Abstract
One of the major topics in plant and animal biology is sexual reproduction. It is, therefore, of great interest to isolate and study germ cells and accessory cells. The male gametophyte of the flowering plant Arabidopsis thaliana (A. thaliana), pollen, is the product of two post-meiotic mitotic divisions. Each mature pollen grain consists of two sperm cells contained within the vegetative cell, the non-reproductive companion cell. The tough pollen wall and its special nested structure make it difficult to study pollen cells separately. Here, we describe a simple and efficient method to fractionate A. thaliana sperm and vegetative cell nuclei by fluorescence activated cell sorting (FACS). Our protocol is based on differences in fluorescence intensity of sperm and vegetative cell nuclei stained with SYBR Green I. 100 plants yield about 1 x 106 sperm and 350,000 vegetative cell nuclei. This method can be used for purifying pollen nuclei of various A. thaliana wild-type accessions and mutant lines, and can, in principle, be adapted for pollen of other plant species.
Keywords: SYBR Green, Fluorescence-activated cell sorting (FACS), Arabidopsis, Pollen, Male gametophyte
Materials and Reagents
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Acknowledgments
This protocol is based on previously published work (Schoft et al., 2009; Schoft et al., 2011; Ibarra et al., 2012; Schoft et al., 2015). We thank the BioOptics Facility of the Research Institute of Molecular Pathology, Vienna, for setting up and optimizing FACS. We thank Gerald Schmauss for measuring sizes of sorted A. thaliana pollen nuclei. We thank Hisashi Tamaru for his support. This work was supported by Austrian Science Fund (FWF) Grants P21389-B03 and P24918-B21.
References
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Hi Dejan,if I remember correctly (I haven't used the protocol since 2014), we didn't do anything special to freeze the pollen. You can, of course, quick freeze them (by using liquid nitrogen) and thawing them on ice is key. But pollen is really tough, therefore we didn't have problems. We anyways wanted to crush them after freezing and by doing FACS, we only collected the intact nuclei (which can be verified by microscopy after the sort). The vegetative nuclei are more fragile than the sperm nuclei due to differences in DNA compaction. We also did ChIP with the sperm nuclei and had a hard time sonicating the nuclei to get the required 500bp fragments for sequencing. I wouldn't worry about the sperm nuclei. If you want to use the vegetative nuclei for a protocol that needs special handling, you will have to develop a freezing/thawing protocol on your own, but you can check the nuclei nicely under the microscope and distinguish sperm and vegetative nuclei by their shape and size.I hope this helps.Best,Vera