Whole-mount immunolocalization to study female meiosis in Arabidopsis.

Abstract

Here we describe a whole-mount immunolocalization protocol to follow the subcellular localization of proteins during female meiosis in Arabidopsis thaliana, a model species that is used to study sexual reproduction in flowering plants. By using confocal microscopy, the procedure allows one to follow megasporogenesis at all stages before differentiation of the functional megaspore. This in particular includes stages that occur during prophase I, such as the installation of the axial and central elements of the synaptonemal complex along the meiotic chromosomes. In contrast to procedures that require microtome sectioning or enzymatic isolation and smearing to separate female meiocytes from neighboring cells, this 3-day protocol preserves the constitution of the developing primordium and incorporates the architecture of the ovule to provide a temporal and spatial context to meiotic divisions. This opens up the possibility to systematically compare the dynamics of protein localization during female and male meiosis. Steps describe tissue collection and fixation, preparation of slides and polyacrylamide embedding, tissue permeabilization, antibody incubation, propidium iodide staining, and finally image acquisition by confocal microscopy. The procedure adds an essential technique to the toolkit of plant meiotic analysis, and it represents a framework for technical adaptations that could soon allow the analysis of plant reproductive alternatives to sexual reproduction.