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URI permanente para esta coleçãohttps://locus.ufv.br/handle/123456789/11846
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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 Downregulation of the δ-subunit reduces mitochondrial ATP synthase levels, alters respiration, and restricts growth and gametophyte development in Arabidopsis(The Plant Cell, 2012-07) Nunes-Nesi, Adriano; Geisler, Daniela A.; Päpke, Carola; Obata, Toshihiro; Matthes, Annemarie; Schneitz, Kay; Maximova, Eugenia; Araújo, Wagner L.; Fernie, Alisdair R.; Persson, StaffanThe mitochondrial ATP synthase (F1Fo complex) is an evolutionary conserved multimeric protein complex that synthesizes the main bulk of cytosolic ATP, but the regulatory mechanisms of the subunits are only poorly understood in plants. In yeast, the δ-subunit links the membrane-embedded Fo part to the matrix-facing central stalk of F1. We used genetic interference and an inhibitor to investigate the molecular function and physiological impact of the δ-subunit in Arabidopsis thaliana. Delta mutants displayed both male and female gametophyte defects. RNA interference of delta resulted in growth retardation, reduced ATP synthase amounts, and increased alternative oxidase capacity and led to specific long-term increases in Ala and Gly levels. By contrast, inhibition of the complex using oligomycin triggered broad metabolic changes, affecting glycolysis and the tricarboxylic acid cycle, and led to a successive induction of transcripts for alternative respiratory pathways and for redox and biotic stress-related transcription factors. We conclude that (1) the δ-subunit is essential for male gametophyte development in Arabidopsis, (2) a disturbance of the ATP synthase appears to lead to an early transition phase and a long-term metabolic steady state, and (3) the observed long-term adjustments in mitochondrial metabolism are linked to reduced growth and deficiencies in gametophyte development.Item Differential root and shoot responses in the metabolism of tomato plants exhibiting reduced levels of gibberellin(Environmental and Experimental Botany, 2019-01) Martins, Auxiliadora O.; Omena-Garcia, Rebeca P.; Oliveira, Franciele S.; Silva, Welder A.; Hajirezaei, Mohammad-Reza; Vallarino, José G.; Ribeiro, Dimas Mendes; Fernie, Alisdair R.; Nunes-Nesi, Adriano; Araújo, Wagner L.The ability to adapt to the environment is crucial for plant survival and thus a refined communication system capable of integrating endogenous and exogenous signals and further relaying this information to different parts of the plant is a key component of such adaptability. Given that they grow in highly distinct environments it is arguably unsurprising that roots and shoots display different responses to a given environmental condition. Accordingly, a higher sensitivity of roots to gibberellins (GAs) allows rapid adjustments in growth and development possibly triggering (a) stress tolerance mechanism(s). Here we investigated the differential metabolic responses between root and shoot following reductions of the endogenous GA content using tomato (Solanum lycopersicum L.) plants deficient in GA biosynthesis (gib3, moderately deficient, gib2, intermediate deficiency and gib1, extremely deficient in GAs). GA depletion impedes shoot growth to a greater extent than root growth in all mutants. Moreover, the greater the reduction in GA content the greater the extent of a disturbance at the metabolic level. Low leaf carbohydrate contents were observed in plants displaying higher root growth, suggesting an enhanced flow of photoassimilate to support root growth. Large increases in amino acids contents of either roots or shoot were observed. The increased amino acid content was coupled to reduced levels of TCA cycle intermediates suggesting that these changes are directly linked to early reactions of nitrogen assimilation. The combined data are discussed in terms of our current understanding of the interaction between GA and primary metabolism and their crosstalk in environmental responses.Item Metabolic diversity in tuber tissues of native Chiloé potatoes and commercial cultivars of Solanum tuberosum ssp. tuberosum L.(Metabolomics, 2018-10) Silva, Franklin Magnum de Oliveira; Machado, Mariana; Nunes-Nesi, Adriano; Inostroza-Blancheteau, Claudio; Durán, Fabiola; Solano, Jaime; Obata, Toshihiro; Fernie, Alisdair R.; Reyes-Díaz, MarjorieThe native potatoes (Solanum tuberosum ssp. tuberosum L.) cultivated on Chiloé Island in southern Chile have great variability in terms of tuber shape, size, color and flavor. These traits have been preserved throughout generations due to the geographical position of Chiloé, as well as the different uses given by local farmers. The present study aimed to investigate the diversity of metabolites in skin and pulp tissues of eleven native accessions of potatoes from Chile, and evaluate the metabolite associations between tuber tissues. For a deeper characterization of these accessions, we performed a comprehensive metabolic study in skin and pulp tissues of tubers, 3 months after harvesting. Specific targeted quantification of metabolites using 96 well microplates, and high-performance liquid chromatography combined with non-targeted metabolite profiling by gas chromatography time-of-flight mass spectrometry were used in this study. We observed differential levels of antioxidant activity and phenolic compounds between skin and pulp compared to a common commercial cultivar (Desireé). In addition, we uncovered considerable metabolite variability between different tuber tissues and between native potatoes. Network correlation analysis revealed different metabolite associations among tuber tissues that indicate distinct associations between primary metabolite and anthocyanin levels, and antioxidant activity in skin and pulp tissues. Moreover, multivariate analysis lead to the grouping of native and commercial cultivars based on metabolites from both skin and pulp tissues. As well as providing important information to potato producers and breeding programs on the levels of health relevant phytochemicals and other abundant metabolites such as starch, proteins and amino acids, this study highlights the associations of different metabolites in tuber skins and pulp, indicating the need for distinct strategies for metabolic engineering in these tissues. Furthermore, this study shows that native Chilean potato accessions have great potential as a natural source of phytochemicals.Item On the role of plant mitochondrial metabolism and its impact on photosynthesis in both optimal and sub-optimal growth conditions(Photosynthesis Research, 2014-02) Araújo, Wagner L.; Nunes-Nesi, Adriano; Fernie, Alisdair R.Given that the pathways of photosynthesis and respiration catalyze partially opposing processes, it follows that their relative activities must be carefully regulated within plant cells. Recent evidence has shown that the components of the mitochondrial electron transport chain are essential for the proper maintenance of intracellular redox gradients, to allow considerable rates of photorespiration and in turn efficient photosynthesis. Thus considerable advances have been made in understanding the interaction between respiration and photosynthesis during the last decades and the potential mechanisms linking mitochondrial function and photosynthetic efficiency will be reviewed. Despite the fact that manipulation of various steps of mitochondrial metabolism has been demonstrated to alter photosynthesis under optimal growth conditions, it is likely that these changes will, by and large, not be maintained under sub-optimal situations. Therefore producing plants to meet this aim remains a critical challenge. It is clear, however, that although there have been a range of studies analysing changes in respiratory and photosynthetic rates in response to light, temperature and CO2, our knowledge of the environmental impact on these processes and its linkage still remains fragmented. We will also discuss the metabolic changes associated to plant respiration and photosynthesis as important components of the survival strategy as they considerably extend the period that a plant can withstand to a stress situation.Item Antisense inhibition of the 2-oxoglutarate dehydrogenase complex in tomato demonstrates its importance for plant respiration and during leaf senescence and fruit maturation(The Plant Cell, 2012-06) Araújo, Wagner L.; Nunes-Nesi, Adriano; Tohge, Takayuki; Osorio, Sonia; Lohse, Marc; Balbo, Ilse; Krahnert, Ina; Sienkiewicz-Porzucek, Agata; Usadel, Björn; Fernie, Alisdair R.Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the gene encoding the E1 subunit of the 2-oxoglutarate dehydrogenase complex in the antisense orientation and exhibiting substantial reductions in the activity of this enzyme exhibit a considerably reduced rate of respiration. They were, however, characterized by largely unaltered photosynthetic rates and fruit yields but restricted leaf, stem, and root growth. These lines displayed markedly altered metabolic profiles, including changes in tricarboxylic acid cycle intermediates and in the majority of the amino acids but unaltered pyridine nucleotide content both in leaves and during the progression of fruit ripening. Moreover, they displayed a generally accelerated development exhibiting early flowering, accelerated fruit ripening, and a markedly earlier onset of leaf senescence. In addition, transcript and selective hormone profiling of gibberellins and abscisic acid revealed changes only in the former coupled to changes in transcripts encoding enzymes of gibberellin biosynthesis. The data obtained are discussed in the context of the importance of this enzyme in both photosynthetic and respiratory metabolism as well as in programs of plant development connected to carbon–nitrogen interactions.Item Evolution and metabolic significance of the urea cycle in photosynthetic diatoms(Nature, 2011-05-12) Allen, Andrew E.; Dupont, Christopher L.; Oborník, Miroslav; Horák, Aleš; McCrow, John P.; Zheng, Hong; Johnson, Daniel A.; Hu, Hanhua; Fernie, Alisdair R.; Bowler, Chris; Nunes-Nesi, AdrianoDiatoms dominate the biomass of phytoplankton in nutrient-rich conditions and form the basis of some of the world’s most productive marine food webs1,2,3,4. The diatom nuclear genome contains genes with bacterial and plastid origins as well as genes of the secondary endosymbiotic host (the exosymbiont5)1,6,7,8,9,10, yet little is known about the relative contribution of each gene group to diatom metabolism. Here we show that the exosymbiont-derived ornithine-urea cycle, which is similar to that of metazoans but is absent in green algae and plants, facilitates rapid recovery from prolonged nitrogen limitation. RNA-interference-mediated knockdown of a mitochondrial carbamoyl phosphate synthase impairs the response of nitrogen-limited diatoms to nitrogen addition. Metabolomic analyses indicate that intermediates in the ornithine-urea cycle are particularly depleted and that both the tricarboxylic acid cycle and the glutamine synthetase/glutamate synthase cycles are linked directly with the ornithine-urea cycle. Several other depleted metabolites are generated from ornithine-urea cycle intermediates by the products of genes laterally acquired from bacteria. This metabolic coupling of bacterial- and exosymbiont-derived proteins seems to be fundamental to diatom physiology because the compounds affected include the major diatom osmolyte proline12 and the precursors for long-chain polyamines required for silica precipitation during cell wall formation11. So far, the ornithine-urea cycle is only known for its essential role in the removal of fixed nitrogen in metazoans. In diatoms, this cycle serves as a distribution and repackaging hub for inorganic carbon and nitrogen and contributes significantly to the metabolic response of diatoms to episodic nitrogen availability. The diatom ornithine-urea cycle therefore represents a key pathway for anaplerotic carbon fixation into nitrogenous compounds that are essential for diatom growth and for the contribution of diatoms to marine productivity.Item Tapping natural variation at functional level reveals allele specific molecular characteristics of potato invertase Pain‐1(Plant, Cell and Environment, 2012-05-24) Nunes-Nesi, Adriano; Draffehn, Astrid M.; Durek, Pawel; Stich, Benjamin; Fernie, Alisdair R.; Gebhardt, ChristianeBiochemical, molecular and genetic studies emphasize the role of the potato vacuolar invertase Pain-1 in the accumu- lation of reducing sugars in potato tubers upon cold storage, and thereby its influence on the quality of potato chips and French fries. Previous studies showed that natural Pain-1 cDNA alleles were associated with better chip quality and higher tuber starch content. In this study, we focused on the functional characterization of these alleles. A genotype- dependent transient increase of total Pain-1 transcript levels in cold-stored tubers of six different genotypes as well as allele-specific expression patterns were detected. 3D modelling revealed putative structural differences between allelic Pain-1 proteins at the molecule’s surface and at the substrate binding site. Furthermore, the yeast SUC2 mutant was complemented with Pain-1 cDNA alleles and enzy- matic parameters of the heterologous expressed proteins were measured at 30 and 4 °C. Significant differences between the alleles were detected. The observed functional differences between Pain-1 alleles did not permit final con- clusions on the mechanism of their association with tuber quality traits. Our results show that natural allelic variation at the functional level is present in potato, and that the heterozygous genetic background influences the manifesta- tion of this variation.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 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.
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