Rôle des phosphatases acides dans le métabolisme et le stockage des sucres dans la vacuole chez Arabidopsis thaliana (approches in vitro et in silico)

Les Purple Acid Phosphatases (PAPs) sont des phosphatases acides présentes dans le règne végétal et animal. Elles sont caractérisées à la fois par la présence de sept acides aminés conservés impliqués dans la structure d un centre bimétallique et par leur couleur rose/violète lorsqu elles se trouvent en solution. Chez les plantes, certaines PAPs présenteraient à la fois une activité phosphatase sur des métabolites (PEP, G6P ) et sur des phospho-peptides (glycosidases ), d une part, et une activité peroxydase, de l autre. Ces PAPs pourraient donc, non seulement être impliquées dans le métabolisme carboné, mais aussi dans la détoxication des espèces réactives de l oxygène. On compte à ce jour 29 gènes codant pour des PAPs chez Arabidopsis thaliana , dont un certain nombre est induit par la carence en Pi et par le stress oxydatif ou la senescence. Parmi eux, le gène de l AtPAP26 (At5g34850) présente de très fortes homologies de séquence avec des orthologues présents chez d autres plantes. La protéine AtPAP26, qui présente in vitro une activité phosphatase sur le PEP et le G6P et une activité peroxydase, est sécrétée même si de nombreux indices laissent penser qu elle pourrait également être localisée dans la vacuole.Notre objectif est d étudier le rôle d AtPAP26 dans le métabolisme et le stockage des sucres dans la vacuole. Notre hypothèse de départ est que la protéine AtPAP26 pourrait intervenir via son activité phosphatase dans un cycle de substrat vacuolaire impliquant le glucose-6-phosphate et le glucose. Nous avons montré que chez le mutant pap26, l activité glucose-6-phosphatase, mesurée in vitro et in organello, chute de manière importante. Parallèlement, chez le mutant gpt2, le transport tonoplastique des hexoses phosphates diminue de façon significative. Des expériences de marquages isotopiques sur du tissu racinaire en culture ont montré qu une part importante du glucose et du fructose ne provient pas de l hydrolyse du saccharose et que AtPAP26 est en partie responsable de cette synthèse. Enfin, par une approche in-silico, nous avons modélisé le métabolisme des sucres dans un système compartimenté impliquant la vacuole et ses transporteurs. La confrontation du modèle avec deux jeux de données expérimentales (concentrations à l état stationnaire et cinétiques de marquage) a permis de confirmer l existence d une activité hydrolytique sur les hexoses phosphates vacuolaires.Purple Acid Phosphatases (PAPs) are acid phosphatases found both in animal and vegetal kingdoms. They are characterized by the presence of seven conserved amino acids involved in a di-metal center and by their pink/purple color in solution. In plants, some PAPs exhibit an acid phosphatase activity on various phospho-esters and on phospho-peptides. An alcalin peroxidase activity has been demonstrated in vitro. Therefore, PAPs could be implicated both in the carbon metabolism and in the scavenging of reactive oxygen species. To date, 29 different PAP-encoding genes were identified in the Arabidopsis thaliana genome. Some of them are induced by phosphate deprivation, oxidative burst or senescence. The gene encoding the protein AtPAP26 (At5g34850) exhibits a high sequence similarity with orthologous from other plants. The AtPAP26 protein, showing both a phosphatase activity on PEP, G6P and F6P, and a peroxidase activity, is one of the most abundant root-secreted acid phosphatases but also appears to be localized in the vacuole. Our aim is to study the implication of AtPAP26 in the metabolism and storage of sugars in the vacuole. Our first hypothesis is that AtPAP26, via its phosphatase activity, could be involved in a glucose / glucose-6-phosphate cycle localized in the vacuole. We showed that the glucose-6-phosphatase activity in the pap26 mutant is highly affected both in vitro and in organello compared to the wild type. At the same time, the hexose phosphate transport capacity of the vacuole is highly affected in the gpt2 mutant compared to the wild-type. Moreover, isotopic labeling experiments performed on cultured root tissues have shown that a significant part of the intracellular glucose and fructose pool does not originate from sucrose and that AtPAP26 is, at least in part, responsible for this synthesis. Finally, we build a kinetic model of sugar metabolism in a compartmented system which has been validated by using two independent experimental data sets (steady state concentrations and kinetics of labeling of soluble sugars). This in silico modeling approach confirmed the involvement of a hexose phosphatase activity localized in the vacuole.BORDEAUX1-Bib.electronique (335229901) / SudocSudocFranceF

Comparison between elementary flux modes analysis and 13C-metabolic fluxes measured in bacterial and plant cells

<p>Abstract</p> <p>Background</p> <p>¹³C metabolic flux analysis is one of the pertinent ways to compare two or more physiological states. From a more theoretical standpoint, the structural properties of metabolic networks can be analysed to explore feasible metabolic behaviours and to define the boundaries of steady state flux distributions. Elementary flux mode analysis is one of the most efficient methods for performing this analysis. In this context, recent approaches have tended to compare experimental flux measurements with topological network analysis.</p> <p>Results</p> <p>Metabolic networks describing the main pathways of central carbon metabolism were set up for a bacteria species (<it>Corynebacterium glutamicum</it>) and a plant species (<it>Brassica napus</it>) for which experimental flux maps were available. The structural properties of each network were then studied using the concept of elementary flux modes. To do this, coefficients of flux efficiency were calculated for each reaction within the networks by using selected sets of elementary flux modes. Then the relative differences - reflecting the change of substrate <it>i.e</it>. a sugar source for <it>C</it>. <it>glutamicum </it>and a nitrogen source for <it>B</it>. <it>napus </it>- of both flux efficiency and flux measured experimentally were compared. For both organisms, there is a clear relationship between these parameters, thus indicating that the network structure described by the elementary flux modes had captured a significant part of the metabolic activity in both biological systems. In <it>B</it>. <it>napus</it>, the extension of the elementary flux mode analysis to an enlarged metabolic network still resulted in a clear relationship between the change in the coefficients and that of the measured fluxes. Nevertheless, the limitations of the method to fit some particular fluxes are discussed.</p> <p>Conclusion</p> <p>This consistency between EFM analysis and experimental flux measurements, validated on two metabolic systems allows us to conclude that elementary flux mode analysis could be a useful tool to complement ¹³C metabolic flux analysis, by allowing the prediction of changes in internal fluxes before carbon labelling experiments.</p

Near-infrared spectroscopy of a heterogeneous turbid system containing distributed absorbers

In most biological tissues, absorbers such as blood in the blood vessels are localized within a low-absorbing background medium. To study the effect of distributed absorbers on the near infrared reflectance, we developed a Monte Carlo code and performed time-domain measurements on heterogeneous tissue-vessel models. The models were made of low absorbing polyester resin mixed with TiO_2 as scatters. A series of tubes with diameters of 3.2 or 6.4 mm were made in the resin sample. The volume ratio of the tubes to the total sample is about 20%. During the measurement, these tubes were filled with turbid fluids with different absorption coefficients to simulate blood in various oxygenation states. We found that the apparent absorption coefficient of the resin/tube system, determined by using the diffusion equation fit, can be approximated by a volume-weighted sum of the absorption coefficients of the different absorbing components. This approximation has to be replaced by a more complex expression if the difference in absorption between the absorbers and background is very large (approximately 20 times). The results of the tissue phantom study are supported by the Monte Carlo simulation. Possible explanations for the photon migration in this kind of heterogeneous system is also presented

Determination of blood oxygenation in the brain by time-resolved reflectance spectroscopy: influence of the skin, skull, and meninges

Near infrared light has been used for the determination of blood oxygenation in the brain but little attention has been paid to the fact that the states of blood oxygenation in arteries, veins, and capillaries differ substantially. In this study, Monte Carlo simulations for a heterogeneous system were conducted, and near infrared time-resolved reflectance measurements were performed on a heterogeneous tissue phantom model. The model was made of a solid polyester resin, which simulates the tissue background. A network of tubes was distributed uniformly through the resin to simulate the blood vessels. The time-resolved reflectance spectra were taken with different absorbing solutions filled in the network. Based on the simulation and experimental results, we investigated the dependence of the absorption coefficient obtained from the heterogeneous system on the absorption of the actual absorbing solution filled in the tubes. We show that light absorption by the brain should result from the combination of blood and blood-free tissue background

Impaired cell growth under ammonium stress explained by modeling the energy cost of vacuole expansion in tomato leaves

Ammonium (NH4+)-based fertilization efficiently mitigates the adverse effects of nitrogen fertilization on the environment. However, high concentrations of soil NH4+ provoke growth inhibition, partly caused by the reduction of cell enlargement and associated with modifications of cell composition, such as an increase of sugars and a decrease in organic acids. Cell expansion depends largely on the osmotic-driven enlargement of the vacuole. However, the involvement of subcellular compartmentation in the adaptation of plants to ammonium nutrition has received little attention, until now. To investigate this, tomato (Solanum lycopersicum) plants were cultivated under nitrate and ammonium nutrition and the fourth leaf was harvested at seven developmental stages. The vacuolar expansion was monitored and metabolites and inorganic ion contents, together with intracellular pH, were determined. A data-constrained model was constructed to estimate subcellular concentrations of major metabolites and ions. It was first validated at the three latter developmental stages by comparison with subcellular concentrations obtained experimentally using non-aqueous fractionation. Then, the model was used to estimate the subcellular concentrations at the seven developmental stages and the net vacuolar uptake of solutes along the developmental series. Our results showed ammonium nutrition provokes an acidification of the vacuole and a reduction in the flux of solutes into the vacuoles. Overall, analysis of the subcellular compartmentation reveals a mechanism behind leaf growth inhibition under ammonium stress linked to the higher energy cost of vacuole expansion, as a result of alterations in pH, the inhibition of glycolysis routes and the depletion of organic acids.TP benefited from a cotutelle PhD (University of Bordeaux and University of the Basque Country) and thanks the University of the Basque Country (UPV/EHU, Spain) for his PhD grant during the execution of this work. This research was financially supported by the Basque Government (IT-932-16) and the Spanish Government (BIO2017-84035-R co-funded by Fondo Europeo para el Desarrollo Regional [FEDER]). Analytics were supported by MetaboHUB (ANR-11-INBS-0010) and PHENOME (ANR-11-INBS-0012) projects. Technical support was provided by Cedric Cassan, Ana Renovales and Mandy Bordas. The authors also thank SGIker (UPV/EHU, FEDER, EU) for the technical and human support provided

Near-infrared spectroscopy of a heterogeneous turbid system containing distributed absorbers

In most biological tissues, absorbers such as blood in the blood vessels are localized within a low-absorbing background medium. To study the effect of distributed absorbers on the near infrared reflectance, we developed a Monte Carlo code and performed time-domain measurements on heterogeneous tissue-vessel models. The models were made of low absorbing polyester resin mixed with TiO_2 as scatters. A series of tubes with diameters of 3.2 or 6.4 mm were made in the resin sample. The volume ratio of the tubes to the total sample is about 20%. During the measurement, these tubes were filled with turbid fluids with different absorption coefficients to simulate blood in various oxygenation states. We found that the apparent absorption coefficient of the resin/tube system, determined by using the diffusion equation fit, can be approximated by a volume-weighted sum of the absorption coefficients of the different absorbing components. This approximation has to be replaced by a more complex expression if the difference in absorption between the absorbers and background is very large (approximately 20 times). The results of the tissue phantom study are supported by the Monte Carlo simulation. Possible explanations for the photon migration in this kind of heterogeneous system is also presented

Determination of blood oxygenation in the brain by time-resolved reflectance spectroscopy: influence of the skin, skull, and meninges

Near infrared light has been used for the determination of blood oxygenation in the brain but little attention has been paid to the fact that the states of blood oxygenation in arteries, veins, and capillaries differ substantially. In this study, Monte Carlo simulations for a heterogeneous system were conducted, and near infrared time-resolved reflectance measurements were performed on a heterogeneous tissue phantom model. The model was made of a solid polyester resin, which simulates the tissue background. A network of tubes was distributed uniformly through the resin to simulate the blood vessels. The time-resolved reflectance spectra were taken with different absorbing solutions filled in the network. Based on the simulation and experimental results, we investigated the dependence of the absorption coefficient obtained from the heterogeneous system on the absorption of the actual absorbing solution filled in the tubes. We show that light absorption by the brain should result from the combination of blood and blood-free tissue background

Controle et regulation du metabolisme energetique de la levure Saccharomyces cerevisiae

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 80329 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Site-Specific Reduction of Oxidative and Lipid Metabolism in Adipose Tissue of 3′-Azido-3′-Deoxythymidine-Treated Rats

Although it is well accepted that treatment with some nucleoside reverse transcriptase inhibitors modifies both fat metabolism and fat distribution in humans, the mechanisms underlying these modifications are not yet known. The present investigation examined whether a decrease in oxidative capacity, induced by a chronic oral administration of 3′-azido-3′-deoxythymidine (AZT) in rats, could be associated with an alteration of the lipogenic capacity of white adipose tissues. The impact of obesity as a factor was then evaluated. Results showed that AZT treatment induced differential effects depending on anatomical localization. Indeed, in the inguinal adipose tissue, the specific activities of cytochrome c oxidase and fatty acid synthase, two rate-controlling enzymes in energy and lipogenic metabolisms, respectively, both decreased under AZT treatment, thus leading to a lowered cell lipid accumulation. Moreover, the AMP-activated protein kinase phosphorylation level tended to increase, thus implying that AZT causes an energy imbalance. Furthermore, the inguinal tissue of obese rats presented a sensitivity to AZT treatment that was higher than that of lean rats. In contrast, for epididymal tissue, no significant change in all these parameters could be detected under AZT treatment, regardless of the nutritional status of the animals. Taken together, these data demonstrate differential effects of AZT on subcutaneous adipose tissue and visceral white adipose tissue. It could be considered that the chronic decreases in energy and lipogenic metabolism of inguinal adipocyte, consecutive to AZT treatment, may lead, in the long term, to adipose tissue atrophy

Le château de Broue (commune de Saint-Sornin).

La tour de Broue (commune de Saint-Sornin) domine de nos jours un paysage à l'histoire contrastée. Située à l'extrémité d'un promontoire qui s'avance dans l'ancien golfe de Saintonge, elle permet d'embrasser les 16 000 hectares d'anciens marais salants reconvertis depuis le début du XIXe siècle en pâturages. Dans cet environnement très spécifique, cette ruine a de tout temps marqué les esprits des visiteurs. Broue était une place fortifiée d'origine comtale mais également un centre économique dont la prospérité reposait sur le commerce du sel. Le château commandait une vaste région côtière, coincée entre les châtellenies de Mornac et de Soubise attestées dès le XIe siècle, soit les estuaires de la Seudre et de la Charente. Elle s'étendait, d'est en ouest, de la forêt de Baconnais jusqu'à l'océan et, du sud au nord, de la Seudre jusqu'au havre de Brouage et même au-delà, jusqu'aux bourgs sauniers de Saint-Symphorien, Saint-Jean-d'Angle et Saint-Agnant. Dès le XIe siècle, ces marais salants devinrent un enjeu économique et commercial majeur pour l'aristocratie de la région, tant religieuse que laïque. La tour de Broue en représente la traduction matérielle, que ce soit du temps de la possession directe par l'administration comtale ou de celui des seigneurs héréditaires, anciens gardiens du château. Symboles de ce pouvoir aristocratique, les restes du site castral de Broue consistent en une tour entourée d'une chemise partiellement conservée