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

Plant Methods

Jörg D Becker¹  Robert A Martienssen²  R Keith Slotkin³  Leonor C Boavida¹  Joseph P Calarco²  Telma Lopes¹  Rui Gardner¹  Filipe Borges¹ 

[1] Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, 2780-156, Oeiras, Portugal;Cold Spring Harbor Laboratory, 1 Bungtown Road, 11724, Cold Spring Harbor, NY, USA;Department of Molecular Genetics, The Ohio State University, 43210, Columbus, OH, USA

关键词: Fluorescence-activated cell sorting;    Arabidopsis;    Plant germline;    Vegetative nucleus;    Sperm cell;    Microspore;    Pollen;   

Others  :  821399
DOI  :  10.1186/1746-4811-8-44

 received in 2012-05-31, accepted in 2012-10-08,  发布年份 2012

【 摘 要 】

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.

【 授权许可】

   
2012 Borges et al.; licensee BioMed Central Ltd.

【 图 表 】

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