Phospholipase activities associated with the tonoplast from Acer pseudoplatanus cells: identification of a phospholipase A1 activity

AbstractThe study of phospholipase activities associated with the tonoplast of Acer pseudoplatanus was performed in vitro with sn-2[14C]acylphosphatidylcholine (PC) as a substrate. The hydrolysis of radiolabelled PC into [14C]phosphatidic acid and [14C]lyso-PC demonstrated the presence of phospholipase D and A1 activities, respectively, associated with the tonoplast of Acer pseudoplatanus. The vacuolar sap did not show any significant phospholipase activity. In a second step, the properties of the phospholipase A1, activity was studied using tonoplast endogenous PC labelled in vivo with [l4C]choline as a substrate. The phospholipase A1 showed an optimal activity at pH about 6–6.5, did not necessarily require divalent cations, but was stimulated by Mg2+ and particularly by Ca2+. This work presents the first evidence for the presence of phospholipases A1 in plant cells

NO signaling functions in the biotic and abiotic stress responses

In the last 1990s, NO became an increasingly popular target of investigation in plants. As in mammals, NO fulfils a broad spectrum of signalling functions in pathophysiological processes in plants. Here we summarize studies published in recent years that provide novel insights into the signalling functions of NO produced by plant cells exposed to abiotic stresses and biotic stress (pathogen-derived elicitors). Particularly, we report that NO emerges as a key messenger governing the overall control of Ca2+ homeostasis. Although the precise signalling functions of NO are poorly understood, its capacity to modulate Ca2+ homeostasis provides an extraordinary and remarkable effective way of conveying information

Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Transbilayer distribution of phosphatidycholine and phosphatidyl-ethanolamine in the vacuolar menbrane of Acer pseudoplatanus cells

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Comparaison de différents éliciteurs des réactions de défense de la vigne (étude du mode d'action de la laminarine sulfatée, un inducteur de résistance à P. viticola)

La vigne, Vitis vinifera, est sensible à de nombreuses maladies cryptogamiques comme le mildiou, la pourriture grise ou l oïdium. La stimulation des défenses naturelles de la vigne après application de molécules d origine naturelle est l une des stratégies alternatives étudiées pour réduire l utilisation des produits phytosanitaires. Ce travail s inscrit dans l étude du mode d action de la laminarine sulfatée, PS3, chez la vigne. En utilisant le modèle de suspensions cellulaires de vigne, nous montrons que PS3, contrairement à la laminarine, n active pas les événements de signalisation normalement induits par les éliciteurs, excepté la dépolarisation de la membrane plasmique. Cependant le traitement foliaire de vigne par PS3 permet de réduire significativement l infection par Plasmopara viticola, agent responsable du mildiou. Cette résistance s observe notamment au niveau microscopique par une limitation drastique de la colonisation in planta et de l inhibition de la sporulation de l oomycète. Les approches complémentaires utilisées dans ce travail montrent que l efficacité de ce b-glucane sulfaté est liée à la potentialisation des voies de défense de la vigne, telles que la production d acide salicylique, le dépôt de callose et de composés phénoliques, la synthèse de phytoalexines et la production d H2O2. Ce travail s intéresse aussi à caractériser les mécanismes moléculaires déterminants pour l établissement de la résistance induite par PS3 contre P. viticola. L étude transcriptomique et l approche pharmacologique nous ont permis d identifier des gènes déterminants pour la résistance induite qui dépendent de l activité de canaux ioniques et de la production d H2O2.Grapevine, Vitis vinifera, is sensitive to a large spectrum of pathogens like downy mildew, grey mould or powdery mildew. Alternative strategies to control grape diseases include activation of defense which relies on the application of inducers of resistance. In this work, we study the action mechanism of sulfated laminarin, PS3, in grapevine. Using grapevine cell suspensions, we demonstrate that PS3 doesn t elicit a wider array of defense responses except plasma membrane potential variations. Likely, grapevine foliar treatment reduces significantly Plasmopara viticola infection, downy mildew pathogen. This resistance can be observed via microscopic approach by a drastic limiting of the colonization in planta and by the inhibition of the oomycete sporulation. By cytological and molecular approaches, we demonstrate that the efficiency of this sulfated b-glucan is associated with some defense responses priming, including salicylic acid production, callose and phenol depositions, phytoalexins synthesis and H2O2 production. This work is focused on the determining mechanisms of the PS3-induced resistance against P. viticola. Transcriptomic and pharmacological approaches lead us to identify determining genes for the induced resistance depending of ion channel activity and reactive oxygen species production.DIJON-BU Sciences Economie (212312102) / SudocSudocFranceF

Caractérisation de protéines kinases nucléaires activées dans des cellules de tabac en réponse à la crytogéine, un éliciteur des réactions de défense

La cryptogéine est un éliciteur protéique des réactions de défense secrété par l oomycète Phytophthora cryptogea, qui active chez le tabac des voies de transduction conduisant à la réponse hypersensible et la résistance systémique acquise. Si les éléments membranaires et cytosoliques de ces voies de transduction sont maintenant bien décrits, les seuls événements nucléaires mis en évidence suite à un traitement par la cryptogéine sont une élévation de la concentration en Ca2+ libre et la modulation de l expression d un certain nombre de gènes impliqués dans la défense. Nous nous sommes ainsi intéressés, au cours de ce travail de thèse, à rechercher et caractériser des activités protéines kinases nucléaires activées en réponse à un traitement par la cryptogéine. Nous avons ainsi montré l activation reproductible de 4 protéines kinases, dénommées d après leur masse moléculaire PK36, PK42, PK47 et PK80. Ces PK présentent des cinétiques et des spécificités d activité différentes. Différentes données suggèrent que la PK42 serait une SnRK2, tandis que des approches de purification ont permis d obtenir sans ambiguïté l identité des PK36 et PK47. La PK36 est ainsi une sous-unité a de CK2 ; la PK47 correspond à SIPK, une MAPK de tabac impliquée dans de nombreux stress et pour laquelle aucune donnée n était disponible quant à son activité nucléaire. Bien que l implication de la PK36 dans la réponse à la cryptogéine n ait pu être étudiée, des liens sont suggérés entre l activité nucléaire de SIPK et l établissement de la mort cellulaire.Cryptogein is a proteinaceous elicitor of plant defense reactions produced by the oomycete Phythophthora cryptogea, which induces a signaling pathway in tobacco cells leading to hypersensitive response and systemic acquired resistance. While the early membrane-localized and cytosolic events are well described, the only nuclear events characterized to date are the variations of free calcium concentration and defence-related genes expression. We were interested, during this thesis work, in the characterization of nuclear protein kinases activated in response to cryptogein. We showed that cryptogein induced the highly reproducible activation of 4 protein kinases, named PK36, PK42, PK47 and PK80, accordingly to their apparent molecular masses. These protein kinases exhibited different activation kinetics and activity specificities. Several data suggested that PK42 might belong to the SnRK2 family, while different purification approaches led us to the identification of PK36 and PK47. PK36 is a catalytic subunit of CK2; PK47 correspond to SIPK, a tobacco MAPK widely involved in stress responses, and for which no data was available on its nuclear activity. The involvement of PK36 in cryptogein signalling pathway could not be investigated, but a link is suggested between nuclear SIPK activity and the appearance of cell death.DIJON-BU Sciences Economie (212312102) / SudocSudocFranceF