Angiosperm sexual reproduction is unique and remarkable. Unlike for other living organisms, fertilisation involves the fusion of two sperm cells to two cell types of female gametophyte, the egg and central cell. This fertilization process is called double fertilisation. Fusion of the egg and the sperm yields a zygote whereas the interaction of the central cell and the sperm gives the nutritive endosperm. This fertilization process has been studied extensively for many years. However, despite these studies, relatively little is known at the molecular level about either of the plant gametes. This is primarily due to the difficulties in plant gamete isolation. Both plant gametes reside within enclosed tissues, pollen grains and embryo sacs. In this study, sperm isolation techniques were successfully developed for Brassica oleracea and Arabidopsis thaliana which ultimately utilised fluorescence-activated cell sorting (FACS) to obtain pure cell samples. Proteomic studies utilising two-dimensional protein electrophoresis and mass spectrometry (MS) were carried out on both semi-purified and FACS-purified sperm cells. In parallel with the proteomic studies a bioinformatics approach was taken which used sperm transcriptome data of maize, Plumbago zeylanica, rice and tobacco to identify homologues in Arabidopsis. Transcriptional analyses, RT-PCR and GFP translational fusion experiments were used to investigate these Arabidopsis sperm cell-expressed gene candidates. As a result, two sperm cell-expressed genes (At1g10090 and At5g39650) were identified and these are being analysed to confirm their functions in the reproductive process. Moreover, the sperm cell-expressed gene candidates derived from the bioinformatics were also screened for roles in plant reproduction by a reverse genetics approach (Arabidopsis T-DNA insertion mutagenesis plant screening) and eight genes were identified. In addition, for the first time, sperm cell size dimorphism was identified for Arabidopsis in this study utilising a GFP-labelled sperm line and confocal microscopy. Overall the techniques for sperm cell-expressed gene candidate selection were proven to be effective and will certainly facilitate further sperm cell-specific gene identification studies. Further the Arabidopsis sperm purification technique successfully developed in this project will surely be useful for any plant sperm cell studies in the future.
|Date of Award||1 Sep 2008|
|Supervisor||James Doughty (Supervisor)|