Cell fate specification and maintenance in the female gametophyte and transcriptional profiling of early embryos in Arabidopsis thaliana

Abstract

The life cycle of flowering plants alternates between a diploid sporophytic and a haploid gametophytic phase. Successful reproduction depends on fate specification of the gametic cells within the embryo sac and on double fertilization. This double fertilization event is initiated by fusion of the central cell with one of the two sperm cells as well as fusion of the egg cell with the other sperm cell, which gives rise to the endosperm and the embryo, respectively. However, the molecular mechanisms of cell fate specification during female gametophyte development remain elusive and gene expression programs in different cell types of early embryos are largely unknown. Here, we show that ALTERED MERISTEM PROGRAM 1 (AMP1) is required to prevent cell fate substitution within the embryo sac in Arabidopsis. Loss-of-function mutations in AMP1 result in supernumerary egg cells at the expense of synergid cells allowing for twin embryo formation. However, generation of twin embryos usually precludes endosperm formation, which eventually leads to ovule abortion. Remarkably, in such a case, ovule abortion can be overcome by delivering functional supernumerary sperm cells in tetraspore (tes) pollen, thereby enabling the formation of twin plants. AMP1 expression in sporophytic tissue is sufficient to suppress the formation of supernumerary egg cells and the twin-embryo phenotype, indicating that one or more mobile factors are involved in synergid fate specification and that the surrounding tissue can contribute to the patterning process of the female gametophyte. This work highlights the importance of specifying two synergids and only one egg cell within the female gametophyte to ensure successful reproduction. The second part of this thesis includes a technique that integrates nuclear sorting by flow cytometry and subsequent microarray analysis to generate transcriptome datasets of different cell types in the Arabidopsis early embryo. The results presented indicate that the majority of genes are similarly expressed in the pro-embryo and the suspensor but that a number of genes with supposedly important functions during embryogenesis showed different expression patterns. This technique and the resource presented will assist further studies of early embryogenesis. In addition, this technique can also be applicable to other inaccessible tissues with a limited number of cells.