In flowering plants, two sperm cells develop in the pollen cytoplasm and are transported through floral tissues to an ovule by a pollen tube, a highly polarized cellular extension. After targeting an ovule, the pollen tube bursts, releasing two sperm that fertilize the egg and central cell producing the embryo and endosperm respectively. The mechanisms responsible for fusion of the two sperm with female cells are unknown. This thesis presents the initial characterization of the Arabidopsis HAP2 gene, demonstrating that it is essential for fertilization and probably directly involved in a deeply conserved gamete fusion mechanism. We also show that HAP2 is required for pollen tube guidance. We searched for proteins that interact with HAP2 through a yeast two-hybrid screen and identified proteins that regulate disulfide bond formation, these data indicate several invariant cysteine residues within the N-terminal region of HAP2 are likely critical for its structure and function. We utilized the tightly regulated sperm-specific expression pattern of HAP2 to disrupt sperm development by expressing the Diphtheria toxin A subunit. By disrupting sperm translation in this manner, single sperm-like cells (SSLCs) were generated that preferentially fertilized the central cell. This finding is contrary to the previously held idea that SSLCs like those created in the cdc2 mutant have a default targeting to the egg. We were able to ectopically express HAP2 in the pollen tube, but were unable to find transformants that express HAP2 in the leaves when expressed from the 35S promoter. These experiments begin to elucidate the expression pattern, proper folding, and function of HAP2 during fertilization.
|Advisor:||Johnson, Mark A.|
|School Location:||United States -- Rhode Island|
|Source:||DAI-B 71/11, Dissertation Abstracts International|
|Subjects:||Molecular biology, Plant biology|
|Keywords:||Fertilization, Membrane fusion, Single sperm-like cells|
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