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Whole-mount immunolocalization to study female meiosis in Arabidopsis

Nature Protocols volume 10, pages 1535–1542 (2015)Cite this article

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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.

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Figure 1: Dissection of meiotic ovules from flower buds of A. thaliana.
Figure 2: Whole-mount immunolocalization of meiotic proteins ASY1 and ZYP1 in developing ovules of A. thaliana.

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Acknowledgements

We are grateful to M. Grelon (INRA Versailles, France) and C. Franklin (University of Birmingham, UK) for the kind gift of ASY1 and ZYP1 antibodies, to M. García-Aguilar for technical advice, and to L. Solorzano and S. García for help with reagent acquisition and purchasing. D.R.L. is the recipient of a graduate scholarship from Consejo Nacional de Ciencia y Tecnología (CONACyT). This research was supported by grants from CONACyT (CON47436), the International Scholar Program of the Howard Hughes Medical Institute (2007–2011) and as part of the DuPont Pioneer regional initiatives to benefit local subsistence farmers.

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  1. Rocio Escobar-Guzmán and Daniel Rodríguez-Leal: These authors contributed equally to this work.

Authors and Affiliations

  1. Grupo de Desarrollo Reproductivo y Apomixis, Unidad de Genómica Avanzada Laboratorio Nacional de Genómica para la Biodiversidad (LANGEBIO). Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Irapuato, Mexico

    Rocio Escobar-Guzmán, Daniel Rodríguez-Leal, Jean-Philippe Vielle-Calzada & Arnaud Ronceret

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  1. Rocio Escobar-Guzmán
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  2. Daniel Rodríguez-Leal
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  3. Jean-Philippe Vielle-Calzada
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Contributions

R.E.-G. performed the experiments and contributed to preparation of the manuscript. D.R.-L. contributed to the development of the protocol, to preparation of the figures and to manuscript writing. A.R. conceived and designed the experiments and contributed to manuscript writing. J.-P.V.-C. conceived and supervised the project, and contributed to manuscript writing.

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Correspondence to Jean-Philippe Vielle-Calzada.

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Escobar-Guzmán, R., Rodríguez-Leal, D., Vielle-Calzada, JP. et al. Whole-mount immunolocalization to study female meiosis in Arabidopsis. Nat Protoc 10, 1535–1542 (2015). https://doi.org/10.1038/nprot.2015.098

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