Environmental signals can directly influence gene expression through epigenetic mechanisms, causing phenotypic changes that can be transmitted to progeny. In plants, this is in part achieved by short interfering RNA (siRNA) which guide covalent modification of DNA, such as cytosine methylation, to specific targets including repetitive sequences and transposable elements. Environmental stress also leads to genome-wide DNA hypomethylation, misregulation of transposable elements and ultimately ‘genomic shock’. Although most stress-induced epigenetic modifications are not thought to be heritable, there is increasing evidence for the inheritance of novel environmentally-induced epigenetic states or ‘environmental epialleles’. The formation of environmental epialleles represents an important source of variation and a powerful driving force of adaptive evolution but the precise mechanism remains unclear. The aim of this thesis is to identify environmental epialleles through computational methods. Analysis of Illumina sequencing data from environmentally stressed maize plants sampled during stress and after a recovery period has so far revealed that a significant proportion of the maize genome is misregulated at both the genetic and epigenetic level. These findings indicate that plants continue to respond after exposure to stress and that this response is likely mediated by at least one epigenetic mechanism, including siRNA-directed DNA modifications.

Colletotrichum acutatum sensu lato includes a number of important pathogens that cause economically significant losses of various crops. The C. acutatum species complex has a wide host range in both domesticated and wild plant species, and its capability to infect different types of hosts such as insects has also been described. Members belonging to this complex are able to develop three different types of interaction with plant hosts including biotrophic, necrotrophic and hemibiotrophic infections and are also capable of surviving on weeds and non-hosts without causing visible symptoms. They are mainly asexual, but some have a teleomorphic state called Glomerella and can be either homothallic or heterothallic. The sexual behaviour in Glomerella is more complicated than in most ascomycetes, and strains within the same species do not show a typical MAT1-1/2 system. The overall aim of this study was to gain an improved understanding of the relationships between the genetic diversity of global populations, host association patterns, geographic distribution and biological and pathological attributes. A database (CaITSdb) containing more than 800 rRNA sequences deposited in GenBank was created along with key biogeographic information, and the data have been analysed in order to investigate genetic diversity and distribution of sub-populations and their evolutionary relationships. The combined evidence was used to assemble a core collection of 120 isolates that are representative of the diversity in host preference, geographic origin, mating behaviour and molecular genetic variation. A multi-locus sequencing approach (based on four neutral loci) has been used to evaluate phylogenetic relationships amongst the isolates in the core collection. A strong relationship was observed between various genetic groups distinguished and their mating behaviour, geographic distribution and host association patterns. Oceania has been identified as a likely geographic origin of this pathogen, as the highest level of variability and groups related to a hypothetical ancestral population are mainly distributed in these countries. All homothallic isolates capable of producing perithecia belonged to the same genetic group A7; whereas all self-sterile heterothallic isolates were classified as either A3 or A5. Isolates derived from the same host tend to cluster together into genetic groups or sub-populations. This evidence is generally in agreement with recent published work on taxonomic re-assessment of Colletotrichum acutatum sensu lato, which indicates at least fifteen new species. This study has provided the evidence for the occurrence of three distinct genetic groups on strawberry in the UK corresponding to three species reported in the literature namely, C. nymphaeae, C. fioriniae and C. godetiae. Isolates belonging to the genetic groups that correspond to C. nymphaeae and C. fioriniae appeared to be the most aggressive on strawberry, followed by C. godetiae, and C. simmondsii (not found in the UK). Representative isolates of other species were less aggressive. The first whole genome sequence an isolate (A9 = C. simmondsii) from the C. acutatum sensu species complex was assembled and analysed using a range of bioinformatics algorithms. An isolate of C. simmondsii was chosen based on its wide host range including strawberry and the phylogeographic position. Genome analyses enabled prediction and annotation of the whole gene set at 13549 including 6 % unique to this species. The data also suggested an interesting expansion of several gene families, such as those encoding carbohydrate-active enzymes, secondary metabolites pathways and effectors which could be associated with the wide host range. The new knowledge and resources developed with the genome analyses along with the results of the population level diversity studies provide a platform for future comparative and functional genomics investigations to advance this research.

Homologous recombination is an integral part of meiosis and is essential for generating crossovers that ensure balanced segregation of homologous chromosomes and establish genetic variation within offspring. It is therefore exceedingly important that meiotic cells employ stringent control mechanisms to safeguard crossover formation. Work in yeast has indicated that the meiotic axis, a proteinaceous structure that tethers meiotic chromosomes into looped arrays, plays a crucial role in many aspects of homologous recombination, from double strand break formation to crossover interference. It has also been suggested that increased crossover interference helps to establish meiotic stability by inhibiting multivalent formation during autopolyploid meiosis. Using immunocytochemistry coupled with super-resolution microscopy, we have further investigated the role played by the meiotic axis protein ASY1 in stabilising meiosis in the established autotetraploid Arabidopsis arenosa. We have also used Arabidopsis arenosa as a model for studying how meiotic interference might operate within an autopolyploid context. Alongside this, experiments using transgenic lines of the model plant Arabidopsis thaliana have helped to shed light on how crossover formation and synapsis are affected by reduced expression of ASY1 and ASY3 and to determine what effect limiting meiotic crossover numbers might have on neopolyploid meiotic stabilisation.

QTL mapping with segregating populations results in poor map resolution which limits the applicability of mapped QTL in further research such as gene cloning. The current research project aimed mainly at developing STepped Aligned Recombinant Inbred Strains (STAIRS) covering the top region of chromosome 3 and demonstrating the feasibility of using STAIRS in high resolution mapping of QTL in Arabidopsis. The top region of chromosome 3 of Arabidopsis had been reported to house QTL related to flowering time. This region was first saturated with 24 polymorphic microsatellite markers and 23 narrow STAIRS were produced within the region via a marker-assisted backcross breeding programme using whole chromosome substitution lines. The analysis of QTL with the narrow STAIRS revealed a major pleiotropic QTL within 2-3 cM affecting flowering time, leaf number at day 20 and rosette and cauline leaf numbers at flowering. A second QTL with less but opposite effect on the same traits were located within 15-20 cM. The search for candidate genes within 2-3 cM of chromosome 3, to locate possible candidate genes revealed COL-2, CONSTANS-Like gene which affects flowering time. Microarray gene expression profiling was performed using the two genotypically closest lines which differ for flowering time to compare the two lines at the same chronological and physiological ages in two experiments respectively. The lists of differentially expressed genes were obtained from the two experiments. Differential expression was observed for the possible candidate gene in the latter experiment. The results emphasized the power of STAIRS in fine mapping of QTL and the possibility of using them in transcriptional profiling to study the expression of genes.

During DNA replication, forks often stall upon encountering obstacles blocking their progression. Cells will act to speedily remove or overcome such barriers, thus allowing complete synthesis of chromosomes. This is the case for R-loops, DNA/RNA hybrids that arise during transcription. One mechanism to remove such R-loops involve DNA/RNA helicases. Here, I have shown that one such helicase, Sen1, associates with replisome components during S phase in the model organism S. cerevisiae. I demonstrate that the N-terminal domain of Sen1 is both sufficient and necessary for the interaction of the protein with the replisome. I also identified Ctf4 as one of at least two replisome interactors of Sen1. By mutational analysis, a mutant of Sen1 (Sen1-3) that cannot interact with the replisome was created. This mutant is healthy on its own but is lethal in the absence of both RNase H1 and H2. Overexpression of the sen1-3 allele from the constitutive ACT1 promoter is able to suppress this synthetic lethality, suggesting that Sen1 travels with replisomes in order to be quickly recruited at sites of R-loops that impair fork progression so as to remove those R-loops. In some cases, cells exploit fork stalling for biologically important processes. This is the case in Sz. pombe, where an imprint prevents complete DNA replication, triggering cell-type switching. This imprint is dependent on Pol1, a component of the replisome. Importantly, a single imprinting-defective allele of pol1 has been identified to date. Using in vitro assays, I have shown that this Pol1 mutant has reduced affinity for its substrates and is a correspondingly poor polymerase. By generating novel alleles of pol1, I have also demonstrated that switching-deficiency correlates with the affinity of Pol1 for its substrates in vivo. Finally, two interactors of Pol1 (Mcl1Ctf4 and Spp1Pri1 ) have been shown to have switching defects. S. cerevisiae and Sz. pombe have similar yet distinct genetic nomenclature conventions. Given that both model organisms were used in this study, it is important to highlight the conventions for both organisms to prevent confusion. In S. cerevisiae, wildtype gene names are expressed as a three letter, uppercase and italic name followed by a number (e.g. SEN1). The three letter name often corresponds to the screen through which the gene in question was originally identified. Mutants are generally designated with the same three letter but in lower case (unless the mutant is dominant) and with an allele designation (e.g. sen1∆, sen1-1 and sen1-2). Because of historical context, the allele designations vary in format (e.g. leu2-3,112 is a mutant of LEU2). Protein names are given as a three letter name with the first letter in uppercase (e.g. Sen1). This is also true for mutant proteins, with the added allele designation (e.g Sen1-1 and Sen1-2). In this study, I have generated constructs of the SEN1 gene and these constructs are referred to as SEN1 (X-Y), where X and Y refer to the first and last residues being encoded for. The corresponding proteins are referred to as Sen1 (X-Y). Different promoters have been used and, where appropriate, the promoters are expressed similarly to their wildtype gene names (e.g. GAL1, SEN1 and ACT1). In Sz. pombe, wildtype gene names are expressed as a three letter, lowercase and italic name followed by a number (e.g. pol1). Mutants are generally designated in the same format but with an allele designation. Like in S. cerevisiae, the allele designation varies widely (e.g. pol1-1, pol1-H4 and pol1-ts13). Additionally, because of the historical context, some (but not all) alleles of pol1 are referred to as swi7 to reflect the fact that they are defective for cell-type switching. Similar to the situation in S. cerevisiae, proteins names are given as a three letter name with the first letter in uppercase for both wildtype and mutants (e.g. Pol1 and Swi7-1). Sometimes, for the sake of comparison, genes or proteins are referred to their S. cerevisiae orthologues (e.g. swi1TOF1 and Swi1Tof1 , respectively). Several protein tags have been used in this study. When written in gene form, they were written in capital letters and italicized, irrespective of the host (e.g. 5FLAG) and when in protein form, they were written in capital, irrespective of the host (e.g. 5FLAG).

Stress responses in plants can be defined as a change that affects the homeostasis of pathways, resulting in a phenotype that may or may not be visible to the human eye, affecting the fitness of the plant. Crosstalk is believed to be the shared components of pathways of networks, and is widespread in plants, as shown by examples of crosstalk between transcriptional regulation pathways, and hormone signalling. Crosstalk between stress responses is believed to exist, particularly crosstalk within the responses to biotic stress, and within the responses to abiotic stress. Certain hormone pathways are known to be involved in the crosstalk between the responses to both biotic and abiotic stresses, and can confer immunity or tolerance of Arabidopsis thaliana to these stresses. Transcriptional regulation has also been identified as an important factor in controlling tolerance and resistance to stresses. In this thesis, networks of regulation mediating the response tomultiple stresses are studied. Firstly, co-regulation was predicted for genes differentially expressed in two or more stresses by development of a novel multi-clustering approach, Wigwams Identifies Genes Working Across Multiple Stresses (Wigwams). This approach finds groups of genes whose expression is correlated within stresses, but also identifies a strong statistical link between subsets of stresses. Wigwams identifies the known co-expression of genes encoding enzymes of metabolic and flavonoid biosynthesis pathways, and predicts novels clusters of co-expressed genes. By hypothesising that by being coexpressed could also infer that the genes are co-regulated, promoter motif analysis and modelling provides information for potential upstream regulators. The context-free regulation of groups of co-expressed genes, or potential regulons, was explored using models generated by modelling techniques, in order to generate a quantitative model of transcriptional regulation during the response to B. cinerea, P. syringae pv. tomato DC3000 and senescence. This model was subsequently validated and extended by experimental techniques, using Yeast 1-Hybrid to investigate the protein-DNA interactions, and also microarrays. Analysis of mutants and plants overexpressing a predicted regulator, Rap2.6L, by gene expression analysis identified a number of potential regulon members as downstream targets. Rap2.6L was identified as an indirect regulator of the transcription factor members of three potential regulons co-expressed in the stresses B. cinerea, P. syringae pv. tomato DC3000 and long day senescence, allowing the confirmation of a predicted gene regulatory network operating in multiple stress responses.

Higher organisms can regulate gene expression through changes in epigenetic marks present on the genome. However, how this regulation takes place in organisms with highly repetitive/complex genomes is not well understood. The acquisition of de novo DNA methylation in plants is mediated by siRNAs through the RNAdirected DNA methylation (RdDM) pathway. The targeted deposition of DNA methylation by this pathway allows for the transcriptional silencing of transposable elements and repeat sequences within the genome, as well as regulating gene expression. In addition, it has been hypothesized that mobile siRNAs may be involved in the epigenetic communication between different seed components. Thus the mobility of non-coding RNAs from extra-embryonic tissues could contribute to epigenetic modifications that could be transmitted to the offspring. The aim of my thesis is to characterise the mechanisms involved in epigenetic gene silencing in maize through the use of a novel transgenic reporter. My work has identified components of the RdDM pathway to be involved in maintenance of gene silencing and show that imprinting and paramutation could be recapitulated using synthetic transgenes. In addition, I developed a novel grafting technique to demonstrate that epigenetic gene silencing could be efficiently transmitted between different seed components. Collectively, this work provides an insight into the complex mechanisms that regulate gene expression in the highly repetitive/complex genome of maize.

Understanding the regulatory DNA sequences are becoming increasingly important in understanding the way plants integrate signalling cues mediated through the actions of the transcription factors (TFs). This thesis presents an interdisciplinary investigations into regulatory elements found in the promoter regions of a model organism Arabidopsis thaliana. The intergenic DNA sequences are studied between sets of orthologous genes in A. thaliana and 3 other related species to uncover hundreds of evolutionary conserved noncoding sequences (CNSs). The CNSs are found to be more skewed towards the annotated transcription start sites (TSSs) and enriched in previously identified transcription factors binding motifs. Furthermore, the nucleosomes are predicted to have strong presence in the uncovered CNS than random intergenic sequences alone. Altogether the evidence presented in the thesis points to the functional nature of the CNSs. Then, the promoters of genes thought to be co-regulated together and transcriptionally active during infection with fungal pathogen Botrytis cinerea are experimentally tested for direct protein-DNA interaction using high-throughput Yeast One-Hybrid (Y1H) library screens against the TFs found in A. thaliana. The resulting predictions were further validated using pairwise Y1H screen to suggest potential common regulation by ORA59, PIF7, ESE1, At4g38900 and ERF14, and uncovering a complex gene regulatory network (GRN) associated with the tested genes. The promoter fragments together with the predictions from the Y1H screens were used in the computational analysis to establish transcription factor specific binding motifs. Some of the newly predicted motifs were mutated and tested again for altered binding of the associated TFs. Furthermore, in planta mutations of the TFs predicted to be interacting with the promoters of the genes in the Y1H screens were found to have significant impact on the susceptibility of A. thaliana to infection with B. cinerea, further informing gene regulatory network active in response to biotic stress.

Circadian clocks are endogenous oscillators that control many physiological processes and confer functional and adaptive advantages in various organisms. These molecular oscillators comprise several interlocked feedback loops at the gene expression level. In plants, the circadian clock was recently shown to be organ specific. The root clock seemed to involve only a morning loop whereas the shoot clock also includes an evening loop in a more complex structure. My work aimed at refining the differences and similarities between the shoot and root clocks, using a combination of experimental and theoretical approaches. I developed an imaging method to obtain more data from the shoot and root clocks over time in various conditions. Some previous results were confirmed: the free running periods (FRPs) are longer in roots compared to shoots under constant light (LL). In addition, the amplitude of clock gene expression rhythms is lower in roots compared to shoots. However, the expression of several evening genes is circadian in roots, contrary to previous conclusions. This was confirmed with qPCR, and was observed in both light- and dark-grown roots. Yet light affects clock gene expression in roots, so an automatic covering system was designed to keep the roots in darkness and obtain data in more physiological conditions. Clock genes behaved differently in shoots and light-grown roots that were in the same environmental conditions, and may be differentially affected by blue and red light. However shoot and root clocks were more similar under constant darkness (DD). My imaging and RT-qPCR data, together with new microarray results and preliminary studies on clock mutants suggest that shoot and root circadian systems may have a similar structure but different input pathways. Entrainment is a fundamental property of circadian systems, which can be reset by cues such as light/dark (LD) cycles. I demonstrated that light can directly entrain the root clock in decapitated plants. The root clock could be entrained by a broad range of T cycles using low light intensity. In addition, rhythms were preferably entrained by low light than by any putative signal from shoots in experiments using conflicting LD cycles of different strengths. My results indicate that direct entrainment by LD cycles could be the main mechanism that synchronise the shoot and root clocks at constant temperature. This is physiologically relevant because dark-grown roots can perceive light channelled by the exposed tissues, in a fibre optic way. I also showed for the first time that clock and output genes could be rapidly entrained by temperature cycles in roots. Several mathematical models of the shoot circadian clock were used to try and fit the root clock data by optimising some parameters. The best set of parameters gave a good qualitative fit to root data under LD, LL and DD. It reproduced the long FRP observed in roots under LL and captured the entrainment under LD with lower amplitude in roots. The parameters that were changed for these simulations were all related to light input, which supports the idea of similar clock structures in shoots and roots but with different input pathways. Together my results confirmed that the plant circadian clock is organ specific and suggest that it is organ autonomous.

Brassica crops are grown worldwide for food, oil, medicinal and crop rotation properties. They suffer from insect pests which cause large yield and economic losses. Application of insecticides is the preferred way of dealing with insect problems, but it is not only hazardous to the environment, it also affects humans as the chemicals easily get incorporated into the food chain. As a result, new more resistant varieties are urgently needed to meet the demand of growing populations. A set of 200 accessions were classified as resistant (non-preferred) or susceptible (preferred) in response to cabbage aphid feeding in the field. Fifteen accessions were further assessed to characterize and identify the level and location of resistance factors by investigating feeding behaviour of cabbage aphid using the Electrical Penetration Graph (EPG) technique. The feeding behaviour assessment revealed the presence of interspecific & intraspecific variation and presence of resistance factors at multiple levels. The transcriptional response of these accessions under presence and absence of aphid feeding for 24h showed that gene expression is highly regulated in response to aphid feeding. Gene ontology (GO) enrichment study helped identify strong candidate genes for aphid resistance. In addition to this, the gene expression differences between CWR and landraces indicated adaptations of landraces during the process of domestication. Lastly, Gene expression data was used to develop models to predict insect resistance status. In conclusion, the combination of EPG and transcriptomics provides an opportunity to assess brassica germplasm for further research into defence mechanisms of cabbage aphids.