Molecular genetic investigation towards functions of imprinted genes in reproduction and seed development in Arabidopsis thaliana

Abstract

In flowering plants and placental mammals a subset of genes known as imprinted genes display full or partial suppression or activation to one of its alleles in a parent of origin dependent manner. During maternal and paternal gametogenesis in plants, epigenetic marks are applied that consequentially alter the regulation of specific alleles and upon fertilisation these marks contribute monoallelic expression patterns. Predominantly, imprinting is considered to occur within the endosperm, a terminal tissue which acts to nourish endosperm development. Reports have demonstrated that a small number of imprinted genes are essential for correct seed development and with the aid of next generation transcriptomic screens the number of discovered candidate imprinted genes has increased. However, functional roles could not be discovered for the majority of these imprinted genes which leads to uncertainty regarding the true evolutionary role towards the selection of genomic imprinting. A portion of this thesis focused on investigating whether changing environments affected the expression and imprinted status of imprinted genes. Of seventeen imprinted genes tested, nine displayed significant expression differences in response to changes in temperature. From these genes, the POLYAMINE UPTAKE TRANSPORTER 1 (PUT1) gene displayed temperature mediated loss of imprinting. In addition, a loss-of-function screen was conducted on imprinted genes which displayed signatures of Positive Darwinian Selection (PDS) in their coding sequences in order to screen for functional roles during seed development. From this screen, one promising candidate mutant emerged named short filaments-1 (sfi-1). sfi-1 displayed a loss of functional filament elongation and decreased seed size compared to wild type (WT) plants. Finally, an efficient targeted mutagenesis strategy was also generated using the Cas9/sgRNA system in order to create novel, null or hypomorphic mutants in Arabidopsis thaliana. Using the system described in this thesis efficient targeted mutations were generated in the METHYLTRANSFERASE (MET1) and TRANSPARENT TESTA GLABRA 1 (TTG1) genes in both a diploid and tetraploid background. Altogether this thesis revealed a potential new role for the imprinted gene SFI as a regulator of mature seed size and filament elongation. In addition, it also provides us with an efficient tool for genome editing in Arabidopsis thaliana for future investigations.