A Simple and Fast Kinetic Assay for the Determination of Fructan Exohydrolase Activity in Perennial Ryegrass (Lolium perenne L.)

International audienceDespite the fact that fructans are the main constituent of water-soluble carbohydrates in forage grasses and cereal crops of temperate climates, little knowledge is available on the regulation of the enzymes involved in fructan metabolism. The analysis of enzyme activities involved in this process has been hampered by the low affinity of the fructan enzymes for sucrose and fructans used as fructosyl donor. Further, the analysis of fructan composition and enzyme activities is restricted to specialized labs with access to suited HPLG equipment and appropriate fructan standards. The degradation of fructan polymers with high degree of polymerization (DP) by fructan exohydrolases (FEHs) to fructosyloligomers is important to liberate energy in the form of fructan, but also under conditions where the generation of low DP polymers is required. Based on published protocols employing enzyme coupled endpoint reactions in single cuvettes, we developed a simple and fast kinetic 1-FEH assay. This assay can be performed in multi-well plate format using plate readers to determine the activity of 1-FEH against 1-kestotriose, resulting in a significant time reduction. Kinetic assays allow an optimal and more precise determination of enzyme activities compared to endpoint assays, and enable to check the quality of any reaction with respect to linearity of the assay. The enzyme coupled kinetic 1-FEH assay was validated in a case study showing the expected increase in 1-FEH activity during cold treatment. This assay is cost effective and could be performed by any lab with access to a plate reader suited for kinetic measurements and readings at 340 nm, and is highly suited to assess temporal changes and relative differences in 1-FEH activities. Thus, this enzyme coupled kinetic 1-FEH assay is of high importance both to the field of basic fructan research and plant breeding

Cloning and characterization of a novel fructan 6-exohydrolase strongly inhibited by sucrose in Lolium perenne

Article de revue (Article scientifique dans une revue à comité de lecture)International audienceMain conclusion The first 6-fructan exohydrolase (6-FEH) cDNA from Lolium perenne was cloned and characterized. Following defoliation, Lp6 - FEHa transcript level unexpectedly decreased together with an increase in total FEH activity. Lolium perenne is a major forage grass species that accumulates fructans, mainly composed of β(2,6)-linked fructose units. Fructans are mobilized through strongly increased activities of fructan exohydrolases (FEHs), sustaining regrowth following defoliation. To understand the complex regulation of fructan breakdown in defoliated grassland species, the objective was to clone and characterize new FEH genes in L. perenne. To find FEH genes related to refoliation, a defoliated tiller base cDNA library was screened. Characterization of the recombinant protein was performed in Pichia pastoris. In this report, the cloning and enzymatic characterization of the first 6-FEH from L. perenne is described. Following defoliation, during fructan breakdown, Lp6-FEHa transcript level unexpectedly decreased in elongating leaf bases (ELB) and in mature leaf sheaths (tiller base) in parallel to increased total FEH activities. In comparison, transcript levels of genes coding for fructosyltransferases (FTs) involved in fructan biosynthesis also decreased after defoliation but much faster than FEH transcript levels. Since Lp6-FEHa was strongly inhibited by sucrose, mechanisms modulating FEH activities are discussed. It is proposed that differences in the regulation of FEH activity among forage grasses influence their tolerance to defoliation.</p

Short-term effects of defoliation intensity on sugar remobilization and N fluxes in ryegrass

International audienceIn grassland plant communities, the ability of individual plants to regrow after defoliation is of crucial importance since it allows the restoration of active photosynthesis and plant growth. The aim of this study was to evaluate the effects of increasing defoliation intensity (0, 25, 65, 84, and 100% of removed leaf area) on sugar remobilization and N uptake, remobilization, and allocation in roots, adult leaves, and growing leaves of ryegrass over 2 days, using a N-15 tracer technique. Increasing defoliation intensity decreased plant N uptake in a correlative way and increased plant N remobilization, but independently. The relative contribution of N stored before defoliation to leaf growth increased when defoliation intensity was severe. In most conditions, root N reserves also contributed to leaf regrowth, but much less than adult leaves and irrespective of defoliation intensity. A threshold of defoliation intensity (65% leaf area removal) was identified below which C (glucose, fructose, sucrose, fructans), and N (amino acids, soluble proteins) storage compounds were not recruited for regrowth. By contrast, nitrate content increased in elongating leaf bases above this threshold. Wounding associated with defoliation is thus not the predominant signal that triggers storage remobilization and controls the priority of resource allocation to leaf meristems. A framework integrating the sequential events leading to the refoliation of grasses is proposed on the basis of current knowledge and on the findings of the present work

Les conditions de travail au Québec : Une analyse différenciée selon les groupes d’âge

Cet article présente les travaux d’une communauté de chercheurs du Réseau de recherche en santé et en sécurité du travail du Québec (RRSSTQ), créée en 2006, laquelle s’intéresse aux questions d’âges et de rapports sociaux en milieux de travail, en lien avec les conditions de travail et la santé et la sécurité du travail. De disciplines diverses (ergonomie, sociologie, psychosociologie, communication, droit, éducation), cette communauté réalise des recherches à partir d’enquêtes et d’études de terrain s’appuyant sur une approche compréhensive des phénomènes. Cette problématique se situe dans un contexte social singulier marqué, entre autres, par la mondialisation des marchés, une introduction massive des nouvelles technologies, une transformation des formes d’emploi, ainsi qu’un vieillissement de la main-d’oeuvre dans les pays industrialisés. Ces transformations créent des conditions particulières de rétention et d’intégration de la main-d’oeuvre dans les milieux de travail. À partir des données de l’Enquête québécoise sur des conditions de travail, d’emploi et de santé et de sécurité du travail (EQCOTESST), réalisée en 2008, auprès d’un échantillon représentatif de 5 071 répondants, l’objectif de cette étude est de tracer un portrait des conditions de travail en fonction de l’âge des travailleurs au Québec. L’article propose l’interprétation de ce portrait à partir d’un cadre d’analyse s’appuyant sur une approche diachronique des dynamiques âge-travail-santé dans le but de mieux orienter les interventions en milieu de travail.This article addresses the issues of age and social relations at work with regards to working conditions and health and safety at work. This study is the results of a group of scholars from disciplines such as ergonomics, sociology, psycho-sociology, communications, law and education using surveys and field studies based on a comprehensive approach of the phenomena in the Quebec Occupational Health and Safety Research Network created in 2006. The issue arises in a specific social context marked, among others, by the globalization of markets, a large influx of new technologies, a transformation of employment patterns as well as an aging work force in industrialized nations. These transformations create particular conditions for the retention and the integration of the work force within the workplace. Using data extracted from the 2008 Étude québécoise sur des conditions de travail, d’emploi et de santé et de sécurité du travail (EQCOTESST, Québec, Survey on Working and Employment Conditions and Occupational Health and Safety) conducted with a representative sampling of 5,071 respondents, this study aims at providing a snapshot of the working conditions of Quebec workers (Canada) by age group. This article offers an interpretation of the profile from an analytical standpoint using a diachronic approach of the age-work-health dynamics in order to better guide interventions in the workplace.Este artículo presenta los trabajos de una comunidad de investigadores de la Red de investigación en salud y seguridad ocupacional del Quebec, que fue creada en 2006 y que se interesa a las cuestiones de edad y de relaciones sociales en los medios de trabajo, en vínculo con las condiciones de trabajo y de la salud y seguridad ocupacional. Incluyendo diversas disciplinas (ergonomía, sociología, psico-sociología, comunicación, derecho, educación), esta comunidad realiza sus investigaciones a partir de encuestas y estudios de terreno, apoyándose en un enfoque comprensivo de los fenómenos. Esta problemática se sitúa en un contexto social singular marcado, entre otros, por la mundialización de mercados, una introducción masiva de nuevas tecnologías, una transformación de formas de empleo, así como por el envejecimiento de la mano de obra en los países industrializados. Estas transformaciones crean condiciones particulares de retención e integración de la mano de obra en los medios de trabajo. A partir de los datos de la Encuesta quebequense sobre las condiciones de trabajo, de empleo y de salud-seguridad ocupacional, realizada en 2008, con una muestra representativa de 5 071 participantes, el objetivo de este estudio es de esbozar un estado de situación de las condiciones de trabajo en función de la edad de los trabajadores en Quebec. El artículo propone la interpretación de este estado de situación a partir de un marco de análisis sustentado por un enfoque diacrónico de las dinámicas edad-trabajo-salud, y esto, con el objetivo de orientar mejor las intervenciones en el medio laboral

Impacts of phosphate limitation and its interactions with nitrogen fertilization on the agronomic performances and nutrient use efficiencies of rapeseed

National audienc

The resilience of perennial grasses under two climate scenarios is correlated with carbohydrate metabolism in meristems

Extreme climatic events (ECEs) such as droughts and heat waves affect ecosystem functioning and species turnover. This study investigated the effect of elevated CO2 on species' resilience to ECEs. Monoliths of intact soil and their plant communities from an upland grassland were exposed to 2050 climate scenarios with or without an ECE under ambient (390 ppm) or elevated (520 ppm) CO2 . Ecophysiological traits of two perennial grasses (Dactylis glomerate and Holcus lanatus) were measured before, during, and after ECE. At similar soil water content, leaf elongation was greater under elevated CO2 for both species. The resilience of D. glomerate increased under enhanced CO2 (+60%) whereas H. lanatus mostly died during ECE. D. glomerate accumulated 30% more fructans, which were more highly polymerized, and 4-fold less sucrose than H. lanatus. The fructan concentration in leaf meristems was significantly increased under elevated CO2. Their relative abundance changed during the ECE, resulting in a more polymerized assemblage in H. lanatus and a more depolymerized assemblage in D. glomerate. The ratio of low degree of polymerization fructans to sucrose in leaf meristems was the best predictor of resilience across species. This study underlines the role of carbohydrate metabolism and the species-dependent effect of elevated CO2 on the resilience of grasses to ECE

Sweet specificities of the root extracellular trap of perennial ryegrass (Lolium perenne), a fructan accumulating plant

International audiencePerennial ryegrass (Lolium perenne) is a fructan-accumulating plant constituting one of the most important grassland species with high herbage production, nutritive value and digestibility for grazing cattle. Although fructans were reported to be involved in plant defense acting as antioxidants or stress signals, their contribution in root protection is still to be explored. In roots, atypical defense is provided by the “Root Extracellular Trap” or “RET” at the root-soil interface. The molecular composition and structural organization of the RET are essential to provide root defense against pathogen attacks and abiotic stresses. The RET was reported to be mainly composed of polysaccharides (homogalacturonan, xylogalacturonan, xyloglucan) and proteoglycans such as arabinogalactan proteins (AGPs). Our aim is to characterize the RET composition of L. perenne using cell imaging techniques and a wide range of monoclonal antibodies directed against epitopes from cell wall glycomolecules and to investigate the potential presence of fructans. Interestingly, we found that both mucilage and cell wall surface of border cells were enriched in AGP epitopes. An increased amount of the AGP-containing mucilage was produced by L. perenne root tip in response to both elicitor and osmotic stress. Fructan epitopes were also detected in root cap cells and appeared to be released in the RET under stress conditions. Taken together our findings suggest that AGPs together with fructans are involved in root response of L. perenne to environmental stresses

Nitrous Oxide: What Do We Know about Its Use Disorder Potential? Results of the French Monitoring Centre for Addiction Network Survey and Literature Review

International audienceBackground: Nitrous oxide (N2O) is used worldwide for analgesia and anesthesia. It is also used for recreational purposes by some people. N2O can have major side effects (myeloneuropathy, delusions, emphysema) when used to excess. In France, N2O is available as an equimolar mixture of oxygen and nitrous oxide (EMONO). Its substance use disorder potential is monitored by the French Monitoring Centre for Addiction (CEIP-A) network. Our objective is to provide an overview of the substance use disorder potential of N2O in general, and of EMONO in particular. Methods: This paper is based on a systematic review of the literature for case reports involving N2O use disorder and on CEIP-A network cases involving EMONO use disorder. We characterized use disorder in accordance with DSM-5 (Diagnostic and Statistical Manual of Mental Disorders, 5th Edition) criteria. Furthermore, we considered data relating to medical history, age, gender, and N2O consumption habits. Results: We identified 59 cases of N2O use disorder in the literature and 17 cases of EMONO use disorder from the CEIP-A network. More than 90% of the cases used N2O in larger quantities and for longer than intended. Conversely, more negative as opposed to positive cases have been documented regarding tolerance and failed attempts to reduce usage. Conclusions: A specific profile of substance use disorder starts to emerge from all the cases studied here. Furthermore, we identified another way N2O use disorder can appear: through exposure for medical purposes