Abstract:
In double fertilization of flowering plants, sperm cells rely on a vegetative cell to germinate a pollen tube which delivers them to the ovary, where one of the sperm cells fuses with an egg cell to form an embryo, and the other fuses with a central cell to form the nourishing endosperm. The de novo zygotic gene expression starts at the late karyogamy stage, followed by clearance of gametic transcripts, thus completing a maternal-to-zygotic transition. While our knowledge of pollen tube guidance and gametic interactions has grown considerably, the molecular signaling events orchestrating the onset of the zygotic genome activation remain elusive, especially those transmitted from the paternal side that has been suggested to be critical in initiating zygotic program. Here, we developed a semi-high throughput mapping-by-sequencing method for paternal-defective mutations. It allows mapping multiple mutants simultaneously with a limited workload. With this method, we corroborated the function of the known paternal genes, CALS5, COBL10, and HAP2, in pollen development, pollen tube guidance, and gametes fusion, respectively. Besides, we revealed a novel paternal gene S-adenosyl-L-homocysteine hydrolase 2 (SAHH2) in pollen tube function. SAHH2 is expressed in the vegetative cell of pollen, and the loss-of-function mutants exhibited reduced male fertility with defective pollen tube elongation in the female transmitting tract, suggesting a role in preovular pollen tube guidance. In addition, the Origin of Replication Complex (ORC) gene ORC1b was selected and characterized using a reverse genetic study for paternal genes in fertilization. ORC1b is a sperm nuclear protein localized in the chromocenters. The depletion of ORC1b in the T-DNA insertion allele orc1b-1 and the knock-out allele orc1b-2 led to reduced male fertility coupled with polytubey, defective gametic nuclear fusion, arrested embryo, abnormal endosperm development and induction of aneuploidy progenies. These results suggest that ORC1 acts as a paternal factor that contributes to gametes fusion, embryo/endosperm development, and genome integrity maintenance. In summary, this thesis provides a novel mapping method and fluorescent marker lines for characterizing fertilization mutations. Assisted by these tools, several paternal contributed genes in fertilization have been studied and these results collectively further our understanding of the paternal contribution to fertilization in flowering plants.
fertilization
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