Investigation into the transcriptional regulation of DUO1: determining the fate of the male germline in flowering plants

The pollen grain is responsible for the production and delivery of sperm cells to the female gametes in flowering plants. Early in pollen development, there is an asymmetric division that is critical for the specification of the male germ cell. In this germ cell, shortly after the division, the transcription factor DUO1 is expressed. DUO1 controls the differentiation of the germline through activating a suite of genes required for cell cycle progression and sperm cell function. Thus, the early germ cell specific expression of DUO1 is a key step in the specification of the male germ line. This work investigated the mechanisms that control DUO1 expression with the hope of gaining a molecular understanding of the asymmetric division. A DUO1 promoter:reporter construct shows the same expression pattern as DUO1, therefore this work focused on transcriptional regulation. Bioinformatic approaches, using DUO1 promoter sequences from a range of flowering plants, identified a region showing a surprising level of conservation for a regulatory region. This region is termed ROD1 and was identified in all eudicot species. Further investigation identified three cis-regulatory elements (CREs) present in ROD1, each with a specific function. The GTGG CREs are required for DUO1 activation. Attempts were made to identify the transcription factor that bound to this CRE but were largely unsuccessful. The AACYG CREs are important for enhancing the expression of DUO1 and are bound directly by DUO1 in an auto activation loop. Lastly, the GAGARAA CRE acts to silence DUO1 expression and is likely bound by the transcription factor BPC2. The process of discovering this new knowledge of the regulation of DUO1 required the development of novel methods. The novel methods as well as the most significant findings of this work have been published