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

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.

Figure 1

Expression pattern of GFP- and RFP-fusion proteins during pollen development. (A) MGH3p::MGH3-eGFP localizes in the generative cell nucleus (GCN) after the first pollen mitosis, and is strongly accumulated in the sperm cell nucleus (SCN) of mature pollen. ACT11p::H2B-mRFP is initially expressed in the GCN until late bicellular pollen (BCP). After the second pollen mitosis the expression of this transgene is down-regulated in the germline, and it becomes strongly expressed in the vegetative nucleus (VN). (B) Merged magnification of a mature pollen grain expressing both transgenes. (C) Population of mature pollen grains from double homozygous plants, confirming strong and stable expression of both transgenes. Scale bars: 10 μm.

Figure 2

Purification of sperm and vegetative nuclei by FACS. (A) Four distinct cell populations are highlighted in the filtrate pre-sorting. Sperm cells (SC) nuclei are identified based on their strong GFP signal (Population A), whereas vegetative nuclei (VN) separate towards the opposite axis based on strong RFP signal (Population B). In addition the filtrate contains empty (Population C) and intact pollen grains (Population D). (B) Purity of sorted SC and VN fractions was confirmed by DIC and fluorescence microcopy; scale bar: 10 μm. Populations C and D were confirmed to represent empty and intact pollen, respectively; scale bar: 30 μm (C) Sorted SC and VN samples were stained with DAPI and run through a flow cytometer to check for purity. Purity is determined by measuring the percentage of SC and VN present within the total number of DAPI positive events, corresponding to all DNA-containing particles present in the sorted sample. (D) RT-PCR analyses confirmed that each fraction is enriched for cell-specific transcripts (MGH3 for SC and VEX1 for VN), and devoid of contaminating RNAs. TUB4 was used as control.

Figure 3

Sperm cell viability before and after sorting. Cell viability assays were performed by staining cells before and after FACS with SYTOX orange dye, which stains only dead cells. Sperm cells (SC) before and after sorting were confirmed to be viable only when prepared in Sperm Extraction Buffer (SEB), while in Galbraith’s buffer (NEB) we observed 100% staining.

Figure 4

Microspore sorting. (A) Pollen population is characterized by an elevated high angle scatter (SSC) and autofluorescence (observed in the GFP channel using a 530/40 nm bandpass filter). Within this population, microspores (right panel, circular gate) can be differentiated from bicellular and tricellular pollen by their characteristic smaller size, captured by a diminished low angle scatter (FSC) and time-of-flight (Pulse Width), as compared to other stages of pollen development. (B) Sorted microspores were inspected by microscopy to show purity and integrity, as revealed by DAPI staining. Scale bars: 30 μm (left panel) and 10 μm (right panels). (C) Sorted microspores stained with DAPI were counted on a wide-field fluorescence microscope to confirm that most sorted cells are Uninucleate Microspores (UNM). BCP - Bicellular Pollen.