Immuno-Glyco-Imaging in Plant Cells: Localization of Cell Wall Carbohydrate Epitopes and Their Biosynthesizing Enzymes

International audienc

Gelatin porous scaffolds fabricated using a modified gas foaming technique: Characterisation and cytotoxicity assessment

The current study presents an effective and simple strategy to obtain stable porous scaffolds from gelatin via a gas foaming method. The technique exploits the intrinsic foaming ability of gelatin in the presence of CO2 to obtain a porous structure stabilised with glutaraldehyde. The produced scaffolds were characterised using physical and mechanical characterisation methods. The results showed that gas foaming may allow the tailoring of the 3-dimensional structure of the scaffolds with an interconnected porous structure. To assess the effectiveness of the preparation method in mitigating the potential cytotoxicity risk of using glutaraldehyde as a crosslinker, direct and in-direct cytotoxicity assays were performed at different concentrations of glutaraldehyde. The results indicate the potential of the gas foaming method, in the preparation of viable tissue engineering scaffolds

Efficient preparation of Arabidopsis pollen tubes for ultrastructural analysis using chemical and cryo-fixation

The pollen tube (PT) serves as a model system for investigating plant cell growth and morphogenesis. Ultrastructural studies are indispensable to complement data from physiological and genetic analyses, yet an effective method is lacking for PTs of the model plant Arabidopsis thaliana. Methods: Here, we present reliable approaches for ultrastructural studies of Arabidopsis PTs, as well as an efficient technique for immunogold detection of cell wall epitopes. Using different fixation and embedding strategies, we show the amount of PT ultrastructural details that can be obtained by the different methods. Results: Dozens of cross-sections can be obtained simultaneously by the approach, which facilitates and shortens the time for evaluation. In addition to in vitro-grown PTs, our study follows the route of PTs from germination, growth along the pistil, to the penetration of the dense stylar tissue, which requires considerable mechanical forces. To this end, PTs have different strategies from growing between cells but also between the protoplast and the cell wall and even within each other, where they share a partly common cell wall. The separation of PT cell walls in an outer and an inner layer reported for many plant species is less clear in Arabidopsis PTs, where these cell wall substructures are connected by a distinct transition zone. Conclusions: The major advancement of this method is the effective production of a large number of longitudinal and cross-sections that permits obtaining a detailed and representative picture of pollen tube structures in an unprecedented way. This is particularly important when comparing PTs of wild type and mutants to identify even subtle alterations in cytoarchitecture. Arabidopsis is an excellent plant for genetic manipulation, yet the PTs, several-times smaller compared to tobacco or lily, represent a technical challenge. This study reveals a method to overcome this problem and make Arabidopsis PTs more amenable to a combination of genetic and ultrastructural analyses

Arabidopsis CSLD1 and CSLD4 are required for cellulose deposition and normal growth of pollen tubes

The cell wall is important for pollen tube growth, but little is known about the molecular mechanism that controls cell wall deposition in pollen tubes. Here, the functional characterization of the pollen-expressed Arabidopsis cellulose synthase-like D genes CSLD1 and CSLD4 that are required for pollen tube growth is reported. Both CSLD1 and CSLD4 are highly expressed in mature pollen grains and pollen tubes. The CSLD1 and CSLD4 proteins are located in the Golgi apparatus and transported to the plasma membrane of the tip region of growing pollen tubes, where cellulose is actively synthesized. Mutations in CSLD1 and CSLD4 caused a significant reduction in cellulose deposition in the pollen tube wall and a remarkable disorganization of the pollen tube wall layers, which disrupted the genetic transmission of the male gametophyte. In csld1 and csld4 single mutants and in the csld1 csld4 double mutant, all the mutant pollen tubes exhibited similar phenotypes: the pollen tubes grew extremely abnormally both in vitro and in vivo, which indicates that CSLD1 and CSLD4 are not functionally redundant. Taken together, these results suggest that CSLD1 and CSLD4 play important roles in pollen tube growth, probably through participation in cellulose synthesis of the pollen tube wall

Étude des mécanismes d'adhésion des tubes polliniques d'Arabidopsis thaliana par une approche de génétique chimique

Au cours de la reproduction sexuée des plantes, la germination du pollen et la croissance des tubes polliniques sont soumises à un contrôle spatio-temporel dans les tissus femelles afin d'acheminer les gamètes mâles jusqu'aux ovules. Le tube pollinique est une cellule à croissance rapide entourée d'une matrice extracellulaire riche en polysaccharides. La croissance polarisée des tubes polliniques et leurs capacités à percevoir des signaux externes permettent de les guider à travers le tissu de transmission de l'organe femelle. Parmi ces signaux, un mécanisme de guidage par adhésion a été postulé chez le lys. Peu de travaux concernent les tubes polliniques de la plante modèle Arabidopsis thaliana. Dans le but d'identifier les interactions impliquées dans l'adhésion des tubes polliniques, nous avons entrepris 1- de caractériser les composés pariétaux des tubes et des cellules du tissu de transmission qui sont la première interface de contact entre ces cellules ; 2 - d'utiliser une approche de génétique chimique pour identifier des molécules impliquées dans l'adhésion cellulaire. Nos résultats indiquent que la paroi des tubes polliniques d'Arabidopsis se compose d'une double paroi riche en callose, pour la paroi interne et en pectines et xyloglucanes pour la paroi externe. Nous avons alors caractérisé la structure des xyloglucanes et mis en évidence que ce polymère est fortement fucosylé et O-acétylé. Cette caractéristique structurale semble être conservée parmi les espèces. L'abondance de l'arabinose provenant des chaînes latérales du rhamnogalacturonane de type 1 a été également mise en évidence. Pour étudier l'adhésion, nous avons reconstitué une matrice à partir de polysaccharides pariétaux extraits de fleurs mais aussi d'organes végétatifs de la plante. Les tubes polliniques adhèrent sur des pectines extraites à l'imidazole de fleurs et de feuilles d'Arabidopsis. Cette matrice nous a permis de constituer un test d'adhésion assez efficace et significatif pour le soumettre à une analyse de génétique chimique. Cette méthode consiste à étudier des processus biologiques en les perturbant via le criblage de petites molécules chimiques. Parmi les 258 molécules en notre possession, cinq ont montré des résultats prometteurs en réduisant significativement l'adhésion des tubes polliniques. De plus, avec certaines molécules, un effet dose-réponse a pu être mis en évidence.During sexual plant reproduction, pollen germination and pollen tube growth require a tight spatial and temporal control while traveling through the female tissues in order to deliver properly the sperm cells to the ovules. Pollen tubes are unique, fast growing cells surrounded by a cell wall enriched in polysaccharides. They are tip-polarized cells able to perceive external signal cues to guide them through the transmitting tract of the female organs. Among these signals, guidance by adhesion has been postulated in lily. Few data are available concerning the cell wall of the pollen tube in the model plant Arabidopsis Thaliana. In order to identify the interactions implicated in pollen tube adhesion, we first characterized the cell wall components from the pollen tube and the transmitting tract tissue. Secondly, we used a chemical genetic approach to disturb cell adhesion. Our data indicate that Arabidopsis pollen tube cell wall is composed of a double cell wall, enriched in callose in the inner layer and pectins and xyloglucans in the outer layer. Characterization of the xyloglucan structure showed that it was highly fucosylated and O-acetylated. These structural features seemed to be preserved among species. In addition, the cell wall is composed of a large amount of arabinosyl residues coming from the rhamnogalacturonan-1 side chains. In order to identify molecules involved on pollen tube adhesion, we have used a chemical genetic approach. This new method uses small molecules to alter a specific biological process and identify protein(s) involved. To this end, we elaborated an artificial matrix where pollen tubes were able to adhere. The matrix constitutes of cell wall pectins extracted with imidazole from Arabidopsis flowers and vegetative organs. Then, the assay was used to screen 258 small molecules from our library. Our data showed that five out of the 258 tested molecules, were able to disturb pollen tube adhesion in a dose-dependent manner

Etude observationnelle de l'utilisation des immunoglobulines polyvalentes par voie intraveineuse, dans la prise en charge du rejet humoral de greffe rénale

PARIS-BIUP (751062107) / SudocSudocFranceF

Bon usage des antifongiques systèmiques dans l'aspergillose pulmonaire

PARIS-BIUP (751062107) / SudocSudocFranceF

De la fleur à la graine : Le fabuleux destin du pollen

International audienc

De la fleur à la graine : Le fabuleux destin du pollen

International audienc