FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei.
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.
Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, 2780-156, Oeiras, Portugal.
Filipe
F
Borges
Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, 2780-156, Oeiras, Portugal.
Rui
R
Gardner
Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, 2780-156, Oeiras, Portugal.
Telma
T
Lopes
Cold Spring Harbor Laboratory, 1 Bungtown Road, 11724, Cold Spring Harbor, NY, USA.
Joseph P
JP
Calarco
Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, 2780-156, Oeiras, Portugal.
Leonor C
LC
Boavida
Department of Molecular Genetics, The Ohio State University, 43210, Columbus, OH, USA.
R Keith
RK
Slotkin
Cold Spring Harbor Laboratory, 1 Bungtown Road, 11724, Cold Spring Harbor, NY, USA.
Robert A
RA
Martienssen
Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, 2780-156, Oeiras, Portugal.
Jörg D
JD
Becker