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Item The mitochondrial thioredoxin system contributes to the metabolic responses under drought episodes in Arabidopsis(Plant and Cell Physiology, 2019-01) Fonseca-Pereira, Paula da; Daloso, Danilo M.; Gago, Jorge; Silva, Franklin Magnum de Oliveira; Condori-Apfata, Jorge A.; Florez-Sarasa, Igor; Tohge, Takayuki; Reichheld, Jean-Philippe; Nunes-Nesi, Adriano; Fernie, Alisdair R.; Araujo, Wagner L.Thioredoxins (Trxs) modulate metabolic responses during stress conditions; however, the mechanisms governing the responses of plants subjected to multiple drought events and the role of Trxs under these conditions are not well understood. Here we explored the significance of the mitochondrial Trx system in Arabidopsis following exposure to single and repeated drought events. We analyzed the previously characterized NADPH-dependent Trx reductase A and B double mutant (ntra ntrb) and two independent mitochondrial thioredoxin o1 (trxo1) mutant lines. Following similar reductions in relative water content (∼50%), Trx mutants subjected to two drought cycles displayed a significantly higher maximum quantum efficiency (Fv/Fm) and were less sensitive to drought than their wild-type counterparts and than all genotypes subjected to a single drought event. Trx mutant plants displayed a faster recovery after two cycles of drought, as observed by the higher accumulation of secondary metabolites and higher stomatal conductance. Our results indicate that plants exposed to multiple drought cycles are able to modulate their subsequent metabolic and physiological response, suggesting the occurrence of an exquisite acclimation in stressed Arabidopsis plants. Moreover, this differential acclimation involves the participation of a set of metabolic changes as well as redox poise alteration following stress recovery.Item Can stable isotope mass spectrometry replace radiolabelled approaches in metabolic studies?(Plant Science, 2016-05-14) Silva, Willian Batista; Daloso, Danilo M.; Fernie, Alisdair R.; Nunes-Nesi, Adriano; Araújo, Wagner L.Metabolic pathways and the key regulatory points thereof can be deduced using isotopically labelled substrates. One prerequisite is the accurate measurement of the labeling pattern of targeted metabolites. The subsequent estimation of metabolic fluxes following incubation in radiolabelled substrates has been extensively used. Radiolabelling is a sensitive approach and allows determination of total label uptake since the total radiolabel content is easy to detect. However, the incubation of cells, tissues or the whole plant in a stable isotope enriched environment and the use of either mass spectrometry or nuclear magnetic resonance techniques to determine label incorporation within specific metabolites offers the possibility to readily obtain metabolic information with higher resolution. It additionally also offers an important complement to other post-genomic strategies such as metabolite profiling providing insights into the regulation of the metabolic network and thus allowing a more thorough description of plant cellular function. Thus, although safety concerns mean that stable isotope feeding is generally preferred, the techniques are in truth highly complementary and application of both approaches in tandem currently probably provides the best route towards a comprehensive understanding of plant cellular metabolism.Item Thioredoxin, a master regulator of the tricarboxylic acid cycle in plant mitochondria(Proceedings of the National Academy of Sciences of the United States of America, 2015-02-02) Daloso, Danilo M.; Müller, Karolin; Obata, Toshihiro; Florian, Alexandra; Tohge, Takayuki; Bottcher, Alexandra; Riondet, Christophe; Bariat, Laetitia; Carrari, Fernando; Nunes-Nesi, Adriano; Buchanan, Bob B.; Reichheld, Jean-Philippe; Araújo, Wagner L.; Fernie, Alisdair R.Plant mitochondria have a fully operational tricarboxylic acid (TCA) cycle that plays a central role in generating ATP and providing carbon skeletons for a range of biosynthetic processes in both heterotrophic and photosynthetic tissues. The cycle enzymeencoding genes have been well characterized in terms of transcriptional and effector-mediated regulation and have also been subjected to reverse genetic analysis. However, despite this wealth of attention, a central question remains unanswered: “What regulates flux through this pathway in vivo?” Previous proteomic experiments with Arabidopsis discussed below have revealed that a number of mitochondrial enzymes, including members of the TCA cycle and affiliated pathways, harbor thioredoxin (TRX)binding sites and are potentially redox-regulated. We have followed up on this possibility and found TRX to be a redox-sensitive mediator of TCA cycle flux. In this investigation, we first characterized, at the enzyme and metabolite levels, mutants of the mitochondrial TRX pathway in Arabidopsis: the NADP-TRX reductase a and b double mutant (ntra ntrb) and the mitochondrially located thioredoxin o1 (trxo1) mutant. These studies were followed by a comparative evaluation of the redistribution of isotopes when 13 C- glucose, 13 C-malate, or 13 C-pyruvate was provided as a substrate to leaves of mutant or WT plants. In a complementary approach, we evaluated the in vitro activities of a range of TCA cycle and associated enzymes under varying redox states. The combined dataset suggests that TRX may deactivate both mitochondrial succinate dehydrogenase and fumarase and activate the cytosolic ATP-citrate lyase in vivo, acting as a direct regulator of carbon flow through the TCA cycle and providing a mechanism for the coordination of cellular function.Item Enhanced photosynthesis and growth in atquac1 knockout mutants are due to altered organic acid accumulation and an increase in both stomatal and mesophyll conductance(Plant Physiology, 2015-11-05) Medeiros, David B.; Martins, Samuel C.V.; Cavalcanti, João Henrique F.; Daloso, Danilo M.; Martinoia, Enrico; Nunes-Nesi, Adriano; DaMatta, Fábio M.; Fernie, Alisdair R.; Araújo, Wagner L.Stomata control the exchange of CO2 and water vapor in land plants. Thus, whereas a constant supply of CO2 is required to maintain adequate rates of photosynthesis, the accompanying water losses must be tightly regulated to prevent dehydration and undesired metabolic changes. Accordingly, the uptake or release of ions and metabolites from guard cells is necessary to achieve normal stomatal function. The AtQUAC1, an R-type anion channel responsible for the release of malate from guard cells, is essential for efficient stomatal closure. Here, we demonstrate that mutant plants lacking AtQUAC1 accumulated higher levels of malate and fumarate. These mutant plants not only display slower stomatal closure in response to increased CO2 concentration and dark but are also characterized by improved mesophyll conductance. These responses were accompanied by increases in both photosynthesis and respiration rates, without affecting the activity of photosynthetic and respiratory enzymes and the expression of other transporter genes in guard cells, which ultimately led to improved growth. Collectively, our results highlight that the transport of organic acids plays a key role in plant cell metabolism and demonstrate that AtQUAC1 reduce diffusive limitations to photosynthesis, which, at least partially, explain the observed increments in growth under well-watered conditions.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 Impaired malate and fumarate accumulation due to the mutation of the tonoplast dicarboxylate transporter has little effects on stomatal behavior(Plant Physiology, 2017-09-12) Medeiros, David B.; Barros, Kallyne A.; Barros, Jessica Aline S.; Omena-Garcia, Rebeca P.; Arrivault, Stéphanie; Sanglard, Lílian M.V.P.; Detmann, Kelly C.; Silva, Willian Batista; Daloso, Danilo M.; DaMatta, Fábio M.; Nunes-Nesi, Adriano; Fernie, Alisdair R.; Araújo, Wagner L.Malate is a central metabolite involved in a multiplicity of plant metabolic pathways, being associated with mitochondrial metabolism and playing significant roles in stomatal movements. Vacuolar malate transport has been characterized at the molecular level and is performed by at least one carrier protein and two channels in Arabidopsis (Arabidopsis thaliana) vacuoles. The absence of the Arabidopsis tonoplast Dicarboxylate Transporter (tDT) in the tdt knockout mutant was associated previously with an impaired accumulation of malate and fumarate in leaves. Here, we investigated the consequences of this lower accumulation on stomatal behavior and photosynthetic capacity as well as its putative metabolic impacts. Neither the stomatal conductance nor the kinetic responses to dark, light, or high CO2 were highly affected in tdt plants. In addition, we did not observe any impact on stomatal aperture following incubation with abscisic acid, malate, or citrate. Furthermore, an effect on photosynthetic capacity was not observed in the mutant lines. However, leaf mitochondrial metabolism was affected in the tdt plants. Levels of the intermediates of the tricarboxylic acid cycle were altered, and increases in both light and dark respiration were observed. We conclude that manipulation of the tonoplastic organic acid transporter impacted mitochondrial metabolism, while the overall stomatal and photosynthetic capacity were unaffected.Item Impaired malate and fumarate accumulation due the mutation of tonoplast dicarboxylate transporter(Plant Physiology, 2017-09-20) Medeiros, David B.; Barros, Kallyne; Barros, Jessica AS; Omena-Garcia, Rebeca P; Detmann, Kelly C; Silva, Willian Batista; DaMatta, Fabio; Nunes-Nesi, Adriano; Araújo, Wagner L.; Arrivault, Stéphanie; Sanglard, Lilian Vincis Pereira; Daloso, Danilo M.; Fernie, Alisdair R.Malate is a central metabolite involved in a multiplicity of plant metabolic pathways, being associated with mitochondrial metabolism and playing significant roles n stomatal movements. Vacuolar malate transport has been characterized at the molecular level and is performed by at least one carrier protein and two channels in Arabidopsis vacuoles. The absence of the Arabidopsis thaliana tonoplast Dicarboxylate Transporter (tDT) in tdt knockout mutant was previously associated with an impaired accumulation of malate and fumarate in leaves. Here, we investigated the consequences of this lower accumulation on stomatal behaviour and photosynthetic capacity as well as ts putative metabolic impacts. Neither the stomatal conductance (g s ) nor the kinetic responses to dark, light or high CO 2 were highly affected in tdt plants. In addition, we did not observe any impact on stomatal aperture following incubation with either bscisic acid (ABA), malate or citrate. Further, an effect on photosynthetic capacity was not observed in the mutant lines. However, leaf mitochondrial metabolism was affected in the tdt plants. Levels of the intermediates of the tricarboxylic acid cycle were altered and increases in both light and dark respiration were observed. We conclude that manipulation of the tonoplastic organic acid transporter impacted mitochondrial metabolism, while the overall stomatal and photosynthetic capacity were unaffected.Item Knockout mutants are due to altered organic acid accumulation and an increase in both stomatal and mesophyll conductance(Plant Physiology, 2003-11-30) Medeiros, David B.; Martins, Samuel C.V.; Cavalcanti, João Henrique F.; Daloso, Danilo M.; Martinoia, Enrico; Nunes-Nesi, Adriano; DaMatta, Fábio M.; Fernie, Alisdair R.; Araújo, Wagner L.Stomata control the exchange of CO 2 and water vapor in land plants. Thus, whereas a constant supply of CO 2 is required to maintain adequate rates of photosynthesis, the accompanying water losses must be tightly regulated to prevent dehydration and undesired metabolic changes. Accordingly, the uptake or release of ions and metabolites from guard cells is necessary to achieve normal stomatal function. The AtQUAC1, an R-type anion channel responsible for the release of malate from guard cells, is essential for efficient stomatal closure. Here, we demonstrate that mutant plants lacking AtQUAC1 accumulated higher levels of malate and fumarate. These mutant plants not only display slower stomatal closure in response to increased CO 2 concentration and dark but are also characterized by improved mesophyll conductance. These responses were accompanied by increases in both photosynthesis and respiration rates, without affecting the activity of photosynthetic and respiratory enzymes and the expression of other transporter genes in guard cells, which ultimately led to improved growth. Collectively, our results highlight that the transport of organic acids plays a key role in plant cell metabolism and demonstrate that AtQUAC1 reduce diffusive limitations to photosynthesis, which, at least partially, explain the observed increments in growth under well-watered conditions.