SYBR green-activated sorting of Arabidopsis pollen nuclei based on different DNA/RNA content

Germ cell epigenetics is a critical topic in plants and animals. The male gametophyte (pollen) of flowering plants is an attractive model in which to study genetic and epigenetic reprogramming during sexual reproduction, being composed of only two sperm cells contained within, its companion, vegetative cell. Here we describe a simple and efficient method to purify SYBR green stained sperm and vegetative cell nuclei of Arabidopsis thaliana pollen using fluorescence activated cell sorting (FACS) to analyze chromatin and RNA profiles. The method obviates generating transgenic lines expressing cell type-specific fluorescence reporters and facilitates functional genomic analysis of various mutant lines and accessions. We evaluate the purity and 2 quality of the sorted pollen nuclei and analyze the technique’s molecular basis. Our results show that both DNA and RNA contents contribute to SYBR green activated nucleus sorting and RNA content differences impact on the separation of sperm and vegetative cell nuclei. We demonstrate the power of the approach by sorting wild-type and polyploid mutant sperm and vegetative cell nuclei from mitotic and meiotic mutants, which is not feasible using cell typespecific transgenic reporters. Our approach should be applicable to pollen nuclei of crop plants and possibly to cell/nucleus types and cell cycle phases of different species containing substantially different amounts of DNA and/or RNA.