Transcriptome dynamics in the Arabidopsis male germline during pollen-pistil interactions

When pollen lands on a receptive stigma, it germinates and extends a tube inside the transmitting tissue of the pistil to deliver the sperm cells for double fertilization. The growth of the pollen tube triggers significant alterations in its gene expression. The extent to which these changes occur in the vegetative cell or extend to the sperm cells transported by the tube is unclear, but important to determine since sperm cells are believed to acquire a competency for fertilization during pollen-pistil interactions. To address these questions, we compared the transcriptomes of Arabidopsis thaliana sperm cells and vegetative nuclei isolated from mature pollen grains with those isolated from in vitro grown pollen tubes. Importantly, we also compared with transcriptomes of sperm cells obtained from pollen tubes grown under semi in vivo conditions where tubes passed through a pistil section. Our data shows that extensive transcriptomic changes occur in sperm cells during pollen tube growth, some of which are elicited only as sperms are carried through the pistil. Their analysis reveals a host of previously unidentified transcripts that may facilitate sperm maturation and gamete fusion. The vegetative cell undergoes even more extensive transcriptomic reprogramming during pollen tube growth, mainly through the upregulation of genes associated with pollen tube growth and vesicle-mediated transport. Interestingly, ATAC-seq data shows that the promoters of genes up-regulated in sperm during pollen tube growth are already accessible in sperm chromatin of mature pollen grains, suggesting pre-configured promoter accessibility. This study's expression data can be further explored here: https://bar.utoronto.ca/eFP-Seq_Browser/. Overall design: 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. Fresh opened flowers were collected upto 5ml in Falcon tubes and using the previously published protocol, the pollen were cracked. Cracked pollen were then filtered through mesh and sperm cells and vegetative nucleus from mature pollen grain were collected and FACS sorted. The pollen were then germinated on plate or grown semi in vivo for 6h, to collect the sperm cells from the pollen tubes. Vegetative nuclei were also collected from pollen tubes grown in vitro.