Whilst not a ‘new’ pest in the UK, Aleyrodes proletella has become an increasing problem for the Brassica industry in recent years, especially on Brussels sprout and kale. The reason for the increasing problem is unknown, but it is believed to be due to a combination of climate change, removal of certain active ingredients from use and later harvest times of crops. Relatively little research has focused on this species as, historically; it has been regarded as a minor pest. Knowledge about the biology of A. proletella is limited and some of what is currently understood about its ecology has been inferred from anecdotal evidence. The overall aim of this project is to understand population trends of A. proletella in the most vulnerable crops, Brussels sprout and kale. This includes understanding the key times of population increase and colonisation of new crops. This information can then be used to inform the development of an integrated control strategy using insecticides and other tools, which might include biological control agents and methods of cultural or physical control. Experiments to investigate the vertical and horizontal distribution of flights by A. proletella showed that A. proletella performs mainly low, short distance flights throughout most of the year and it is these flights that are mostly responsible for colonisation of new vulnerable crops, which can be achieved by overwintering females early in the season. Monitoring of field populations on kale has shown that populations of whitefly develop without regulation by predators or parasitoids, with increases in numbers mostly determined by the development of further generations from the initial immigrants to the crop. The size of a population of A. proletella that can be achieved within a crop seems to be governed by the number of generations that can develop before the onset of diapause in September, which prevents further reproduction. A new fungal pathogen, which causes epizootics within the field, has been observed. This killed up to >90% of adult A. proletella. Of all potential natural enemies this pathogen had the largest potential to reduce A. proletella infestations and offers potential for the development of a new method of biological control.

With an increasing world population and finite resources for growing crops, producing plants that can adapt well to adverse conditions is crucial to enable adequate food production. The development of crops that have enhanced disease resistance form a core part of this strategy. We investigate the potential of Alternative splicing (AS), the mechanism whereby the same pre-mRNA can lead to different mRNA transcripts, as a mechanism for plants to regulate their defence. We show that Botrytis cinerea infection has wide ranging effects on the Arabidopsis transcriptome, with approximately one third of intron-containing genes displaying B. cinerea-mediated differential alternative splicing (DAS), including a wide variety of defence-related genes from all stages of the defence pathway. Our work suggests, that in-line with known AS mechanisms, B. cinerea-mediated DAS influences the plant defence response to B. cinerea by enabling the plant to mount a rapid response to pathogens via circumventing the time required for transcript initiation and mRNA accumulation, altering signal transduction by affecting functional domains of proteins, as well as altering regulatory feedback loops. For one gene, encoding a leucine-rich repeat receptor-like kinase, we demonstrate that B. cinerea-mediated DAS occurs, and that expression of this gene influences resistance to B. cinerea. We determine that DAS of this gene is altered in loss-of-function mutants of one component of the MOS4 associated complex (MAC), AtCDC5, which also has a defence phenotype to B. cinerea. With genome-wide investigations of MAC loss-of-function mutants indicating that splicing factor associated genes form highly interconnected networks. We add substantially to the body of evidence showing genome-wide DAS occurs in response to stress. We identify some putative DAS stress core genes, and splicing regulatory element motifs, which with additional work, could be used to help design crops with added disease resistance, contributing towards food security in the 21st century.

The plant endoplasmic reticulum (ER), a highly dynamic membrane-bound organelle, is not only the site of secretory protein production and lipid synthesis, but also responsible for calcium storage. It is currently hypothesised that the shape of the ER network relates to these functions. The sheets, large at areas of network are proposed to be the sites of protein production and the tubules, thin, highly-mobile and interconnected, the regions of lipid production and calcium storage. The reticulon protein family has been shown to bend the ER lipid bilayer to form tubules and the edges of sheets. Identifying protein interactors to reticulons may help to understand how the morphology of the ER is controlled or in uenced. Mass spectrometry and co-immunoprecipitation techniques were used to identify protein interactors to the Arabidopsis thaliana seed-specific reticulon, RTN13. Five non-reticulon proteins were found to interact with RTN13 in developing A. thaliana seed; GTP-binding protein 2, lysophospholipase 1, NADH: Cytochrome B5 Reductase 1, sterol methyltransferase 2 and synaptotagmin a. Microscopy analysis of the ER in over-expression lines and T-DNA insertions lines for each putative interactor, showed that only sterol methyltransferase 2 and synaptotagmin a influenced the ER morphology. Additionally, the morphology of the ER was analysed during seed development and germination through confocal microscopy. An image analysis macro was used to determine the percentage of sheet morphology in the network. Significant changes in the amount of sheet morphology were recorded in the ER of cotyledon cells during seed development and over the first six days of germination. Wild type embryos were also compared to mutants known to have altered ER morphology. The analysis suggested that the amount of sheet morphology is maximal at times of maximum protein production, highlighting the link between ER form and function.

Self-incompatibility (SI) prevents pollination by “self” pollen and promotes outbreeding. SI is a widespread mechanism among angiosperms. Grass SI is known to be controlled by two loci, S and Z, mapped in linkage group 1 and 2 respectively but the mechanism remains elusive. SI in perennial ryegrass (Lolium perenne L.) is studied at the gene level for the S-locus. Using a fine-mapping approach, followed by the sequencing of the genomic S-locus region and the transcripts from pollen and stigma, the project identified four stigma S-candidate genes and potentially six pollen S-candidate genes. Moreover, using closely linked markers, the allelic diversity in a breeding population was assessed for both S and Z. This assay demonstrated that allelic diversity is maintained in a half-sib family recurrent selection programme and that even if the SI genes are unknown, it is possible to predict their genotypes. Finally, self-compatibility has been reported in many grasses. Using self-compatible perennial ryegrass populations, two additional loci, F and T, have been investigated, by a mapping approach as well as observation of self-pollination under the microscope. For both loci, a distortion segregation was observed on linkage group 3 (F) and 5 (T) and initial marker recombination maps were created.

Flowering plants have evolved complex genetic mechanisms of self-incompatibility (SI) to overcome the problem of self-fertilization. SI is a cell-cell recognition system where the interaction of genetically linked pollen and pistil S-determinants prevents self-fertilization. In Papaver rhoeas, the pistil S-determinant is PrsS, a secreted protein of around 15 kDa. The pollen determinant, PrpS, encodes a novel transmembrane protein of around 20 kDa. Upon the interaction of incompatible PrsS and PrpS variants, the SI response is triggered, activating a signalling network. Rapid increases in cytosolic free calcium ([Ca\( {2+}\)]\(_i\)) are followed by changes to the actin cytoskeleton and activation of a DEVDases, resulting in programmed cell death (PCD). Within this thesis, three inter-related studies are described. Initially, we investigated the role of the ubiquitin-proteasomal system during SI in Papaver, the second study focused on the PrpS protein. Thirdly, we also created transgenic Arabidopsis thaliana lines expressing PrpS and PrsS, in order to investigate if the Papaver SI system might be functionally transferable to other plant species. We have demonstrated that PrpS binds the PrsS in an S-specific manner, while the functional analysis “in vitro” revealed that PrpS expressed in A.thaliana is functional and that just PrpS and PrsS are sufficient for a fully functional SI response in A.thaliana pollen.

I examined the effects of different carbohydrates on floral determination in the short day (SD) plant, Pharbitis nil by excising apices at various times after an inductive 48 h dark period, explanting onto culture media under non-inductive conditions and examining them for flowering 4 weeks later. Cultured apices forming floral organs in non-inductive conditions were determined. The determination time for the sepal, petal and stamen whorls was 1 day whereas it was 5 days for the carpel whorl on medium supplemented with sucrose (2%), on 4-7% (w/v)(-0.29 to 0.51 MPa) it was 3 days but partial replacement of sucrose with mannitol (-0.29 to 0.51 MPa) lenghtened it to 5-6 days; thus, the effect was not caused by the osmotic potential per se. A similar reduction in carpel whorl determination time occurred on 1:1 glucose:fructose and fructose. Again, none of these changes occurred in mannitol treatments. Remarkably, glucose treatment (2, 3 or 6%), resulted in a carpel determination time of one day demonstrating that glucose specified carpels alongside other whorls. DNA replication was measured in the shoot meristems of Silene coeli-rosa (long day (LD) plant) and P.nil during floral determination. The plants were subjected to florally inductive or non-inductive treatments, exposed to tritiated thymidine and apical domes were prepared as fibre autoradiographs. In S. coeli-rosa, replicon size was 10-15um in SD (non-inductive) and 0-5um in LD (inductive) while in P.nil it was 10-15um in the 48 h dark interrupted by red light, 5-10um in continuous light (both non-inductive) but was reduced to 0-5um in the 48 h dark treatment (inductive). Therefore, recruitment of additional initiations points occurred in both a LD and SD plant during floral determination. In the discussion these findings are integrated into unifying models of floral determination.

The primary objective of this study was to characterise the Alternaria alternata species group. Particular focus was put on understanding the pathogens Alternaria mali and Alternaria gaisen, responsible for leaf spot diseases of apple and pear and of phytosanitary importance in Europe. Understanding evolutionary relationships is important in defining the genetic and biological characteristics associated with plant pathogens in this species group. This will inform management strategies and facilitate the development of reliable detection tools for important plant diseases. Evolutionary relationships within the A. alternata species group were established using a phylogenetic approach based on functional genes. Highly variable loci (endoPG, Alta1, L152 and three novel loci) identified three major lineages within the A. alternata which were supported by isolate morphology. These were considered to represent subspecies within A. alternata. The presence of toxin-synthesis genes, required for the production of host-selective toxins (HSTs) in apple and pear pathotypes, was established within A. alternata isolates. Isolates carrying apple HST-genes were only present in ssp. tenuissima, while isolates carrying pear HST-genes were present in ssp. gaisen. Virulence assays showed that apple HST-genes are required for pathogenicity on apple leaf. The presence of different mating type genes in isolates was used to assess evidence for recombination within the A. alternata species group. Distribution of mating type idiomorphs indicated that recombination must have occurred in this putatively asexual species. Analysis of whole genome sequence data indicated that A. alternata possesses the genes required for meiotic recombination, supporting a theory of recombination and possible sexuality within this group. This work provides insight into the evolution and causal agents of A. alternata plant diseases. Furthermore, whole genome sequencing data was generated during the course of this study and represents valuable genetic resource that can be used for future research, including development of pathotype-specific molecular markers.

Seed dormancy is an important trait refined by evolution, to aid survival in adverse environments and to time germination and thereby select the correct habitat and climate space for subsequent plant growth and reproduction. Depth of dormancy changes continuously in response to the environment surrounding the seed and is therefore a relative rather than an absolute condition. In nature, these changes are triggered by seasonally characteristic environmental signals that are integrated by the seed over time to select the optimum conditions for germination. The mechanisms by which environmental signals influence this dormancy cycling have been studied in the present work using a combination of eco-physiology and molecular biology. Two contrasting Arabidopsis thaliana ecotypes Cape Verdi Isle (Cvi) and Burren (Bur) have been compared. They are adapted to a hot dry (Cvi) and a cool damp (Bur) climate and exhibit winter and summer annual phenotypes respectively. Experimental work in the laboratory, controlled environment and field has focussed on the effect of temperature, light and nitrate during seed maturation and subsequent imbibition. The work was also extended to studying other life cycle events such as the transition from vegetative growth to reproductive growth, flowering and seed maturity. This work has extended our understanding of the responses of life cycle traits to environmental signals. However, climates are changing and further data was collected in a series of experiments in a unique thermal gradient tunnel to provide insight into the impact of predicted global warming scenarios on these traits. The results presented indicate the plasticity of the plant life cycle and the extent to which global warming might affect this in Arabidopsis, and how increased temperature is likely to affect different annual phenotypes.

UV-B is a natural component of the sunlight spectrum. As a result of the potentially harmful effects of this radiation, plants have evolved a highly effective suit of protective and repair mechanisms. However, the signalling pathways that control such responses are not yet well known. For example while the photoreceptors responsible for red and blue light responses are well characterised, no such UV-B photoreceptor has yet been identified. Despite this particularly large gap in our knowledge, previous work identified the first UV- B specific signalling component which, unlike the more general stress-associated pathways often seen at high doses, specifically regulates expression of genes in response to even very low fluence rates of UV-B. This protein, UV-RESISTANCE LOCUS 8 (UVR8) regulates the induction of a number of photoprotective genes mostly via the transcription factors ELONGATED HYPOCOTYL 5 (HY5) and HY5 HOMOLOGUE (HYH). The end result of this pathway is the production of photoprotective compounds such as the flavonoids which enhance a plants ability to withstand UV-B stress. Thus UVR8 promotes plant fitness under these conditions. While we know that UVR8 binds to chromatin in the promoter region of HY5 and that it accumulates in the nucleus under UV-B, many other questions about this particular protein remain unanswered. For example, we do not yet know if UVR8-mediated UV-B signalling involves other factors which interact with UVR8 nor do we understand the mechanism by which UVR8 localisation is mediated. In addition, although we are aware of the importance of UVR8 in UV-B acclimation, it is unclear what roles might be played by other genes and proteins acting independently of this pathway. Therefore, the aims of this study were to investigate low fluence UV-B pathways that may act independently of UVR8 and to further examine the UVR8 protein itself both in terms of its interactions with other proteins and also in the role of the N-terminal region in regulation of its localisation. To achieve the first of these aims, RNA samples derived from plants treated with low fluence UV-B were submitted for microarray analysis. It was initially determined that the total number of genes induced was roughly equal in both low fluence treated samples and also to that found in the previous microarray performed by Brown et al. (2005) at a comparatively higher fluence. Thus, as only 72 genes have currently been linked to UVR8, there do appear to be many low-fluence UV-B induced pathways besides that regulated by UVR8. Several genes were analysed further using RT-PCR and qPCR methods in order to confirm their independence from the UVR8 signalling pathway components as well as assess their dependence on other hypothesised UV-B sensory mechanisms. It was found that while some genes did seem to be expressed independently of known photoreceptors, DNA damage signals as well as UVR8, HY5, HYH and COP1; one gene was expressed in a COP1-dependent but UVR8 independent manner. It therefore appears that at least four classes of genes are induced by UV-B; low fluence UVR8/HY5/HYH independent COP1 dependent, low fluence UVR8/HY5/HYH/COP1 dependent, low fluence UVR8/HY5/HYH/COP1 independent and finally high fluence non- specific signalling. The second portion of this thesis examined the structure and function of UVR8 in greater detail. To assist in this analysis, the BLAST sequence homology tool was used to probe both the Arabidopsis genome and available green plant sequences. It was found that 23 UVR8-like sequences exist in Arabidopsis but none of these appear to have similar N or C-terminal sequences to UVR8. As these two regions have previously been shown to be of vital importance in UVR8 function (Kaiserli and Jenkins, 2008; Kaiserli unpublished data) it is unlikely that any are acting in a redundant fashion to UVR8. A number of similar proteins to UVR8 can be found in other plant species. These potential homologues however fall into two categories based on their closer similarity with either UVR8 or its close homologue in humans REGULATOR OF CHROMATIN CONDENSATION 1 (RCC1). The wide variety of plant species that did show UVR8-like proteins suggests that this particular means of UV-B acclimation may have arisen relatively early with the colonisation of land plants. Interestingly, many of these likely homologues had a conserved N terminal. The N-terminal of UVR8 has previously been show to have a role in UV-B dependent nuclear accumulation (Kaiserli and Jenkins, 2008). This was examined further in Chapter 4 through the generation of a number of deletion and addition constructs in both a stable Arabidopsis uvr8-1 background as well as transiently in tobacco. From analysis of localisation of these constructs via confocal microscopy it was determined that the first 12 amino acids are sufficient but not necessary for nuclear accumulation, while the first 20 appear to be both necessary and sufficient. Indeed, it was shown that the initial 32 amino acids also confer constitutive localisation of a GFP tag in the nucleus regardless of light condition and despite the presence of a nuclear exclusion signal (NES). It therefore appears that this region, which shows strong conservation with UVR8-like proteins in other plant species, is of vital importance to the nuclear accumulation seen under UV-B. Finally, in Chapter 5, the possibility that UVR8 may be acting as part of a complex was explored. This involved use of size exclusion chromatography to provide approximate sizes of the UVR8 protein complex. It was found that native UVR8 appears to exist in a complex of about 70-90 kDa in size. This suggests that at least one other protein interacts stably with UVR8. Other fusion constructs were also analysed in this way, however the results were more difficult to interpret due the apparent artificial dimerisation of the GFP tag. In summary, the work presented here has shown that although UVR8 dependent pathways are predominant, a variety of low fluence UV-B induced genes and pathways may exist. Homology searches and mutational analyses suggest that the N-terminal region of UVR8 plays a critical role in its function and localisation. Finally, size exclusion chromatography suggests that UVR8 forms a complex in vivo with as yet uncharacterised partner proteins. In total these results provide further insight into the mechanisms UVR8 action and highlight new avenues for both UVR8 dependent and independent UV-B signalling.

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