A study of mechanisms of cell cycle regulation in the male gametophyte of Arabidopsis thaliana

The haploid male gametophyte of angiosperms has an integral role in the production of twin sperm cells necessary for the double fertilization, the essence of flowering plants. However, the mechanisms regulating sperm cell formation and cell fate specification has yet to be identified. In this study, the thesis investigates the function of key cell cycle regulators and presents characterisation of a novel pollen division mutant of Arabidopsis (duo3) that fails to produce twin sperm cells. In addition, the project also examines the activity of small RNA (smRNA) pathways as a potential mechanism that modulates native gene expression. Pollen cell-specific vectors were constructed to drive the expression of hairpin double stranded RNA (hp-dsRNA) as tools for investigating the activity of smRNA pathways, and their efficacy was tested by manipulating expression of key cell cycle regulators in Arabidopsis. Indeed, expression of hp-dsRNA intended to knockdown transcripts of Cyclin B1 members, revealed a putative role Cyclin B1 in microspore and germ cell division. Furthermore, analysis of a Cyclin B1;1 reporter led to the identification of DUO1 (a pollen specific R2R3 MYB protein) but not DUO3 as a germ cell-specific regulator of Cyclin B1;1 expression. This interaction was further verified by rescuing mutant duo1 plants with Cyclin B1;1. Analysis of DUO3 expression revealed restricted patterns confined predominantly in dividing tissues. Moreover, study of Cyclin B1;1 reporter revealed mutant duo3 cells to be impaired in degrading Cyclin B1;1 protein, suggesting a role in modulating Cyclin B1;1 activity. In summary, this work has highlighted a potential role of the Cyclin B1 family in the development of the male gametophyte. Use of Cyclin B1;1 marker has demonstrated a first example of germ cell specific integrator of cell division and cell differentiation and a putative role of DUO3 in germ cell division. A significant progress has been achieved in understanding smRNA pathways and the vectors generated will be exploited to gain more insight into the development of the male gametophyte.