FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei

Borges, F., Gardner, R., Lopes, T., Calarco, J. P., Boavida, L. C., Slotkin, R. K., Martienssen, R. A., Becker, J. D. (October 2012) FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei. Plant methods, 8 (1). p. 44. ISSN 1746-4811 (Electronic)1746-4811 (Linking)

[thumbnail of Paper]
PDF (Paper)
Martienssen Plant Methods 2012.pdf - Published Version

Download (3MB) | Preview
URL: http://www.ncbi.nlm.nih.gov/pubmed/23075219
DOI: 10.1186/1746-4811-8-44


ABSTRACT: BACKGROUND: The male germline in flowering plants differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a second mitosis to originate two sperm cells. The vegetative cell and the sperm cells activate distinct genetic and epigenetic mechanisms to control pollen tube growth and germ cell specification, respectively. Therefore, a comprehensive characterization of these processes relies on efficient methods to isolate each of the different cell types throughout male gametogenesis. RESULTS: We developed stable transgenic Arabidopsis lines and reliable purification tools based on Fluorescence-Activated Cell Sorting (FACS) in order to isolate highly pure and viable fractions of each cell/nuclei type before and after pollen mitosis. In the case of mature pollen, this was accomplished by expressing GFP and RFP in the sperm and vegetative nuclei, respectively, resulting in 99% pure sorted populations. Microspores were also purified by FACS taking advantage of their characteristic small size and autofluorescent properties, and were confirmed to be 98% pure. CONCLUSIONS: We provide simple and efficient FACS-based purification protocols for Arabidopsis microspores, vegetative nuclei and sperm cells. This paves the way for subsequent molecular analysis such as transcriptomics, DNA methylation analysis and chromatin immunoprecipitation, in the developmental context of microgametogenesis in Arabidopsis.

Item Type: Paper
Subjects: organism description > plant > Arabidopsis
organism description > plant
CSHL Authors:
Communities: CSHL labs > Martienssen lab
School of Biological Sciences > Publications
Depositing User: Matt Covey
Date: 17 October 2012
Date Deposited: 31 Jan 2013 20:50
Last Modified: 22 Sep 2014 20:29
PMCID: PMC3502443
Related URLs:
URI: https://repository.cshl.edu/id/eprint/26909

Actions (login required)

Administrator's edit/view item Administrator's edit/view item
CSHL HomeAbout CSHLResearchEducationNews & FeaturesCampus & Public EventsCareersGiving