GAMETEFUSION

Molecular and functional characterization of sperm-expressed proteins with potential fusogenic roles during double fertilization in Arabidopsis thaliana

 Partecipanti

Mappa

 Word cloud

Esplora la "nuvola delle parole (Word Cloud) per avere un'idea di massima del progetto.

 Obiettivo del progetto (Objective)

'Sexual reproduction is a fundamental biological process common among eukaryotes. In angiosperms, fertilization although associated with pollination, is actually a fairly late phenomenon in the process of reproduction and involves a double fusion event between two male and two female gametes. In contrast to animal fertilization, the molecular mechanisms underlying this process in flowering plants are poorly understood and the molecular players unknown. With this project we intend to explore recent advances in the field, namely the availability of expression profiles of plant gametes to identify the molecular players which mediate sperm-egg cell/central cell fusion in Arabidopsis thaliana. We used bioinformatics approaches to identify plasma membrane proteins with expression in sperm cells and sharing structural similarities to known fusogenic peptides. The first candidates are two sperm-specific proteins which belong to the conserved family of tetraspanins. Functional analysis of the candidate proteins will be performed in knockout, amiRNA and overexpression transgenic lines. Interacting proteins in sperm cells and in the female partner cells will be identified using yeast-two hybrid system and validated by functional analysis. Candidate proteins will be assayed for specific functions during double fertilization, such as preferential gamete targeting, fusogenic properties, polyspermy block and fertilization barriers using manipulation of gene expression and proteomic approaches coupled to in vivo and in vitro assays. We also intend to develop an in vivo fluorescent based method to detect and track specific sperm-expressed mRNAs, in order to understand the importance of intercellular communication during double fertilization and initiation of embryogenesis. This project is addressing fundamental questions involving one of the most complex biological processes in nature and might provide novel tools for biotechnology applications and crop improvement.'

Introduzione (Teaser)

Unlike animal sexual reproduction, flowering plants feature a double fertilisation event involving two male and two female sex cells. Identifying the molecular players in this complex procedure promises to open doors to crop improvement.

Descrizione progetto (Article)

With EU funding, the GAMETEFUSION project has uncovered signalling mechanisms in double fertilisation. The importance of this process cannot be understated as one fertilisation gives rise to the embryo and the other a nutrient-rich food source for the developing plant.

Studying the model plant Arabidopsis thaliana, the researchers looked at two sperm-enriched tetraspanins (TETs), also signalling mediators in diverse processes in animal systems: adhesion and gamete fusion. Gene expression analysis confirmed that TETs are membrane-associated proteins in specific reproductive tissues including gametes.

Similar to their animal counterparts, TETs' functions in plants may depend on the assembly of multi-molecular membrane complexes that rely on presence of potential binding partners. The unique localisation pattern found for the TETs in the interface of both types of sperm cell suggests a membrane scaffold could be involved in mediating sperm-sperm cell adhesion or communication.

The GAMETEFUSION team identified two potential TET binding partners. Using knockout mutants, fertility defects occurred, such as a single fertilisation. This finding is relevant in particular as a heightened understanding of cellular processes behind gamete differentiation and double fertilisation may contribute to possible divergent and conserved mechanisms in plants and animals.

Researchers developed a fluorescence-activated cell sorting tool to isolate individual subcellular components of the male gametophyte and analysed their repertoires of expressed messenger RNAs (mRNAs). Researchers plan to detect and track sperm mRNAs during male gamete differentiation and pollen tube growth. The experimental system represents the first application of an in vivo system to image mRNA movement in pollen. Scientists expect to reveal molecular events during double fertilisation and early embryogenesis.

GAMETEFUSION has developed several unparalleled molecular tools that are already or will become available to the scientific research community. Ability to manipulate the fertilisation process has powerful applications in the field of plant breeding.