In Situ/Subcellular Localization of Arabinogalactan Protein Expression by Fluorescent In Situ Hybridization (FISH).

The arabinogalactan proteins are highly glycosylated and ubiquitous in plants. They are involved in several aspects of plant development and reproduction; however, the mechanics behind their function remains for the most part unclear, as the carbohydrate moiety, covering the most part of the protein core, is poorly characterized at the individual protein level. Traditional immunolocalization using antibodies that recognize the glycosidic moiety of the protein cannot be used to elucidate individual proteins' distribution, function, or interactors. Indirect approaches are typically used to study these proteins, relying on reverse genetic analysis of null mutants or using a reporter fusion system. In the method presented here, we propose the use of RNA probes to assist in the localization of individual AGPs expression/mRNAs in tissues of Arabidopsis by fluorescent in situ hybridization, FISH. An extensive description of all aspects of this technique is provided, from RNA probe synthesis to the hybridization, trying to overcome the lack of specific antibodies for the protein core of AGPs.

Immunolocalization of AGPs and Pectins in Quercus suber Gametophytic Structures.

The arabinogalactan proteins (AGPs) are highly glycosylated proteins, ubiquitous in plants that have been linked to numerous aspects of sexual reproduction in several plant species, including the monoecious tree species Quercus suber. AGPs are found in cell membranes and cell walls of all types of tissues, including reproductive cells and organs. Pectins are cell wall components that also have been shown to change in composition and quantity during the maturations of the male and female gametophyte in cork oak. These findings were only possible to reveal, due to the histological study of AGP and pectins epitopes by immunolabeling. The immunofluorescence microscopy technique uses antibodies linked to fluorophores and relies on the specificity of the antibody binding to its antigen, labeling the epitope with a fluorescent dye.In the method presented here, we explore the immunolocalization technique performed in male and female flowers of Quercus suber, using London Resin (LR-White) as the embedding medium, after vacuum fixation with formaldehyde/glutaraldehyde. An extensive description of all the aspects of this technique is provided, from the plant material developmental stages selection to the critical analysis of results performed, continuously supported by troubleshooting recommendations.

Growth Media Induces Variation in Cell Wall Associated Gene Expression in Arabidopsis thaliana Pollen Tube.

The influence of three different pollen germination media on the transcript profile of Arabidopsis pollen tubes has been assessed by real-time PCR on a selection of cell wall related genes, and by a statistical analysis of microarray Arabidopsis pollen tube data sets. The qPCR assays have shown remarkable differences on the transcript levels of specific genes depending upon the formulation of the germination medium used. With the aid of principal component analysis performed on existing microarray data, a subset of genes has been identified that is more prone to produce diverging transcript levels. A functional classification of those genes showed that the clusters with higher number of members were those for hydrolase activity (based in molecular function) and for cell wall (based in cellular component). Taken together, these results may indicate that the nutrient composition of the pollen germination media influences pollen tube metabolism and that caution must be taken when interpreting transcriptomic data of pollen tubes.