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HEIndividual fellowship2023–2024

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

Horizon Europe — Marie Skłodowska-Curie Actions

Duration
2023-01-01 → 2024-09-30
EU contribution
€137,181
Participants
1
Scheme
HORIZON-TMA-MSCA-PF-EF

Lines connect the coordinator with its partners.

Results in brief

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

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).

Data: CORDIS, © European Union

Project objective

Human societies are sustained by flowering plants in different ways but especially as a key food supplier. However, it is known that increasing global temperatures strongly affect plant reproduction and the growing human population will require more food in short-middle term. Thus, to create new crop varieties more resistant to heat stress and with enhanced yield, a deeper understanding of the mechanism of plant sexual reproduction will be crucial for increasing the efficiency of plant breeding programs. Plant reproduction relies on a double fertilization process, in which two sperm cells from a pollen grain reach the ovule and one fertilize the egg cell while the other the central cell. In some species with dimorphic sperm cells a preferential fertilization of each sperm cell has been described, but for Arabidopsis thaliana as well as for many economically important crops with isomorphic sperm cells this question remains unclear. The main goal of this project is to identify by single cell RNA-seq analysis potential transcriptomic differences between the two Arabidopsis sperm cells. To do that, individual sperm cells from pollen tubes growing through female stylar transmitting tissue (semi in vivo) will be isolated by micromanipulation using a specially designed microfluidic chamber on an inverted microscope. The identification of differentially expressed genes would enable for the first time the creation of a sperm single-cell marker line to eventually explore a hypothetical preferential fertilization in Arabidopsis. These results can be adapted for plant breeding programs to develop impactful biotechnological tools, like haploid inducer lines with genetically modified sperm cells to specifically deliver components in the egg cell to induce in vivo gynogenesis. Additionally, the results will be a valuable scientific resource to address other topics beyond the preferential fertilization, like exploring hybridization barriers based on gamete recognition.

Original text from CORDIS.

Participants

  • UNIVERSIDADE NOVA DE LISBOA · LisboaCoordinatorPortugal

Links

Data: CORDIS, © European Union