Analysis of stage-specific gene perturbations and characterisation of two novel F-box genes during flower development in Arabidopsis thaliana

Abstract

The model plant Arabidopsis thaliana has been used for past three decades to study the genetic and molecular processes underlying floral organogenesis. Flowers of this small plant consist of four concentric whorls, containing four sepals, four petals, six stamens, and a pair of fused carpels. Numerous studies in this field led to the proposal of the ABCE model of flower development, in which the combinatorial activities of four classes of master regulator transcription factors direct the formation of the floral organs. Extensive research has delineated the roles these transcription factors have with regard to specifying the floral organs, although gaps in our knowledge still remain concerning the downstream targets of these important regulators.

The first part of this thesis is focused on the development and optimisation of transgenic lines that can be used to reduce the activities of some of these homeotic genes at any given point during flower development, in order to study the gene networks that are regulated by these transcription factors at discrete developmental stages. This has been achieved through the use of artificial microRNAs (amiRNAs), designed to target and degrade mRNA transcripts of either the B-class regulator APETALA3 (AP3) or the C-class regulator AGAMOUS (AG). These amiRNAs were controlled by a hormone-activated system, such that transcription of the amiRNA precursors only occurred upon application of the correct inducer. These were used in tandem with a second hormone-inducible construct, which allows for the user-defined initiation of hundreds of synchronously-developing floral buds, facilitating the collection of large amounts of stage-specific tissue that can be used for downstream global analyses. These lines were used to perform a series of gene perturbations, at different stages during flower development, in order to analyse changes to global expression patterns using next generation sequencing technologies. From this it was possible to examine the different genes and processes these transcription factors regulated depending on the flower?s developmental stage, providing a greater insight into the dynamic roles of the floral homeotics.

The second part of the thesis focused on characterising two novel F-box genes that were thought to genetically interact with the F-box gene, and transcriptional co-regulator of B-class homeotic gene activity, UNUSUAL FLORAL ORGANS (UFO). F-box genes encode for proteins that typically provide substrate specificity to multi-subunit E3 ligase complexes, which participate in protein modification and turnover through the conjugation of small ubiquitin moieties. Two closely-related genes, named NANNY (NAN) and GRANNY (GRA), were identified through searches for F-box genes possessing similar expression profiles to that of UFO, in order to determine if they shared any functions related to flower development. Transcriptional reporter lines, and in situ hybridisations were performed, to establish the expression profiles of these genes, and their ability to interact with other subunits of E3 ligase complexes was verified. Several over-expression constructs were generated and studied, to attempt to place the genes in some developmental pathway, and finally the genome-editing technology CRISPR/Cas9 was employed to obtain full knockouts of both genes, separately and together, to learn more about any shared functions these genes might have.