Cell-type specific alternative splicing in the Arabidopsis germline

During sexual reproduction in flowering plants, the two haploid sperm cells embedded within the cytoplasm of a growing pollen tube are carried to the embryo sac for double fertilization. Pollen development in flowering plants is a dynamic process that encompasses changes at transcriptome and epigenome level. While the transcriptome of pollen and sperm cells in Arabidopsis thaliana is well documented, previous analyses were mostly based on expression at gene level. In-depth transcriptome analysis, particularly the extent of alternative splicing at the resolution of sperm cell and vegetative nucleus was still lacking. Therefore, we performed RNA-seq analysis to generate a spliceome map of Arabidopsis sperm cells and vegetative nuclei isolated from mature pollen grains. Based on our de-novo transcriptome assembly we identified 58039 transcripts, including 9681 novel transcripts, of which 2091were expressed in sperm cells and 3600 in vegetative nuclei. Our data from sperm cell and vegetative nucleus identified 468 genes that were regulated both at gene and splicing level, with many having functions in mRNA splicing, chromatin modification, and protein localization. Moreover, a comparison with egg cell RNA-seq data uncovered sex-specific regulation of transcription and splicing factors. Our study provides novel insights into a gamete specific alternative splicing landscape at unprecedented resolution. Seeds of a transgenic marker line harbouring MGH3p::MGH3-GFP and ACT11p::H2B-mRFP were sown on soil in short-day conditions (8-h light at 21–23 °C) for 8 weeks and then transferred to long-day conditions (16-h light) to induce flowering. Flowers were collected, pollen cracked and FACS sorting of sperm cells and vegetative nucleus from mature pollen grain (MPG) was performed. 3 Biological replicates for each sperm cell and vegetative nuclei sample were used.