Single cell RNA-seq analysis of individual sperm cells to identify transcriptomic differences potentially involved in preferential fertilization.

Project Information

SAMEorNOT

Grant agreement ID: 101059247

DOI

10.3030/101059247

Project closed

EC signature date 14 December 2022

Start date 1 January 2023

End date 30 September 2024

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

€ 137 181,24

EU contribution

€ 137 181,24

Coordinated by

UNIVERSIDADE NOVA DE LISBOA
Portugal

Periodic Reporting for period 1 - SAMEorNOT (Single cell RNA-seq analysis of individual sperm cells to identify transcriptomic differences potentially involved in preferential fertilization.)

Reporting period: 2023-01-01 to 2024-09-30

Summary of the context and overall objectives of the project

Sexual reproduction in flowering plants requires a pollen grain with two sperm cells and a vegetative nucleus to create a pollen tube. The two immobile sperm cells travel attached to the vegetative nucleus inside the pollen tube, growing through the female tissue towards the ovary until it reaches an ovule. Once inside, the pollen tube bursts and releases the two sperm cells in contact with the female gametes. Finally, one sperm cell will fuse with the egg cell producing the embryo and future plant, while the other will fuse with the central cell, producing the nurturing tissue for the embryo to develop.
Interestingly, it remains unclear if both sperm cells are the same or not, if they keep a travelling order or if they have preferential fertilization of either the egg or the central cell. These key questions are of paramount importance for plant breeders, because the targeted delivery of components either in the egg or in the central cell, would represent a valuable
biotechnological tool to increase the efficiency of plant breeding programs.

It is known that some species produces two different sperm cells (e.g. Plumbago zeylanica), and this difference is related not only to their travelling order but also to their preference to fertilize either the egg cell or the central cell. Species like Arabidopsis and many relevant crops (e.g. maize or wheat) produce, in terms of morphology, identical sperm cells, but if there are differences at other level (transcriptomic, proteomic, metabolomic) is a question that remains open.
Therefore, the main goal of this project is to explore potential transcriptomic differences between the two sperm cells. If any, the idea is to use this difference to make “visible” with fluorescence this difference and be able to see each sperm cell inside the ovule and identify which cell they fuse with. In this way, this project will be useful to explore hypothetical preferential fertilization events in Arabidopsis.
The specific objectives of the research proposal are:

· O1: The creation of a microdevice to germinate pollen in contact with female tissue and enable a separate growth and manipulation of each pollen tube (WP1).

· O2: The manual isolation of single sperm cells from the same tube, according to their travelling order (WP2).

· O3: Transcriptomic analysis of isolated sperm cells to detect differentially expressed genes (WP3).

· O4: The creation of a sperm single-cell fluorescent plant based on putative differentially expressed genes to evaluate a hypothetical preferential fertilization (WP4).

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

We created a unique prototype of microdevice to germinate pollen in contact with female tissue and to observe and manipulate it in the confocal microscope. We also design a protocol for manual single sperm cell isolation and obtained an accurate transcriptomic data from single sperm cells. Finally we detected several genes that were differentially expressed and obtained a list of candidate genes for further work.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The transcriptomic differences found in Arabidopsis sperm cells represent a historical opportunity to develop a sperm single-cell marker line, in which one cell is labelled to be distinguished from the other. This marker would constitute a major breakthrough in plant reproduction, because it would enable preferential fertilization assays in a physiological context. Additionally, this marker can be used to isolate large amounts of each sperm cells by FACS to perform other studies that require large amounts of sample (e.g. proteomics, metabolomics, small RNA content, epigenetics) that would push significantly forward our current knowledge of plant male gametes.