of these plants have provided an almost complete view of the distribution of control in this important pathway. Our studies suggest that citrate synthase, aconitase, isocitrate dehydrogenase, succi- nyl CoA ligase, succinate dehydrogenase, fumarase and malate dehydrogenase have control coefficients flux for res- piration of -0.4, 0.964, -0.123, 0.0008, 0.289, 0.601 and 1.76, respectively; while 2-oxoglutarate dehydrogenase is esti- mated to have a control coefficient of 0.786 in potato tubers. These results thus indicate that the control of this pathway is distributed among malate dehydrogenase, aconitase, fumarase, succinate dehydrogenase and 2-oxoglutarate dehydrogenase. The unusual distribution of control esti- mated here is consistent with specific non-cyclic flux mode and cytosolic bypasses that operate in illuminated leaves. These observations are discussed in the context of known regulatory properties of the enzymes and some illustrative examples of how the pathway responds to environmental change are given.Item Growth inhibition by selenium is associated with changes in primary metabolism and nutrient levels in Arabidopsis thaliana(Plant, Cell and Environment, 2016-06-25) Ribeiro, Dimas M.; Silva Júnior, Dalton D.; Cardoso, Flávio Barcellos; Martins, Auxiliadora O.; Silva, Welder A.; Nascimento, Vitor L.; Araújo, Wagner L.Although Selenium (Se) stress is relatively well known for causing growth inhibition, its effects on primary metabolism remain rather unclear. Here, we characterized both the modu- lation of the expression of specific genes and the metabolic adjustments in Arabidopsis thaliana in response to changes in Se level in the soil. Se treatment culminated with strong inhibition of both shoot and root growth. Notably, growth inhibition in Se‐treated plants was associated with an incom- plete mobilization of starch during the night. Minor changes in amino acids levels were observed in shoots and roots of plants treated with Se whereas the pool size of tricarboxylic acid (TCA) cycle intermediates in root was not altered in response to Se. By contrast, decreased levels of organic acids involved in the first part of the TCA cycle were observed in shoots of Se‐treated plants. Furthermore, decreased expression levels of expansins and endotransglucosylases/endohydrolases (XHTs) genes were observed after Se treatment, coupled with a significant decrease in the levels of essential elements. Collec- tively, our results revealed an exquisite interaction between energy metabolism and Se‐mediated control of growth in Arabidopsis thaliana to coordinate cell wall extension, starch turnover and the levels of a few essential nutrients.Item Phosphonate analogs of 2-oxoglutarate perturb metabolism and gene expression in illuminated Arabidopsis leaves(Frontiers in Plant Science | Plant Physiology, 2012-06-04) Araújo, Wagner L.; Tohge, Takayuki; Nunes-Nesi, Adriano; Daloso, Danilo M.; Nimick, Mhairi; Krahnert, Ina; Bunik, Victoria I.; Moorhead, Greg B. G.; Fernie, Alisdair R.Although the role of the 2-oxoglutarate dehydrogenase complex (2-OGDHC) has previously been demonstrated in plant heterotrophic tissues its role in photosynthetically active tissues remains poorly understood. By using a combination of metabolite and transcript profiles we here investigated the function of 2-OGDHC in leaves of Arabidopsis thaliana via use of specific phosphonate inhibitors of the enzyme. Incubation of leaf disks with the inhibitors revealed that they produced the anticipated effects on the in situ enzyme activity. In vitro experiments revealed that succinyl phosphonate (SP) and a carboxy ethyl ester of SP are slow-binding inhibitors of the 2-OGDHC. Our results indicate that the reduced respiration rates are associated with changes in the regulation of metabolic and signaling pathways leading to an imbalance in carbon-nitrogen metabolism and cell homeostasis. The inducible alteration of primary metabolism was associated with altered expression of genes belonging to networks of amino acids, plant respiration, and sugar metabolism. In addition, by using isothermal titration calorimetry we excluded the possibility that the changes in gene expression resulted from an effect on 2-oxoglutarate (2OG) binding to the carbon/ATP sensing protein PII. We also demonstrated that the 2OG degradation by the 2-oxoglutarate dehydrogenase strongly influences the distribution of intermediates of the tricarboxylic acid (TCA) cycle and the GABA shunt. Our results indicate that the TCA cycle activity is clearly working in a non-cyclic manner upon 2-OGDHC inhibition during the light period.Item Evolution and functional implications of the tricarboxylic acid cycle as revealed by phylogenetic analysis(Genome Biology and Evolution, 2014-09-25) Cavalcanti, João Henrique Frota; Esteves-Ferreira, Alberto A.; Quinhones, Carla G.S.; Pereira-Lima, Italo A.; Nunes-Nesi, Adriano; Fernie, Alisdair R.; Araújo, Wagner L.The tricarboxylic acid (TCA) cycle, a crucial component of respiratory metabolism, is composed of a set of eight enzymes present in the mitochondrial matrix. However, most of the TCA cycle enzymes are encoded in the nucleus in higher eukaryotes. In addition, evidence has accumulated demonstrating that nuclear genes were acquired from the mitochondrial genome during the course of evolution. For this reason, we here analyzed the evolutionary history of all TCA cycle enzymes in attempt to better understand the origin of these nuclear-encoded proteins. Our results indicate that prior to endosymbiotic events the TCA cycle seemed to operate only as isolated steps in both the host (eubacterial cell) and mitochondria (alphaproteobacteria). The origin of isoforms present in different cell compartments might be associated either with gene-transfer events which did not result in proper targeting of the protein to mitochondrion or with duplication events. Further in silico analyses allow us to suggest new insights into the possible roles of TCA cycle enzymes in different tissues. Finally, we performed coexpression analysis using mitochondrial TCA cycle genes revealing close connections among these genes most likely related to the higher efficiency of oxidative phosphorylation in this specialized organelle. Moreover, these analyses allowed us to identify further candidate genes which might be used for metabolic engineering purposes given the importance of the TCA cycle during development and/or stress situations.Item 2-Oxoglutarate: linking TCA cycle function with amino acid, glucosinolate, flavonoid, alkaloid, and gibberellin biosynthesis(Frontiers in Plant Science, 2014-10-05) Araújo, Wagner L.; Martins, Auxiliadora O.; Fernie, Alisdair R.; Tohge, TakayukiThe tricarboxylic acid (TCA) cycle intermediate 2-oxoglutarate (2-OG) is used as an obligatory substrate in a range of oxidative reactions catalyzed by 2-OG-dependent dioxygenases. These enzymes are widespread in nature being involved in several important biochemical processes. We have recently demonstrated that tomato plants in which the TCA cycle enzyme 2-OG dehydrogenase (2-ODD) was antisense inhibited were characterized by early senescence and modified fruit ripening associated with differences in the levels of bioactive gibberellin (GA). Accordingly, there is now compelling evidence that the TCA cycle plays an important role in modulating the rate of flux from 2-OG to amino acid metabolism. Here we discuss recent advances in the biochemistry and molecular biology of 2-OG metabolism occurring in different biological systems indicating the importance of 2-OG and 2-OG dependent dioxygenases not only in glucosinolate, flavonoid and alkaloid metabolism but also in GA and amino acid metabolism. We additionally summarize recent findings regarding the impact of modification of 2-OG metabolism on biosynthetic pathways involving 2-ODDs.