Regulatory mechanisms controlling male germline development in Arabidopsis thaliana

In flowering plants, the male gametophyte plays a vital role in plant fertility through the generation and delivery of two sperm cells to the embryo sac. A strict male germline is only established after meiosis when haploid microspores divide asymmetrically to form a small germ cell and large vegetative cell. This germ cell goes on to differentiate and divide once again to produce the functional twin sperm cells required for double fertilisation. Despite its importance in plant fertility and crop production, the mechanism integrating germ cell proliferation and specification during male gametogenesis has remained elusive. DUO1 is an evolutionary conserved unique R2R3-type MYB transcription factor that is specifically expressed in the male germline. DUO1 is the major determinant of male germline fate, regulating the expression of key specification genes required for fertilisation whilst integrating this with germ cell cycle progression through the regulation of the G2/M regulator CYCB1;1. DUO1 constitutes the major focus of this thesis and as such it addresses several aspects of DUO1 regulatory network. The first chapter of the thesis explores upstream regulatory mechanisms controlling male germline restricted expression in Arabidopsis. The expression of several genes, including DUO1, does not depend on a previously proposed derepression mechanism whilst DUO1 expression involves only positive promoter elements. The second chapter involves functional characterisation of the DUO1 protein, which has helped to delimit the transactivation domain of DUO1. This analysis has also provided insights into the evolutionary conserved supernumerary lysine residue present in the DUO1 MYB domain, which is likely to play a role in attenuating the rate of target gene transcription. The third part describes the analysis and verification of novel target genes in the DUO1 regulatory network. A mechanism for direct transactivation of target genes is also described whereby DUO1 binds to MYB sequences in target gene promoters. The final chapter concerns the functional analysis of two redundant DUO1-activated zinc finger (DAZ) EAR repressor proteins. Their intermediate role in coordinating male germ cell cycle progression is demonstrated by their ability to complement the cell cycle defect in the duo1 mutant. Furthermore, DAZ1 and DAZ2 also influence sperm cell specification, indicating that the DAZ subregulon also integrates specification with cell cycle progression. Together this data has provided compelling insights into the scale and architecture of the DUO1 male germline regulatory network controlling the production of functional sperm cells in flowering plants.