Fucosylation and defucosylation in A. thaliana

Topic 1, a1,2-fucosyltransferases: cDNAs encoding the xyloglucan a1,2-fucosyltransferase homologues from A. thaliana shall be cloned and expressed. The recombinant proteins shall be tested for their enzymatic activity. Potential promoter sequences of the 10 xyloglucan fucosyltransferases family members shall be cloned and used to supply information on the expression pattern of the respective genes at spatial and temporal levels. The effect of biotic and abiotic stresses and hormones on the expression pattern of these putative fucosyltransferase genes shall be investigated. Insertion mutants shall be studied considering their phenotype. A special focus shall be put to the stage of development and the tissues for which the promoter fusion studies had revealed gene expression in wild type plants. For these developmental stages and organs, a biochemical and electron microscopy analysis of cell wall components will be undertaken. Topic 2, the a1,2-fucosidase: This fucosidase, recently identified and proven to be active on xyloglucans in our lab, shall be assayed with arabinogalactan proteins and rhamnogalacturonans. Its gene expression shall be analyzed by promoter studies and the enzyme expression and subcellular localization shall be analyzed by western blot and immuno-localization by electron microscopy after raising of a specific antibody. Taking advantage of the knowledge acquired thereby, we will focus on the relevant developmental stages and tissues to compare the xyloglucan structures found in wild type and insertion mutants. The effect of hormones and of the nonasaccharide XXFG on mutants and wild type will be investigated. Overexpression of the a1,2-fucosidase shall be performed in A. thaliana under the control of 35S promoter via Agrobacterium transformation. The xyloglucan structure found in the over-expressing mutant shall be analysed by mass spectrometry after digestion with endo-(1,4)-ß-D-glucanase. The phenotype of the over-expressing mutant shall be observed during growth under normal conditions and after treatment with hormones or stresses. Topic 3, the a1,3/4-fucosidase: The A. thaliana a1,3/4-fucosidase promoter will be studied by promoter fusion in order to know whether it possesses an expression pattern compatible with that of Lewis a carrying N-glycans. Western blot and immuno-localization will be performed on different A. thaliana tissues with a specific polyclonal antibody obtained by immunization of a rabbit with a1,3/4-fucosidase produced as a recombinant protein in E. coli. N-glycan analysis of the mutant will be compared with results obtained with wild type plants.

Topic 1, a1,2-fucosyltransferases: cDNAs encoding the xyloglucan a1,2-fucosyltransferase homologues from A. thaliana shall be cloned and expressed. The recombinant proteins shall be tested for their enzymatic activity. Potential promoter sequences of the 10 xyloglucan fucosyltransferases family members shall be cloned and used to supply information on the expression pattern of the respective genes at spatial and temporal levels. The effect of biotic and abiotic stresses and hormones on the expression pattern of these putative fucosyltransferase genes shall be investigated. Insertion mutants shall be studied considering their phenotype. A special focus shall be put to the stage of development and the tissues for which the promoter fusion studies had revealed gene expression in wild type plants. For these developmental stages and organs, a biochemical and electron microscopy analysis of cell wall components will be undertaken. Topic 2, the a1,2-fucosidase: This fucosidase, recently identified and proven to be active on xyloglucans in our lab, shall be assayed with arabinogalactan proteins and rhamnogalacturonans. Its gene expression shall be analyzed by promoter studies and the enzyme expression and subcellular localization shall be analyzed by western blot and immuno-localization by electron microscopy after raising of a specific antibody. Taking advantage of the knowledge acquired thereby, we will focus on the relevant developmental stages and tissues to compare the xyloglucan structures found in wild type and insertion mutants. The effect of hormones and of the nonasaccharide XXFG on mutants and wild type will be investigated. Overexpression of the a1,2-fucosidase shall be performed in A. thaliana under the control of 35S promoter via Agrobacterium transformation. The xyloglucan structure found in the over-expressing mutant shall be analysed by mass spectrometry after digestion with endo-(1,4)-ß-D-glucanase. The phenotype of the over-expressing mutant shall be observed during growth under normal conditions and after treatment with hormones or stresses. Topic 3, the a1,3/4-fucosidase: The A. thaliana a1,3/4-fucosidase promoter will be studied by promoter fusion in order to know whether it possesses an expression pattern compatible with that of Lewis a carrying N-glycans. Western blot and immuno-localization will be performed on different A. thaliana tissues with a specific polyclonal antibody obtained by immunization of a rabbit with a1,3/4-fucosidase produced as a recombinant protein in E. coli. N-glycan analysis of the mutant will be compared with results obtained with wild type plants.