1. Female gametophyte development and double fertilization
Plant gametes are, in contrast with animal gametes, not direct products of meiosis but differentiate within multicellular haploid generations, the male and the female gametophytes. In flowering plants (angiosperms), the gametophytic generations are reduced to a few cells that are located within the diploid sporophytic tissues of the flower.
The female gametophyte (or embryo sac) is enclosed by the ovule that is located within the ovary. In Arabidopsis, the female gametophyte originates from a single haploid spore (functional megaspore), which undergoes three free nuclear divisions to develop into an eight-nucleate syncytium. Following cellularization, the distinct haploid cell types of the female gametophyte are formed: Two female gametic cells, the egg cell and the central cell, which are flanked by accessory cells. The large vacuole of the central cell occupies the central region of the female gametophyte. Two synergid cells neighbour the egg cell at the micropylar end of the female gametophyte, while three antipodals are adjacent to the central cell at the chalazal end (Sprunck & Groß-Hardt, 2011).
Double fertilization
Fertilization in flowering plants is unique among all known organisms in that not one, but two, female reproductive cells are fertilized in a process called double fertilization. A complex mechanism involving two male gametes (sperm cells) and two female gametes (egg cell and central cell) results in two distinct fertilization products, the diploid embryo and the triploid endosperm. Both fertilization products are required to achieve successful seed development.
During double fertilization, the two non-motile sperm cells are transported as a pair by the growing pollen tube, which is guided through the style and the transmitting tract of the pistil to the ovule (A). Finally, the pollen tube grows through the micropyle of the ovule into the embryo sac (B), where two female gametes (egg cell and central cell) are located. After pollen tube burst, the sperm cell pair is released towards a cleft between the egg and central cell. One sperm cell fuses with the egg cell, while the second sperm cell fuses with the central cell (C), resulting in two distinct fertilization products: the embryo and the endosperm.
In contrast with the molecular events involved in guided pollen tube growth and sperm cell delivery into the female gametophyte, almost nothing is known about later processes, covering the phase from sperm cell release until gamete fusion (plasmogamy) (Sprunck, 2010). Within minutes after a sperm cell pair is delivered, each one of the sperm cells fuse with one of the female gametes to initiate seed development in a coordinated manner. Attempts to elucidate these issues at the cellular and molecular levels are hampered by the fact that angiosperm gamete interactions are short-lived events, taking place deeply embedded in the maternal tissues of the ovule and ovary.
2. Monitoring male-female gamete interactions in Arabidopsis
Using our expression data derived from egg cells and central cells from wheat (Sprunck et al., 2005), we identified gamete-specific genes and their putative orthologs in Arabidopsis. We used the corresponding promoter regions to generate ß-glucuronidase (GUS) and green fluorescent protein (GFP) marker lines for the Arabidopsis egg cell, the central cell, and synergid cells. The green fluorescent ARO1 marker (Gebert et al., 2008) labels the egg cell cytoplasm (EC) in ovules at maturity, while the green fluorescent EC1.1p:NLS-3xGFP marker is specifically expressed in the egg cell nucleus (ECN). These and new membrane marker lines now serve as important tools to investigate gamete interactions on the cellular and molecular level. The interaction of the gametes can be observed microscopically, when our marker lines are pollinated with sperm cell membrane marker lines (Sprunck et al., 2012), or the previously published red fluorescent sperm nucleus marker line HTR10-mRFP1 (Ingouff et al., 2009).
Currently, these marker lines are used to study mutants that have been generated in our group, showing defects during double fertilization.
3. Cell surface molecules involved in gamete recognition and/or fusion
Male-female gamete recognition, adhesion, and fusion encompass the most intimate steps during fertilization. Nevertheless, the knowledge about molecules that act on the cell surface of flowering plant gametes is scarce.
We identified a cluster of egg cell-specific transcripts in our expression data from wheat egg cells (EC-1) that encode secreted small cysteine-rich EC1 proteins (Sprunck et al., 2005). Five genetically redundant EC1 genes (EC1.1 to EC1.5) are present in the genome of Arabidopsis thaliana. Functional studies revealed that egg cell-secreted EC1 proteins are essential for the sperm cells to gain fusion competence (Sprunck et al., 2012, Rademacher and Sprunck, 2013).
Currently, we are analyzing our transcriptome and proteome data to identify more cell surface proteins which are involved in male-female gamete recognition, adhesion, and fusion.
The main aims and research questions of this project are:
- Which molecules on the cell surface of female and male gametes mediate gamete recognition, adhesion and fusion in flowering plants?
- How do the egg cell-secreted EC1 proteins act on the molecular level?
- Are there species-specific gamete interaction mechanisms in flowering plants?