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

Filipe Borges, Rui Gardner, Telma Lopes, Joseph P. Calarco, Leonor C.

Boavida, R. Keith Slotkin, Robert A. Martienssen, Jörg D. Becker

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Filipe Borges, Rui Gardner, Telma Lopes, Joseph P. Calarco, Leonor C. Boavida, et al. FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei. Plant Methods, 2012, 8 (1), pp.1-8. 10.1186/1746-4811-8-44. hal-02649137

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

Borges *et al.*
<figure><img src="image_4.png" /></figure>

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

Filipe Borges<fnref n="1" />†, Rui Gardner<fnref n="1" />†, Telma Lopes<fnref n="1" />, Joseph P Calarco², Leonor C Boavida<fnref n="1" />, R Keith Slotkin³, Robert A Martienssen² and Jörg D Becker<fnref n="1" />*

**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*.

**Keywords:** Pollen, Microspore, Sperm cell, Vegetative nucleus, Plant germline, *Arabidopsis*, Fluorescence-activated cell sorting

**Background**

Plant germ cells differentiate late in ontogeny within gametophytes. In the male gametophyte (pollen grain), this process requires post-meiotic microspores to undergo two subsequent mitotic divisions, giving rise to the male germ unit (MGU) that is composed of a vegetative cell nucleus (VN) and two sperm cells (SC). The vegetative cell arrests cell cycle progression upon pollen mitosis I (PM I), while the two sperm cells originated from pollen mitosis II (PM II) are specified into gametes [1]. The germ cell lineage is immersed in the pollen vegetative cell, being dependent on this companion cell

for transportation towards the embryo sac. However, the gametes are known to have their own unique molecular repertoire [2,3]. Several studies reported different methodologies to isolate generative cells of *Lilium longiflorum* (lily) [4,5] and sperm cells of *Oryza sativa* (rice) [6,7], *Zea mays* (maize) [8], and more recently from *Nicotiana tabacum* (tobacco) [9] and the dimorphic sperm cells of *Plumbago zeylanica* [10]. In *Arabidopsis*, technical difficulties in obtaining sufficient amounts of pure biological material constituted a major problem for purification of sperm cells, especially because of their small size. However, Fluorescence Activated Cell Sorting (FACS) has been successfully used to purify *Arabidopsis thaliana* sperm cells [2,11].

Microgametogenesis in *Arabidopsis* has proven to be an excellent model to identify novel mechanisms controlling cell cycle transitions, cell fate specification and

<footnote>* Correspondence: jbecker@igc.gulbenkian.pt</footnote>

<footnote>†Equal contributors</footnote>

<footnote n="1">¹Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, 2780-156, Oeiras, Portugal</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
epigenetic reprogramming (reviewed in [12]). Such studies
also highlighted the importance of analyzing the compo-
nents of the male germ unit independently, as they acti-
vate different transcriptional machineries and establish
distinct epigenetic states [2,11,13]. The VN may partici-
pate actively in controlling heritable epigenetic modifica-
tions in the germline, as it activates expression of
transposable elements (TEs) and produces a specific class
of small interfering RNAs (siRNAs) that accumulate in the
gametes [13]. Therefore, in order to understand epigenetic
reprogramming throughout pollen development a simple
and powerful method to co-purify the two differentiated
types of nuclei in mature pollen, as well as their precursor
microspore, was needed.

Here we describe a fast and reliable method to isolate *Arabidopsis* SC, VN and microspores, based on further development of previously reported techniques to isolate mature pollen using high-speed cell sorting [2,14]. Our first study described a fluorescent marker line specifically labeling differentiated SC in mature pollen, which allowed their FACS-purification and genome-wide transcriptional profiling [2]. Even though this method allowed obtaining pure and viable sperm cell fractions, it was laborious, time consuming and inefficient, considering the amount of plants needed as starting material. In addition, the need for DRAQ5 or other DNA dyes may become problematic for certain down-stream applications such as chromatin IPs, as it is known to interfere with chromatin condensation and nucleosome positioning [15,16].

This method was improved significantly by using strong-
ger fluorescent markers and more efficient methodologies
for pollen disruption, resulting in larger amounts of highly
pure material at very high rates. As such, it also allowed
co-purifying the VN from the same cell population.

**Results**

Co-purification of sperm cell and vegetative nuclei

In order to improve and simplify the SC-sorting method
and to additionally co-purify the VN from the same gen-
etic background, we generated a transgenic line expres-
sing distinct fluorescent proteins in both nuclei. The
*ACT11* promoter driving histone H2B fused to mRFP
was used as a VN marker [17]. A homozygous plant har-
boring the *ACT11p::H2B-mRFP* transgene in Col-0 back-
ground was crossed with a sperm-specific marker line of
the same ecotype, harboring a *MGH3p::MGH3-eGFP*
construct, which encodes a male germline-specific his-
tone variant [18,19]. *MGH3* is expressed as early as
bicellular pollen, and is highly abundant in the sperm
nuclei of mature pollen (Figure 1A, B). Progeny resulting
from this cross were allowed to segregate by self-pollin-
ation, in order to obtain stable double homozygous
plants (Figure 1C). Studying the expression pattern of both

transgenes throughout pollen development revealed that *ACT11p::H2B-mRFP* is initially expressed in the generative cell (GC) of bicellular pollen, but preferentially expressed in the VN at the mature pollen stage (Figure 1A, B). We did not observe any pollen phenotypes in homozygous lines for both transgenes, and the plants are fully fertile, thus indicating that ectopic expression of an additional *H2B* gene in the VN and an extra copy of *MGH3* in the sperm cells did not result in any significant change in chromatin structure and dynamics in both nuclei.

Pollen grains were collected from open flowers by
vortexing with Galbraith buffer [20] (see Materials and
Methods), and released pollen was then disrupted by add-
itional vortexing with glass beads. Enriched filtrates con-
taining SC nuclei and VN released from broken pollen
were co-purified by FACS based on their distinct fluores-
cence properties (Figure 2A). We inspected purity of
sorted samples by microscopy (Figure 2B) and RT-PCR on
SC and VC-specific transcripts. *MGH3* is only expressed
in the SC and was not detected in the sorted VN frac-
tion, while *VEX1*, a VC-expressed gene, was equally not
detected in the SC fraction (Figure 2D), indicative of
two pure populations. Re-analyzing sorted populations
stained with 4',6-diamidino-2-phenylindole (DAPI) con-
firmed that the SC and VN populations were consist-
ently more than 99% pure (Figure 2C). Furthermore, the
ratio SC/VN before sorting was consistently 2:1, demon-
strating a good recovery for both types of nuclei after
pollen disruption (data not shown).

As Galbraith's buffer was originally conceived to sort
and analyze nuclei by flow cytometry [20], we tested our
FACS method using a sperm extraction buffer (SEB) that
has been previously used to sort intact and viable sperm
cells [2]. In order to test sperm cell viability before and
after sorting, we stained the cells with SYTOX Orange
dye that stains DNA of dead cells. As expected, sorted
GFP positive events prepared in Galbraith buffer stained
100%, while only 19% of the cells stained after isolation
in SEB (Figure 3). This result indicates that whenever in-
tact and viable sperm cells are required for downstream
applications, the cells should be prepared in SEB instead
of Galbraith buffer. We could also confirm that this buf-
fer is suitable for VN sorting, although the efficiency is
significantly lower (data not shown). SYTOX staining
can be additionally used to exclude dead sperm cells
from sorted fraction.

The procedure was optimised to obtain higher yields
of VN and SC nuclei/cells, maximising the number of
sorted events per second, though the whole procedure
can be scaled-down to approximately 150mg of fresh
weight material in a 2 ml eppendorf tube. 0.8-1.5 million
SC can be obtained from a 10 ml flower batch, while a 2
ml batch of open flowers yields approximately 100.000
sperm cells.
<figure><img src="image_5.png" /><figcaption>**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 germine, 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.</figcaption></figure>

Microspore purification by FACS

To understand the genetic and epigenetic mechanisms
that control VN and SC differentiation, analysis of their
precursor microspore cell is essential. Available methods
to isolate *Arabidopsis* microspores relied on Percoll dens-
ity gradients [21], and would not provide a sufficiently
pure fraction suitable for genomic analysis at the DNA
level, besides the problem of relatively low yield. Previous
attempts to isolate microspores by flow cytometry were
successfully applied in *Brassica napus* [22], however,
to our knowledge these same methods were not tried in
*Arabidopsis*. We used closed flower buds of wild type
plants to collect all different stages of pollen development,
by grinding in pollen extraction buffer (PEB) (see details
in Materials and Methods). The method to purify SC and
VN revealed that intact pollen grains have a high amount
of autofluorescence (population D in Figure 2A). As such,

we explored this property in addition to the smaller size of
microspores in comparison with other stages of pollen de-
velopment (Figure 4A). The sorted population was ana-
lyzed by microscopy and confirmed to contain mostly
microspores (97.7%) (Figure 4B and C). A very small frac-
tion (2.3%) of early bicellular pollen was also observed
(Figure 4C). As these are probably cells that have just gone
through the first pollen mitosis, they have identical size
and autofluorescent properties as microspores, and are
therefore impossible to distinguish with our FACS settings.

Discussion

Gametogenesis and fertilization involve a series of gen-
etic and epigenetic reprogramming events, often charac-
terized by transcriptional activation of germline genes
and repression of somatic gene expression, as well as dy-
namic changes of DNA methylation on a genome-wide
<figure><img src="image_6.png" /></figure>

scale. A comprehensive interpretation of such phenomena relies on analyzing isolated cell populations, and as such it demands methods for efficient and robust purification of each cell type involved in the process. We found that FACS coupled to nuclei-specific fluorescent markers provides fast and reliable results to isolate *Arabidopsis* sperm cell nuclei and vegetative nucleus fractions that are consistently more than 99% pure. Sorted nuclei are suitable for most molecular analysis such as DNA methylation profiling and chromatin immunoprecipitation. We have shown that our method allows the isolation of intact and viable sperm cells when prepared in SEB buffer, providing roughly 80% of viable cells in the sorted population. Moreover, as SYTOX dye stains DNA of dead cells, it can be used to sort only intact sperm cells.

Our results show that using fluorescent proteins under the control of strong cell-specific promoters eliminates the need for DNA dyes, e.g. SYBR Green as used previously to isolate VN and SC nuclei by flow cytometry [11,23]. The use of such dyes may enhance noise and interfere with downstream DNA analysis.

Besides the isolation of VN and SC, we developed a FACS-based protocol allowing the isolation of microspores from wild-type plants with unprecedented purity, accomplished again without the use of DNA dyes. These methods in combination with recent improvements in sample preparation, will allow fast and robust genome-wide analyses at the transcriptome and methylome levels from a very limited number of cells. As such, the genetic and epigenetic characterization of the *Arabidopsis* male gametophyte will provide the necessary data depth and resolution to boost our understanding of the key pathways involved in microgametogenesis in higher plants. Small RNA activity and epigenetic reprogramming of transposable elements in the germline [24] are particularly interesting for their defined role during post-fertilization processes such as genomic imprinting and heterosis.

## Methods

### Plant material, growth conditions and transgene cloning

Transgenic *Arabidopsis* plants were used for both sperm cell and pollen isolation. Plants were sown on soil and grown for 8 weeks in short-day conditions (8 h light at
<figure><img src="image_7.png" /><figcaption>**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.</figcaption></figure>

21°C-23°C) and then transferred to long-day conditions (16 h light) to induce flowering. *MGH3p::MGH3-eGFP* was obtained by cloning 1.2 kb upstream of the MGH3 transcriptional start site, together with the MGH3 genomic sequence without the stop codon, into the pMDC107 vector [25] by gateway cloning. The *MGH3* coding sequence contains all endogenous introns, which might help for stable transcription and accumulation to high levels. Primers used for *MGH3p::MGH3-eGFP* cloning are presented in Additional file 1. Double homozygous plants harbouring *MGH3p::MGH3-eGFP* and *ACT11p::H2B-mRFP* [17] transgenes, were obtained by crossing individual homozygous lines. This seed stock is available from ABRC under stock number CS67829.

## Purification of sperm cells, vegetative nuclei and sperm nuclei

Open flowers were collected into a 50 ml falcon tube, filled roughly with a volume of 10 ml of fresh material. Flowers were vortexed at medium speed (Fisher Scientific, TOP-mix 3 IKA, speed 2000) in 10 ml of Galbraith buffer (45 mM MgCl₂, 30 mM Sodium Citrate, 20 mM MOPS, 1% Triton-100, pH to 7.0) for 3 minutes, at room temperature. This crude fraction was then filtered through a Miracloth mesh (Calbiochem) to remove flower parts and centrifuged for 1 minute at 2600 g to pellet pollen. The supernatant was carefully removed with minimal disturbance and the pollen enriched pellet was resuspended in approximately 1.5 ml of fresh buffer. This pollen enriched fraction was then transferred to a

1.5 ml eppendorf tube containing 100 µl of acid-washed glass beads (425–600 µm, Sigma), and vortexed continuously at maximum speed (2500) for 4 minutes in order to break mature pollen grains. The fraction containing the released nuclei was then filtered through a 28µm mesh (SEFAR) to exclude unbroken and hydrated pollen. The nuclei enriched solution was ready for FACS at this point, which included debris from broken pollen. Hydrated pollen that remained intact and thus was retained in the filter can be recovered by washing the 28 µm mesh in new buffer and used in a second extraction step with the glass beads.

If intact and viable SC are desired for downstream applications (e.g. RNA extraction or *in vitro* manipulation) the whole procedure should be performed with sperm extraction buffer (SEB) (1.3 mM H₃BO₃, 3.6 mM CaCl₂, 0.74 mM KH₂PO₄, 438 mM sucrose, 5.83 mM MgSO₄, 7 mM MOPS at pH 6), which assures that 75-80% of the sorted sperm cells are viable and intact.

## Purification of Microspores from young flower buds

Closed flower buds (approximately 20 ml) were gently ground using mortar and pestle in 10 ml of pollen extraction buffer (PEB) (10 mM CaCl₂, 2 mM MES, 1 mM KCl, 1% H₃BO₃, 10% Sucrose, pH 7.5) in order to release the spores. This crude fraction was initially filtered through Miracloth (Calbiochem) to remove bigger debris, and concentrated by centrifugation (800 g, 5 min) in 15 ml falcon tubes. The resulting yellowish pellet enriched in microspores was resuspended in 1.5 ml of
<figure><img src="image_8.png" /><figcaption>**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.</figcaption></figure>

PEB, and filtered through a 20 µm mesh (Partec, Cell-Trics) before FACS to further enrich for microspores and exclude tricellular and mature pollen.

Fluorescence activated cell sorting

Fluorescent activated cell sorting was carried out with a MoFlo (Beckman Coulter, Fort Collins, USA) with a 488 nm laser (200 mW air-cooled Sapphire, Coherent) at 140 mW used for scatter measurements (Low angle or Forward Scatter, and High angle or Side Scatter; FSC and SSC, respectively) and for GFP excitation, and a 561 nm laser (50 mW DPSS, CrystaLaser) at 38 mW for RFP excitation. GFP and RFP were detected using a 530/40 nm and a 630/75 nm bandpass filters, respectively. FSC was used for triggering, and threshold had to be low to avoid missing the sperm cell population, whose size is on average 2.5 µm in diameter. Phosphate Buffer Saline (PBS) was used as sheath, and run at a constant pressure

of 400 kPa (~60 psi). Frequency of drop formation was approximately 96,000 Hz. Even though pollen was present in the sample (broken and intact) it did not interfere with drop formation or break-off, as we were able to sort pollen under the same conditions as for SC and VN. Sorting rates were typically 2 million SC and 1 million VN per hour, i.e. an average rate of 500 SC and 250 VN per second, respectively.

Viability tests were performed with approximately 50.000 sorted SC and VN by staining with SYTOX Orange (Molecular Probes, Invitrogen) at a final concentration of 25nM. Sperm cells negative for SYTOX Orange and positive for GFP were considered viable (intact membrane) and considered compromised or to represent bare nuclei (only the nuclei stained), if positive for both SYTOX and GFP. SYTOX Orange was excited with the 488 nm laser and detected with a 580/20 nm bandpass filter in the MoFlo.
Purity was determined by running aliquots of sorted cells in a CyAn ADP flow cytometer (Beckman Coulter, Fort Collins, USA). A 488 nm laser was used to excite both GFP and RFP, detected with 530/30 and 616/21 nm bandpass filters, respectively. 1 µM DAPI (Sigma) was added to the sorted cells and incubated on ice for 5 min, to discriminate between nuclei containing cells and electronic noise. DAPI was excited with a 405 nm laser and detected with a 450/40 nm bandpass filter.

Microspores were also purified by FACS using a Moflo
high-speed cell sorter. The machine was used in a stand-
ard configuration, using a 100 µm ceramic nozzle with
PBS running at a constant pressure of 200 kPa (~30 psi),
and a drop-drive frequency of approximately 30,000 Hz.
The 488 nm laser line was used for scatter measurements
and autofluorescence excitation, which was detected in
the GFP channel using a 530/40 nm bandpass filter.
Microspores and other stages of pollen development were
identified by their elevated high angle scatter (SSC) and
autofluorescence properties (observed in the GFP chan-
nel). Within this population, microspores were selected by
their characteristic smaller size, captured by a diminished
low angle scatter (FSC) and time-of-flight (Pulse Width).

RT-PCR

Semi-quantitative RT-PCR was performed with total RNA isolated from approximately 100,000 cells/nuclei that were sorted directly into Tri Reagent LS (Sigma). First-strand cDNA (Oligo-dT primed) was synthesized in 25 µl reactions using the MLV reverse transcriptase - RNaseH minus (Promega) according to manufacturer instructions. 2 µl of non-diluted cDNA was used as a template for 30 PCR cycles. Primers used are listed in Additional file 1.

**Additional file**

Additional file 1: List of primers used in this study.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

JDB initiated the project; FB, JDB, RG and RKS designed the experiments for isolation of sperm cells and vegetative nuclei; FB, JDB, RG, JPC and RM developed the strategy for isolation of microspores; FB, RG, TL, JPC, LCB and RKS carried out the experiments; FB, JDB and RG wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank Frédéric Berger for providing the ACT11p::H2B-mRFP transgenic line. This work was supported by grants PTDC/AGR-GPL/103778/2008 and PTDC/BIA-BCM/103787/2008 from Fundação para a Ciência e a Tecnologia (FCT), Portugal. FB acknowledges FCT PhD fellowship SFRH/BD/48761/2008. JPC acknowledges a graduate student fellowship from NSERC and a grant from the Fred C. Gloeckner Foundation.

Author details

Spring Harbor, NY, USA. ³Department of Molecular Genetics, The Ohio State University, 43210, Columbus, OH, USA.

Received: 31 May 2012 Accepted: 8 October 2012

Published: 17 October 2012

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doi:10.1186/1746-4811-8-44

Cite this article as: Borges et al.: FACS-based purification of *Arabidopsis* microspores, sperm cells and vegetative nuclei. *Plant Methods* 2012 8:44.

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