Characterisation of the floral transcriptome of Sinapis alba and analysis of PRC2 accessory proteins in Arabidopsis thaliana

Abstract

Flower development has been studied in the model organism Arabidopsis thaliana for decades. Years of research into the development of the floral organs - carpels, stamens, petals and sepals - led to the formation of the ABCE model of flower development. This model elegantly describes the combinatorial roles of certain sets of genes to form the floral organs, primarily the MADS-box gene family. With the advent of genome-wide transcriptomic studies, it has been found that this model is upheld in other angiosperms, even those far diverged from Arabidopsis thaliana. It has become clear since the discovery of this model that there are a multitude of molecular factors at play in the timing of flowering and the development of the individual organs. Many studies have aimed to explore the multimeric protein complexes, epigenetic mechanisms and the post- transcriptional modifications that regulate the development of flowers and have contributed to the overall understanding of these mechanisms in the spatial and temporal regulation of genes. All of the work in this thesis will make use of one or more genome-wide experiments to investigate changes in gene expression across different species and genotypes. The first part of this thesis was focused on the flowers of Sinapis alba. Genome assemblies using Nanopore sequencing were conducted to establish a reference genome to base subsequent studies on. RNA-seq and cryo-scanning electron microscopy allowed for the generation of the first in-depth floral transcriptome of this species by characterising the MADS box gene family’s expression and levels of conservation relative to Arabidopsis thaliana and Brassica rapa.. A highly conserved floral transcriptome was identified and established. An upregulation of some positive regulators of trichome development and a notable depletion of some negative regulators was found. This led to the identification of several candidate genes for this phenotype by comparisons to closely related species within the Brassicaceae family. While no definitive cause for this trichome phenotype was established, it provided a strong platform for future experiments and significantly improved annotation for a species without much prior data available for it. The second part of this thesis explores the functions of the domesticated transposases ALP1 and ALP2 in the deposition of histone modification marks by Polycomb Repressive Complex (PRC2) in Arabidopsis thaliana. The H3K27me3 of the histone methyltransferase mutant, clf, was explored using clf alp1 and clf alp2 lines - both of which showed significant restoration of the H3K27me3 mark, highlighting the inhibition of PRC2 by the ALP1/2 proteins in wild-type Arabidopsis thaliana. The same was done in a histone demethylase mutant background, ref6, to see if the changes in H3K27me3 were compounded by introducing the alp mutants. H3K27me1 deposition changes were also scrutinized as it was unknown whether the ALP proteins inhibit the deposition of this mark as well as H3K27me3. Analysis of the magnitude of the H3K27me3 changes as well as the shapes of the peaks found at certain sites were used in tandem with corresponding RNA- seq data to establish what role the ALP proteins were playing in PRC2 inhibition. It was found that the ALP proteins do not inhibit the deposition of the H3K27me1 mark but have impacts on the deposition of the H3K27me3 mark genome wide. The effects of these changes in H3K27me3 were small but seemed to have impacts on the expression of genes involved in the floral transition. The final part of this thesis examined the differential gene expression in different mutants of the floral organ boundary gene, SUPERMAN (SUP). Microarray data of sup-5 mutants at different floral developmental stages were analysed. Follow-up studies involving different mutant phenotypes, namely sup-1 and a novel CRISPR allele generated during this study, were analysed and compared phenotypically to sup-5. The differentially expressed genes of sup-5 and sup-1 were compared by making use of the floral induction system (FIS) and RT-qPCR. Through this, candidate genes for the notably divergent phenotypes of the two alleles were identified - such as an upregulation of the C2H2 zinc finger transcription factor, URO and a potential role for changes in chromatin conformation playing a role in the distinctive sup-5 phenotype. Taken together, the aim of this thesis was to improve the understanding of gene expression throughout plant development in different contexts and species. Making use of genome wide approaches and bioinformatic analysis provided a comprehensive set of data that gave insights into floral initiation and development of Sinapis alba and Arabidopsis thaliana.