A study of molecular and genetic mechanisms mediating the formation of twin sperm cells in Arabidopsis thaliana

In flowering plants, the male gametophyte produces a pair of functional sperm cells that are transported to the embryo sac for double fertilisation. Asymmetric division of the microspore establishes the germline and division of the germ cell results in two sperm cells, however, the molecular mechanisms governing the germ cell specification and division are yet to be uncovered. DUO1 has been identified as a germline-specific MYB binding transcription factor that coordinates germ cell division with gamete specification. One of the major objectives of this thesis is the characterization of an EMS-induced germ cell division mutant termed as duo pollen 5 (duo5) in Arabidopsis. Mutant duo5 germ cells were shown to elongate and enter mitosis but fail to complete the division cycle. Genetic analysis showed that duo5 is an incompletely penetrant gametophytic mutation that has reduced transmission thorough the male. Map based cloning defined duo5 to a genetic interval of ~250 kb region on the lower arm of chromosome IV. The thesis also explores the expression and regulation of two novel DUO1-activated zinc finger genes, DAZ3 and DAZ3L. The reduction in the activity of both promoters in duo1 germ cells suggested that DUO1 is required for their activation in the male germline. Analysis suggests that DUO1 possibly employ both direct and indirect mechanisms to activate DAZ3 and DAZ3L. Furthermore, analysis of protein fusion constructs demonstrated that DAZ3 and DAZ3L expression is predominantly localized in the sperm cell cytoplasm and this expression pattern persists in the developing pollen tubes. The analysis of DAZ3 expression in germ cell division mutants revealed that late activation of DAZ3 promoter is independent of germ cell division. The work demonstrated, will further add to the knowledge of male gametophyte development and will provide new opportunities to understand molecular and genetic mechanisms involved in the production of two plant sperm cells.