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Dive into the research topics where Danilo M. Daloso is active.

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Featured researches published by Danilo M. Daloso.


Plant Cell and Environment | 2015

Tobacco guard cells fix CO2 by both Rubisco and PEPcase while sucrose acts as a substrate during light-induced stomatal opening.

Danilo M. Daloso; Werner C. Antunes; Daniela Pereira Pinheiro; Jardel P. Waquim; Wagner L. Araújo; Marcelo Ehlers Loureiro; Alisdair R. Fernie; Thomas C.R. Williams

Transcriptomic and proteomic studies have improved our knowledge of guard cell function; however, metabolic changes in guard cells remain relatively poorly understood. Here we analysed metabolic changes in guard cell-enriched epidermal fragments from tobacco during light-induced stomatal opening. Increases in sucrose, glucose and fructose were observed during light-induced stomatal opening in the presence of sucrose in the medium while no changes in starch were observed, suggesting that the elevated fructose and glucose levels were a consequence of sucrose rather than starch breakdown. Conversely, reduction in sucrose was observed during light- plus potassium-induced stomatal opening. Concomitant with the decrease in sucrose, we observed an increase in the level as well as in the (13) C enrichment in metabolites of, or associated with, the tricarboxylic acid cycle following incubation of the guard cell-enriched preparations in (13) C-labelled bicarbonate. Collectively, the results obtained support the hypothesis that sucrose is catabolized within guard cells in order to provide carbon skeletons for organic acid production. Furthermore, they provide a qualitative demonstration that CO2 fixation occurs both via ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPcase). The combined data are discussed with respect to current models of guard cell metabolism and function.


Plant Physiology | 2016

Enhanced Photosynthesis and Growth in atquac1 Knockout Mutants Are Due to Altered Organic Acid Accumulation and an Increase in Both Stomatal and Mesophyll Conductance

David B. Medeiros; Samuel C. V. Martins; João Henrique F. Cavalcanti; Danilo M. Daloso; Enrico Martinoia; Adriano Nunes-Nesi; Fábio M. DaMatta; Alisdair R. Fernie; Wagner L. Araújo

Impaired stomatal closure in a anion channel mutant is accompanied by increased mesophyll conductance, photosynthesis, and leaf area, ultimately enhancing biomass accumulation under controlled conditions. 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.


New Phytologist | 2016

Roles of sucrose in guard cell regulation

Danilo M. Daloso; Letícia dos Anjos; Alisdair R. Fernie

The control of stomatal aperture involves reversible changes in the concentration of osmolytes in guard cells. Sucrose has long been proposed to have an osmolytic role in guard cells. However, direct evidence for such a role is lacking. Furthermore, recent evidence suggests that sucrose may perform additional roles in guard cells. Here, we provide an update covering the multiple roles of sucrose in guard cell regulation, highlighting the knowledge accumulated regarding spatiotemporal differences in the synthesis, accumulation, and degradation of sucrose as well as reviewing the role of sucrose as a metabolic connector between mesophyll and guard cells. Analysis of transcriptomic data from previous studies reveals that several genes encoding sucrose and hexose transporters and genes involved in gluconeogenesis, sucrose and trehalose metabolism are highly expressed in guard cells compared with mesophyll cells. Interestingly, this analysis also showed that guard cells have considerably higher expression of C4 -marker genes than mesophyll cells. We discuss the possible roles of these genes in guard cell function and the role of sucrose in stomatal opening and closure. Finally, we provide a perspective for future experiments which are required to fill gaps in our understanding of both guard cell metabolism and stomatal regulation.


Plant Physiology | 2016

Relationships of Leaf Net Photosynthesis, Stomatal Conductance, and Mesophyll Conductance to Primary Metabolism: A Multispecies Meta-Analysis Approach

Jorge Gago; Danilo M. Daloso; Carlos M. Figueroa; Jaume Flexas; Alisdair R. Fernie; Zoran Nikoloski

Multispecies meta-analysis of metabolic profiles reveals conserved and species-specific relationships between metabolism, leaf conductances, and net photosynthesis. Plant metabolism drives plant development and plant-environment responses, and data readouts from this cellular level could provide insights in the underlying molecular processes. Existing studies have already related key in vivo leaf gas-exchange parameters with structural traits and nutrient components across multiple species. However, insights in the relationships of leaf gas-exchange with leaf primary metabolism are still limited. We investigated these relationships through a multispecies meta-analysis approach based on data sets from 17 published studies describing net photosynthesis (A) and stomatal (gs) and mesophyll (gm) conductances, alongside the 53 data profiles from primary metabolism of 14 species grown in different experiments. Modeling results highlighted the conserved patterns between the different species. Consideration of species-specific effects increased the explanatory power of the models for some metabolites, including Glc-6-P, Fru-6-P, malate, fumarate, Xyl, and ribose. Significant relationships of A with sugars and phosphorylated intermediates were observed. While gs was related to sugars, organic acids, myo-inositol, and shikimate, gm showed a more complex pattern in comparison to the two other traits. Some metabolites, such as malate and Man, appeared in the models for both conductances, suggesting a metabolic coregulation between gs and gm. The resulting statistical models provide the first hints for coregulation patterns involving primary metabolism plus leaf water and carbon balances that are conserved across plant species, as well as species-specific trends that can be used to determine new biotechnological targets for crop improvement.


Trends in Plant Science | 2017

The Unprecedented Versatility of the Plant Thioredoxin System

Peter Geigenberger; Ina Thormählen; Danilo M. Daloso; Alisdair R. Fernie

Thioredoxins are ubiquitous enzymes catalyzing reversible disulfide-bond formation to regulate structure and function of many proteins in diverse organisms. In recent years, reverse genetics and biochemical approaches were used to resolve the functions, specificities, and interactions of the different thioredoxin isoforms and reduction systems in planta and revealed the most versatile thioredoxin system of all organisms. Here we review the emerging roles of the thioredoxin system, namely the integration of thylakoid energy transduction, metabolism, gene expression, growth, and development under fluctuating environmental conditions. We argue that these new developments help us to understand why plants organize such a divergent composition of thiol redox networks and provide insights into the regulatory hierarchy that operates between them.


Plant Cell and Environment | 2015

Utilizing systems biology to unravel stomatal function and the hierarchies underpinning its control

David B. Medeiros; Danilo M. Daloso; Alisdair R. Fernie; Zoran Nikoloski; Wagner L. Araújo

Stomata control the concomitant exchange of CO2 and transpiration in land plants. While a constant supply of CO2 is need to maintain the rate of photosynthesis, the accompanying water losses must be tightly regulated to prevent dehydration and undesired metabolic changes. The factors affecting stomatal movement are directly coupled with the cellular networks of guard cells. Although the guard cell has been used as a model for characterization of signaling pathways, several important questions about its functioning remain elusive. Current modeling approaches describe the stomatal conductance in terms of relatively few easy-to-measure variables being unsuitable for in silico design of genetic manipulation strategies. Here, we argue that a system biology approach, combining modeling and high-throughput experiments, may be used to elucidate the mechanisms underlying stomata control and to determine targets for modulation of stomatal responses to environment. In support of our opinion, we review studies demonstrating how high-throughput approaches have provided a systems-view of guard cells. Finally, we emphasize the opportunities and challenges of genome-scale modeling and large-scale data integration for in silico manipulation of guard cell functions to improve crop yields, particularly under stress conditions which are of pertinence both to climate change and water use efficiency.


Frontiers in Plant Science | 2012

Phosphonate Analogs of 2-Oxoglutarate Perturb Metabolism and Gene Expression in Illuminated Arabidopsis Leaves

Wagner L. Araújo; Takayuki Tohge; Adriano Nunes-Nesi; Danilo M. Daloso; Mhairi Nimick; Ina Krahnert; Victoria I. Bunik; Greg B. G. Moorhead; Alisdair R. Fernie

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.


Oncotarget | 2016

Inhibition of mitochondrial 2-oxoglutarate dehydrogenase impairs viability of cancer cells in a cell-specific metabolism-dependent manner

Victoria I. Bunik; Garik Mkrtchyan; Aneta Grabarska; Henry Oppermann; Danilo M. Daloso; Wagner L. Araújo; Małgorzata Juszczak; Wojciech Rzeski; Lucien Bettendorff; Alisdair R. Fernie; Jürgen Meixensberger; Andrzej Stepulak; Frank Gaunitz

2-Oxoglutarate dehydrogenase (OGDH) of the tricarboxylic acid (TCA) cycle is often implied to be inactive in cancer, but this was not experimentally tested. We addressed the question through specific inhibition of OGDH by succinyl phosphonate (SP). SP action on different cancer cells was investigated using indicators of cellular viability and reactive oxygen species (ROS), metabolic profiling and transcriptomics. Relative sensitivity of various cancer cells to SP changed with increasing SP exposure and could differ in the ATP- and NAD(P)H-based assays. Glioblastoma responses to SP revealed metabolic sub-types increasing or decreasing cellular ATP/NAD(P)H ratio under OGDH inhibition. Cancer cell homeostasis was perturbed also when viability indicators were SP-resistant, e.g. in U87 and N2A cells. The transcriptomics database analysis showed that the SP-sensitive cells, such as A549 and T98G, exhibit the lowest expression of OGDH compared to other TCA cycle enzymes, associated with higher expression of affiliated pathways utilizing 2-oxoglutarate. Metabolic profiling confirmed the dependence of cellular SP reactivity on cell-specific expression of the pathways. Thus, oxidative decarboxylation of 2-oxoglutarate is significant for the interdependent homeostasis of NAD(P)H, ATP, ROS and key metabolites in various cancer cells. Assessment of cell-specific responses to OGDH inhibition is of diagnostic value for anticancer strategies.


Oncotarget | 2015

Specific inhibition by synthetic analogs of pyruvate reveals that the pyruvate dehydrogenase reaction is essential for metabolism and viability of glioblastoma cells

Victoria I. Bunik; Artem Artiukhov; Alexey V. Kazantsev; Renata L.S. Goncalves; Danilo M. Daloso; Henry Oppermann; Elena Kulakovskaya; N. V. Lukashev; Alisdair R. Fernie; Martin D. Brand; Frank Gaunitz

The pyruvate dehydrogenase complex (PDHC) and its phosphorylation are considered essential for oncotransformation, but it is unclear whether cancer cells require PDHC to be functional or silenced. We used specific inhibition of PDHC by synthetic structural analogs of pyruvate to resolve this question. With isolated and intramitochondrial PDHC, acetyl phosphinate (AcPH, KiAcPH = 0.1 μM) was a much more potent competitive inhibitor than the methyl ester of acetyl phosphonate (AcPMe, KiAcPMe = 40 μM). When preincubated with the complex, AcPH also irreversibly inactivated PDHC. Pyruvate prevented, but did not reverse the inactivation. The pyruvate analogs did not significantly inhibit other 2-oxo acid dehydrogenases. Different cell lines were exposed to the inhibitors and a membrane-permeable precursor of AcPMe, dimethyl acetyl phosphonate, which did not inhibit isolated PDHC. Using an ATP-based assay, dependence of cellular viability on the concentration of the pyruvate analogs was followed. The highest toxicity of the membrane-permeable precursor suggested that the cellular action of charged AcPH and AcPMe requires monocarboxylate transporters. The relevant cell-specific transcripts extracted from Gene Expression Omnibus database indicated that cell lines with higher expression of monocarboxylate transporters and PDHC components were more sensitive to the PDHC inhibitors. Prior to a detectable antiproliferative action, AcPH significantly changed metabolic profiles of the investigated glioblastoma cell lines. We conclude that catalytic transformation of pyruvate by pyruvate dehydrogenase is essential for the metabolism and viability of glioblastoma cell lines, although metabolic heterogeneity causes different cellular sensitivities and/or abilities to cope with PDHC inhibition.


Scientific Reports | 2017

Resolving the central metabolism of Arabidopsis guard cells

Semidán Robaina-Estévez; Danilo M. Daloso; Youjun Zhang; Alisdair R. Fernie; Zoran Nikoloski

Photosynthesis and water use efficiency, key factors affecting plant growth, are directly controlled by microscopic and adjustable pores in the leaf—the stomata. The size of the pores is modulated by the guard cells, which rely on molecular mechanisms to sense and respond to environmental changes. It has been shown that the physiology of mesophyll and guard cells differs substantially. However, the implications of these differences to metabolism at a genome-scale level remain unclear. Here, we used constraint-based modeling to predict the differences in metabolic fluxes between the mesophyll and guard cells of Arabidopsis thaliana by exploring the space of fluxes that are most concordant to cell-type-specific transcript profiles. An independent 13C-labeling experiment using isolated mesophyll and guard cells was conducted and provided support for our predictions about the role of the Calvin-Benson cycle in sucrose synthesis in guard cells. The combination of in silico with in vivo analyses indicated that guard cells have higher anaplerotic CO2 fixation via phosphoenolpyruvate carboxylase, which was demonstrated to be an important source of malate. Beyond highlighting the metabolic differences between mesophyll and guard cells, our findings can be used in future integrated modeling of multi-cellular plant systems and their engineering towards improved growth.

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Wagner L. Araújo

Universidade Federal de Viçosa

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Adriano Nunes-Nesi

Universidade Federal de Viçosa

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David B. Medeiros

Universidade Federal de Viçosa

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