The primary aim of this project was to identify and characterise Fusarium species associated with the basal rot of Allium species and internal fruit rot of sweet peppers in the UK. The secondary objective was to develop quick molecular markers to identify Fusarium oxysporum f. sp. cepae (FOC) causing onion basal rot. Isolates representing diverse Fusarium species taken from onions, garlic, shallot and leeks obtained from different production and processing sites in the UK were collected. F. proliferatum was found for the first time to be a causal agent of onion basal rot in the UK, but F. oxysporum was by far the most common species and F. oxysporum isolates belonged to at least two different genotypes based on a sequence comparison of several “housekeeping” genes, and overall, appeared to be polyphyletic. None of the housekeeping genes studied correlate with pathogenicity. Secreted in xylem (SIX) genes offer more promise for the specific identification of F. oxysporum formae speciales (Lievens et al., 2009a) and a homologue of the SIX7 gene was found only in a few FOC isolates suggesting that SIX7 is not absolutely necessary for pathogenicity. Whole genome sequencing of a FOC isolate was carried out in order to understand pathogenicity and identify novel effector genes. This work revealed the presence of further homologues of published SIX genes, namely SIX3, SIX5 and SIX9. The presence of SIX3 and SIX5 has only been reported from F. oxysporum f. sp. lycopersici previously. Additionally, screening of eleven new candidate effector genes suggested that FOC isolates have different gene sets which correspond to the continuous variation of aggressiveness found within the FOC population. Fusarium lactis, F. proliferatum and F. solani were identified in association with internal fruit rot of sweet pepper obtained from three different production sites in the UK.

The use of crop protection products (CPPs) is essential to improve crop production levels and feed the rising global population. Safety testing of CPPs is also essential to ensure that their use does not adversely affect human health or the environment. Currently, laboratory-based studies typically over-estimate the environmental persistence of CPPs in the environment. The inclusion of environmental variables that are currently omitted from laboratory studies, such as non-UV light, could reduce the disparity between laboratory and field degradation studies. The inclusion of light resulted in a significant reduction in extractable parent compound for benzovindiflupyr, chlorotoluron, prometryn, imidacloprid, and fludioxonil compared to dark conditions. In contrast, a significantly slower rate of cinosulfuron transformation was observed under light compared to dark conditions. In a separate experiment, terminal restriction fragment length polymorphism showed that under light conditions, the soil surface (0-3 mm) harboured distinct phototroph, bacterial and fungal communities compared to the underlying bulk soil (3-12 mm), or dark incubated soil. 454 pyrosequencing revealed that light selected for diazotrophs at the soil surface, including Nostoc punctiforme, in addition to heterotrophic bacteria, particularly within the phylum Firmicutes. Metatranscriptomic analysis showed that light also selected for actively transcribing photosynthetic eukaryotes at the soil surface, such as Chlorophyceae and Saccharomycetes, in addition to heterotrophs, such as Nostocaceae. Finally, light selected for major soil functions such as photosynthesis, and reduced the alpha and beta diversity of predicted protein coding regions at the soil surface. This work has important implications for CPP regulatory studies and soil surface management practices.

Upland headwater streams are important sources of freshwater in mountainous temperate to sub-arctic latitude European countries like Scotland. Yet much less is known about the ecology of small, characteristically oligotrophic, mountain streams supporting periphyton and aquatic bryophyte dominated vegetation, and their potential bioindicator capacity of environmental water quality, than lowland rivers impacted by anthropogenic disturbance, in this context. This scarcity of knowledge has significant implications for the success of the recently implemented Water Framework Directive (WFD: 2000/60/EC). The WFD is a major piece of environmental legislation for water policy and sustainable water management in Europe. New contributions are fundamental to environment agencies, such as the Scottish Environment Protection Agency (SEPA), tasked with the responsibility of enforcing WFD statutory requirements and developing effective biomonitoring tools for assessing water quality status in Scotland. A major aim of the WFD is to achieve at least ‘good’ ecological status of inland waterbodies by 2015. Further, in doing so, to ascertain ecological benchmark communities of near-pristine (or minimally-impacted) reference conditions as indicators of high water quality status. The objective is to improve understanding of the environmental processes driving the production and diversity of freshwater plant species-assemblages in upland streams. Such information can be used for assessing perturbations threatening the ecological integrity of rivers impacted by anthropogenic disturbances (human pressure). This enables environment agencies such as SEPA, to respond appropriately by implementing corrective measures and sustainable management strategies. This project monitored a range of near-pristine headwater streams of contrasting underlying geology in the Scottish Highlands. The approach adopted was compatible with current WFD river characterisation and biomonitoring strategies. These were used to investigate the structural and functional response of freshwater plant communities (chiefly diatoms and other algal groups; aquatic bryophyte and vascular submerged macrophyte vegetation) to environmental drivers (e.g. flow, substrate morphology, nutrient inputs, water chemistry, underwater light availability). The work was carried out with the aim of contributing to future development of baseline monitoring tools for assessing upland stream habitat quality in Scotland.

With the increasing evidence of climate change in the coming decades, adaptive mechanisms present in nature may permit crop survival and growth on marginal or saline soils and is considered an important area of future research. Some subspecies of Restharrow; O. repens subsp. maritima and O. reclinata have developed the remarkable ability to colonise sand dunes, shingle beaches and cliff tops. α-onocerin is a major component within the roots of Restharrow (Ononis) contributing up to 0.5% dry weight as described by Rowan and Dean (1972b). The ecological function of α-onocerin is poorly understood, with suggestions that it has waterproofing properties, potentially inhibiting the flow of sodium chloride ions into root cells, or preventing desiccation in arid environments. The fact that alpha-onocerin (a secondary plant metabolite) biosynthesis has evolved a number of times in distantly related taxa; Club mosses, Ferns and Angiosperms, argues for a relatively simple mutation from non-producing antecedents. No direct research has been reported to have investigated the biosynthetic mechanism towards α-onocerin synthesis via a squalene derived product originally characterised by Dean, and Rowan (1972a). A bi-cyclisation event of 2,3;22,23-dioxidosqualene by an oxidosqualene cyclase, may provide plants with an alternative mechanism for synthesising a range of triterpene diol products via alpha-onocerin (Dean, and Rowan, 1972a). This mechanistic possibility presented the opportunity to investigate the biosynthesis of α-onocerin using a multi-disciplinary approach. This thesis presents supporting data, that α-onocerin is a derivative of 2,3;22,23-dioxidosqualene via an oxidosqualene cyclase. Genetic markers were developed for Ononis versions of squalene cyclase, squalene epoxidase and a putative oxidosqualene cyclase. It was determined that squalene epoxidase; At4g37760 (SQE3), from A. thaliana showed the highest level of amino acid sequence conservation with the O. spinosa version. SQE3 is known to cyclise 2,3;22,23-dioxidosqualene (Rasbery et al., 2007). Based on amino acid sequence alignments and predictive protein modelling a partial putative oxidosqualene cyclase isolated from O. repens III subsp. maritima is likely to be an Ononis version of β-amyrin synthase rather than a multifunctional oxidosqualene cyclase. Further functional characterisation studies are needed. Methods were developed for analysing the transcriptome and metabolome in Restharrow which will aid future functional characterisation studies. Within O. spinosa root SQE3 was highly expressed. In contrast SQE3 was expressed at low levels in O. spinosa leaf and O. pusilla root and leaf. This data was supported by metabolomic profiling of five species of Restharrow; O. spinosa, O. repens, O. repens subsp. maritima, O. pusilla and O. rotundifolia. Triterpenes α-onocerin and 2,3;22,23-dioxidosqualene were not present in O. rotundifolia and O. pusilla. Where 2,3;22,23-dioxidosqualene was present in plant extracts, α-onocerin accumulation was also detected. O. pusilla and O. spinosa can be utilised for studying the occurrence of alpha-onocerin within plants. The data presented in this thesis provides the necessary background information providing targets for functional expression studies of squalene epoxidases and oxidosqualene cyclases from Restharrow. In summary the results in this thesis support the hypothesis proposed by Dean and Rowan (1971). There is good evidence to suggest the ability of Restharrow to cyclise alpha-onocerin, may be dependant on the availability of 2,3;22,23-dioxidosqualene as the primary precursor. This was shown in development, tissue specific, ecotype and cell free enzyme analytical chemistry assays. There was little evidence to suggest a single specific oxidosqualene cyclisation event was primarily responsible for alpha-onocerin biosynthesis. The work also presents evidence to suggest that differences in the squalene epoxdiase sequence and transcription signals may affect the plants ability to cyclise alpha-onocerin. This may have ecological implications and allow plants to adapt to their environment by providing and alternative route to biosynthesising membrane constituents via an alternative substrate specific mechanism.

Sporophytic self-incompatibility is a genetically controlled inbreeding prevention mechanism, which is prevalent in the Brassicaceae, and has been reported in a variety of high profile species. Despite the benefits of preventing self-fertilization in terms of maintaining genetic diversity, variation in the strength of self-incompatibility (SI) has also been well documented, as has a shift from SI to inbreeding at the species and population levels. An important underlying driving force behind a switch to inbreeding could be the reproductive assurance provided by not requiring an unrelated mating partner for sexual reproduction. This could be beneficial for a species undergoing rapid colonization, because only a single individual is required to begin a sexually reproducing colony after a long-distance dispersal event (Baker’s law), which is characteristic of the plight of many species after the last glacial maxima. The purpose of my thesis was to evaluate the effects of variation in mating system on post-glacial colonization, using two model species that show intraspecific variation in outcrossing rates. The first, Arabidopsis lyrata, represents an excellent model system to assess post-glacial colonization history because it exhibits broad geographical and ecological ranges, and has a recently completed genome sequence. In North America, A. lyrata has further benefits as a model system, namely it exhibits variation in the strength of SI and shift to SC at the population level, which is not observed in Europe. The second species is Arabis alpina, which also appears to show population level variation in mating system strength in Europe based on variation in FIS. This has been putatively linked to colonization history after the last glacial maxima. Unlike in A. lyrata however, its mating system has not been characterized. Mating system delimitation in A. alpina has the potential to aid the interpretation of patterns of ecological genetic diversity, which may in part be influenced by local or regional stochastic changes to mating system variation. My first objective was to identify if A. alpina had a functioning SI system based on both self-fertilization experiments, and allozyme based outcrossing rate estimations. I found strong evidence to suggest the presence of a functional barrier preventing self-fertilization in A. alpina. I identified multiple putative SRK alleles (the female determinant of self-incompatibility), suggesting that the same type of sporophytic system seen in other Brassicaceae species governs SI in this species. I also demonstrated linkage of SI phenotype to some SRK genotypes by diallel crosses, strengthening the case for a functional SI system in this species. Further to this I demonstrate variation in mating system strength between populations, and autonomous inbreeding was seen in a single population. I note that the potential changes in SI status coincide with areas suspected to differ in post-glacial history based on allozyme diversity reported in previous work. While the number of populations sampled was insufficient to link mating system variation to colonization history in A. alpina, mating system variation has been more extensively characterized in North American A. lyrata, allowing more fine-scale resolution of population structure and post-glacial colonization history; an underlying objective of my thesis. I used three molecular marker systems (cpDNA, nuclear micro-satellites and allozymes) to assess these phylogeographic questions, and present evidence of three putative colonization routes for the Great Lakes region. These putative routes are congruent with those described in other species, particularly amphibians and reptiles. Further to this I considered the possible location of glacial refugia, and likelihood that plant taxa may have survived during Pleistocene glaciation in close proximity to the Laurentide Ice Sheet, particularly in Illinois, Indiana, Wisconsin and Minnesota, which may also be true for some animal taxa. I examined patterns of population structure, and scenarios that may have influenced this, and present support for the previously documented theory of multiple breakdowns in SI in this geographic region. My final objective was to assess the suitability of the three marker systems for phylogeographic reconstruction in A. lyrata, by comparing and contrasting the patterns of population structure, and colonization history suggested by each system. Levels of variation observed between the marker systems used varied, and I explored how these patterns complemented and contradicted each other. As expected, the nuclear micro-satellite loci represent the system with the greatest genetic diversity, but do not allow meaningful conclusions to be drawn regarding colonization history because of low levels of shared variation between populations. Conversely, the allozyme loci presented much lower levels of genetic diversity, but support population structuring conclusions based on both cpDNA data and previous studies of A. lyrata and other taxa in this area. The cpDNA marker (trnF) represents a somewhat contentious system to use for phylogeography in A. lyrata, as it contains a tandem array of highly variable, but complexly evolving duplications (pseudogenes). I concluded that these structural changes could be phylogenetically informative when pseudogene evolutionary relationships can be resolved This was based on variation in patterns of diversity, and the subsequent population structure change that occurred when using different methods of assessing trnF variation.

The aim of this project was to try to find the mode of action of azadirachtin in cultured cells, and to compare its cytotoxicity with some well-known phytochemical pesticides. The results presented here showed that azadirachtin was toxic to the cultured insect cells used (Sf9 and C6/36, derived respectively from Spodoptera frugiperda and Aedes albopictus) even in very low concentrations with an EC50 for the Spodoptera cells estimated at 5x10-9 M, but that the mammalian fibroblast cell line L929 was little affected except at concentrations greater than 10-4M. The other major neem terpenoids, nimbin and salannin, showed low toxicity towards the cultured cells. The neurotoxic pyrethrum showed little effect against the cultures, except for some slight stimulatory effect on growth at 10-8 M. Rotenone, known to inhibit the electron transport chain, effectively inhibited the growth of both insect and mammalian cells. Nicotine, another neurotoxic phytochemical, had little effect on the growth of the cultured cells. It was concluded that while cell growth assessment is not appropriate for all phytochemical pesticides, it is useful for those, such as azadirachtin and rotenone, whose effect is on the essential mechanisms of insect cells in general. Rotenone was used as a positive control to investigate if azadirachtin had its effect on respiration of the cells. Only at the highest feasible concentration of azadirachtin, was there a slight but significant (15%) reduction of respiration which was the same in both insect and mammalian cells. As expected, rotenone inhibited both insect and mammalian cells even at concentrations as low as 10-11 M. When the effects of azadirachtin on the cell cycle were examined by means of cell cytometry, it was shown that the compound arrested the cell-cycle in G2/M phase, and that the effect was related to the concentration. Microscopy confirmed that there was a three-fold increase in the mitotic index after 2 hours of exposure of 2x10-6 M azadirachtin. The similarity of the of the nuclear profiles and cell-cycle distribution to Sf9 cells treated with colchicine, a well-known antimitotic phytochemical, suggested there was a similarity of action between the two compounds.

The global success of plants depends largely on their ability to perceive and respond to light, mainly in two regions of the electromagnetic spectrum. Phytochromes are light sensors for the red and far-red wavelengths of light while cryptochromes, phototropins and members of the ZTL/ADO family respond to blue and UV-A wavelengths of light. Phototropins are UV-A/blue-light receptor kinases found ubiquitously in plants from the unicellular green alga Chalmydomonas reinhardtii through bryophytes and pteridophytes up to angiosperms. The model plant Arabidopsis possesses two phototropins (phot1 and phot2) and is the subject of the work presented in this thesis. The general structure of the phototropin protein comprises a photosensory region at the N-terminal that contains two LOV (light, oxygen and voltage sensing) domains and a C-terminal kinase domain belonging to the large AGC family of protein kinases. The LOV domains form a covalent adduct with the chromophore flavin mononucleotide (FMN) in response to illumination with blue light which in turn leads to structural changes throughout the protein resulting in autophosphorylation of the N-terminal region by the kinase domain. Phototropins function redundantly to mediate a number of physiological responses in planta which serve to promote plant fitness and maximise photosynthetic potential. Phototropism, chloroplast accumulation, blue light-induced stomatal opening, leaf expansion and leaf movements can be induced through the activation of both phot1 and phot2 in response to different intensities of light, with phot1 being more light sensitive than phot2. In addition to the functionally redundant responses, phot1 alone is responsible for destabilisation of certain mRNA transcripts and the rapid inhibition of hypocotyl elongation when etiolated seedlings are transferred to blue light, while phot2 is solely responsible for the high light induced chloroplast avoidance response. While much is known about the mechanisms of light perception by the phototropins at the molecular level, and the responses mediated by them have been well described, little is known about their methods of signalling to induce these physiological responses upon photoactivation by blue light. Therefore, the aims of this study were to identify novel phot-interacting proteins and to investigate the modes of phot1 signalling by structure/function analyses in order to better understand the way phototropins elicit signal transduction to downstream components in order to bring about the responses described above. Initially, a yeast two-hybrid screen was carried out to try to identify immediate interacting partners for phot1. The results of the yeast two-hybrid screen are described in Chapter 3. One hundred and thirty yeast colonies containing putative phot1-interacting proteins were identified from the screen and preliminary characterisation of six of these proteins are described in this chapter. Two of the proteins investigated are members of the ADP-ribosylation family which is involved in the regulation of membrane trafficking. The ARF proteins identified show a blue-light-sensitive interaction with phot1 and also interact with phot2. These proteins are of interest given the subcellular movement of phototropins from the plasma membrane after exposure to blue light. The C-terminal kinase domain of phot1 was found to interact with p-glycoprotein 19 (PGP19), a protein involved in polar auxin transport. The interaction between these proteins is interesting because of the role auxin plays in phot1-mediated responses such as phototropism and leaf expansion, and preliminary characterisation of the interaction in vitro is shown in Chapter 3. The implications of a direct link between phototropins and the proteins involved in auxin transport are discussed. A further two proteins identified from the screen are members of the NPH3/RPT2-Like (NRL) family. RPT2 has already been identified as a phot1-interacting protein and identification of this protein increased confidence in the efficacy of the screen to identify genuine interacting proteins. A novel member of the NRL family, designated NPH3-L, was also identified from the screen. Chapter 4 describes the tissue specific and subcellular localisation of NPH3-L and contains results of preliminary investigations into the function of NPH3-L in planta. 14-3-3λ was identified from the screen using full-length phot1 as bait. A 14-3-3 protein has been shown previously to bind to autophosphorylated phototropin in Vicia faba (Kinoshita et al., 2003). Chapter 5 details the localisation of 14-3-3λ at tissue and subcellular levels and shows that 14-3-3λ binding to plant-derived phot1-GFP is both light dependent and induced by receptor autophosphorylation. Creation of GFP-14-3-3λ overexpressing lines in wild-type and phot1-5phot2-1 backgrounds allowed investigation into the roles that light and phototropins play in regulating the subcellular localisation of 14-3-3λ. It is shown that light-induced movement of 14-3-3λ at the plasma membrane is dependent on the presence of endogenous phototropins. Physiological implications of this interaction are discussed. Finally, in order to determine the modes of phototropin signalling, structure/function studies were carried out by expressing different regions of phot1 in a variety of Arabidopsis backgrounds. The results of the structure/function studies are described in Chapter 6. Known phot1-mediated responses were investigated in the transgenic plants to determine the effects of individual domains of phot1. Particular attention was paid to the role of receptor autophosphorylation in phot1-mediated responses to light. A transgenic line overexpressing the LOV2-kinase region of phot1 demonstrates that phot1 autophosphorylation is not the primary signalling event involved in phot1-mediated responses to light and shows that the truncated version of phot1 is sufficient to complement most phot1-mediated responses. This also shows that the LOV1 domain is dispensable and suggests phot1 may signal through phosphorylation of substrates. Comparisons are drawn between phot1 kinase overexpressing lines and inactive phot1 kinase overexpressing lines. Preliminary observations of a transgenic line overexpressing phot1 in a wild-type background indicate that overexpression of phot1 may alter polar auxin transport. Together these studies provide new insights into possible mechanisms of phot1 signalling and the function of major domains of phot1.

Eukaryotic cells maintain a compartmental cellular organization of membrane-enclosed organelles that communicate with each other through the exchange of trafficking vesicles. Members of a superfamily of membrane proteins, the so-called SNAREs, are essential for the necessary fusion of vesicle membranes to the membrane of target organelles. SNAREs are needed to overcome the energy barrier that prevents spontaneous membrane fusion events. A number of studies from the past decade indicated that SNARE proteins might fulfill a function beyond merging membranes. The mammalian plasma membrane SNARE Syntaxin1A was shown to directly interact with and through this interaction modify the activity of, for example, a calcium ion channel and a potassium ion channel. In its classical function as SNARE protein, Syntaxin1A mediates specialized vesicle fusion events such as synaptic transmission in neurons or secretion of insulin from pancreatic cells. These specialized vesicle fusion events require precise timing that is controlled by intracellular signaling events. These intracellular signaling events involve the coordinated action of members from different families of ion channels. Current models suggest that the dual functions of a SNARE protein in ion channel regulation and membrane fusion serve to fine-tune highly regulated vesicle fusion events. This thesis provides evidence for the first direct interaction between a SNARE protein and an ion channel from plants and suggests a function for this interaction in Arabidopsis potassium nutrition. Three different protein-protein interaction assays for full-length membrane proteins that comprised a yeast mating based split-ubiquitin assay, co-immunoprecipitation after expression in insect cells and bi-molecular fluorescence complementation after transient Arabidopsis root transformation, confirm that the Arabidopsis plasma membrane SNARE SYP121 interacts in vitro and in vivo with the Shaker ion channel subunit KC1. Furthermore, the interaction between KC1 and SYP121 is specific over the closest homologue of Syp121, namely SYP122. Shaker channels are plasma membrane proteins with four subunits that transport the essential macronutrient potassium in response to changes in membrane voltage. The KC1 subunit is unique among the Shaker channels. It can only act as a regulatory subunit that modifies channel properties when forming heterotetramers with other Shaker subunits such as AKT1, not as functional homotetramer. AKT1 is expressed predominantly in the root epidermis, i.e. root hairs, where it overlaps with the more broadly expressed KC1 and SYP121. Previous publications showed that a low external potassium concentration combined with high levels of ammonium that is used to block all root potassium uptake systems apart from AKT1, causes akt1 null mutants to display strongly reduced main root length as well as whole plant potassium content compared to wild type plants. It is shown here that the phenotype of both syp121 and kc1 null mutants is identical to the akt1 mutant under these growth conditions. The design of new antibodies against native AKT1 and KC1 and an optimized protocol for root plasma membrane protein enrichment and solubilisation allowed for the first time visualization of native Arabidopsis AKT1 protein. This technical advance made it possible to confirm that both Shaker channel subunits are present in equal amounts in the plasma membrane of roots cells from syp121 mutant and wild type plants. It is concluded that the potassium uptake phenotype of the syp121 mutant is not caused by the absence of channel proteins from the plasma membrane due to a disruption of the vesicle trafficking function of the SNARE SYP121. An alternative function for SYP121 in potassium nutrition that involves direct interaction with AKT1-KC1 heterotetrameric channels is supported by electrophysiological measurements after heterologous expression in Xenopus leavis oocytes. SYP121 modifies the voltage-dependent potassium uptake characteristics of AKT1-KC1 heterotetramers in a way most easily understood in context of a conformational change within the voltage sensing protein parts of the Shaker channel that are caused by the direct interaction with the SNARE protein. It is concluded that the identical potassium uptake phenotype of the akt1, kc1 and syp121 mutants is caused by the inability to form a functional tripartite complexes. As KC1 is able to form heterotetrameric channels with several different Shaker channel subunits, for example KAT1 that is highly expressed in guard cells, it is likely that this novel interaction between KC1 and SYP121 might modulate channel activities in different tripartite complexes to affect various cellular functions.

UV-B is an integral component of the daylight spectrum that regulates plant gene expression and development, but very little is known about how plants perceive UV-B. Although UV-B-induced damage and repair have been extensively investigated, the mechanisms by which UV-B is perceived as a signal, which mediates physiological and protective responses is not yet clearly understood neither in mammals, nor in higher plants. Low fluence rates of UV-B induce the expression of genes involved in UV-protective responses such as flavonoid biosynthesis and promote plant survival in UV-B. The aim of this study is to contribute to the elucidation of the signal transduction events that lead to the acclimation of plants in response to non-damaging levels of UV-B (< 3.5 μmol m-2 s-1). In particular, the characterisation of UVR8 (UV-RESISTANCE LOCUS 8), a UV-B specific signalling component, is carried out at the protein level. The function of UVR8 involves the orchestration of the expression of a range of genes mediating vital UV-protective responses, including those encoding light-regulated transcription factors HY5 and HYH, enzymes involved in the phenylpropanoid pathway, antioxidant and stress proteins (Brown et al., 2005). UVR8 shows 30% sequence identity to the human regulator of chromatin condensation (RCC1) but differs both in activity and function. The phenotype of uvr8 mutant plants is characterised by an increased susceptibility to UV-B and the lack of the UV-B-specific induction of genes involved in UV-protection, such as CHS (encoding the flavonoid biosynthetic enzyme chalcone synthase) and the transcription factor HY5. The UVR8-mediated regulation of transcription in response to UV-B seems to occur via the association of UVR8 with chromatin via histones in the promoter region of HY5 (Brown et al., 2005) and other genes involved in light signalling. In this study, further investigation of the mechanism by which UVR8 acts as a UV-B specific signalling component is performed by employing a number of approaches including: spatial, temporal protein analysis, subcellular localisation studies, structure-function analyses, and the yeast-two-hybrid assay for the identification of UVR8 interacting proteins. To study spatial, temporal and wavelength specific UVR8 protein abundance anti-UVR8 peptide antibodies were generated. Western blot analyses showed that UVR8 is ubiquitously expressed in all plant tissues from the very early stages of development and at every light treatment tested (dark, white light, UV-B). The subcellular localisation of UVR8 analysed by confocal fluorescence microscopy revealed that a fusion of UVR8 with green fluorescent protein (GFP) is localised in the cytoplasm and the nucleus of various plant tissues (leaf, hypocotyl, root, flower) and under various light fluence rates and qualities (white, red, UV-A, UV-B). Interestingly, a treatment of low fluence rates of UV-B led to an increase of GFP-UVR8 protein accumulation in the nucleus, which was confirmed by western blot analysis based on protein fractionation studies in wild-type plants. The wavelength specificity, the kinetics and the fluence-rate sensitivity of GFP-UVR8 nuclear accumulation suggest that this response is UV-B specific, rapid (10 min UV-B) and very sensitive to very low fluence rates of UV-B (0.1 μmol m-2 s-1). Protein synthesis does not seem to be involved in this process, as there is no change in the protein levels before and after a UV-B irradiation. To assess the importance of the presence of UVR8 in the nucleus and the cytoplasm of the plant cell, uvr8-1 transgenic plants were produced expressing either constitutively nuclear localised GFP-UVR8 fused to a nuclear localisation signal (NLS), or cytosolically retained GFP-UVR8 fused to a nuclear export signal (NES). Nuclear exclusion of NES-GFP-UVR8 fusion protein was sustained under most light conditions apart from UV-B, which induced nuclear import of the protein. This indicates that the mechanism involved in the nuclear accumulation of UVR8 can overcome an export signal either by masking it or by simply superseding it. Furthermore, the NES-GFP-UVR8 construct was functional after UV-B treatment, since it rescued the mutant uvr8 phenotype. None of the inhibitor treatments tested (staurosporine, cycloheximide, cantharidin) was successful in blocking the UV-B induced nuclear import of NES-GFP-UVR8, although they impaired the UVR8 regulated induction of CHS expression. Thus, no evidence is presented for a specific protein modification, which could control this response. Constitutive nuclear localisation of NLS-GFP-UVR8 had no effect on the function of the protein according to complementation analyses. Furthermore, no change in localisation, fluorescence intensity or protein abundance was observed in response to white light or after a UV-B irradiation. These results indicate that the constitutive nuclear localisation of UVR8 is not sufficient for constitutive activation of UVR8 regulated gene expression and that a UV-B stimulus is still necessary to trigger these responses. Unfortunately, based on the current data it cannot be concluded whether the UV-B signal perception occurs in the nucleus or in the cytosol of the plant cell. To investigate the structure-function relationship within the UVR8 protein, deletion analyses followed by complementation studies in transgenic plants were performed. More specifically, deletion of the first 23 amino acids at the N-terminus of UVR8 impaired its nuclear accumulation in response to UV-B. Deletion of a 27 amino acid region near the C-terminus had no effect on the UV-B dependent re-localisation of the protein, but abolished UVR8 regulated gene expression. In addition, a highly basic sequence at the extreme C-terminal of UVR8, resembling a putative monopartite nuclear localisation signal, was deleted. Subcellular localisation and complementation analyses suggest that this sequence does not serve as a nuclear localisation signal, it is not involved in the UV-B induced nuclear accumulation and its absence does not affect UVR8 protein function. Chromatin immunoprecipitation assays show that none of the regions deleted is required for chromatin association and none of the deletions affects subcellular localisation in white light. In order to identify interacting partners for UVR8, the yeast-two hybrid system was used. Unfortunately no interacting proteins have been identified, neither from a screen, nor by directed-interaction studies. A different approach could be employed in the future involving size exclusion chromatography of protein extracts from plants in order to establish whether UVR8 functions as part of a complex in vivo.

Post harvest discolouration in lettuce is an increasingly important problem due to the shift in the market for prepacked processed salads. Variation in post harvest discolouration was recorded in a lettuce diversity set of 28 accessions representative of the lettuce genepool. The parents of the WHRI lettuce mapping population, Saladin and Iceberg were included in the diversity set. They showed significantly different responses for discolouration and the difference between them was representative of a major part of the variation seen in the diversity set. F7 RILs derived from a cross between Saladin and Iceberg were suitable for genetic analysis of post harvest discolouration. As a precursor to the genetic analysis, a good quality linkage map based on the F7 Sal x Ice population was generated. Significant genetic variation in the post harvest response was demonstrated for these RILs. Twenty-one significant QTL were identified for post harvest discolouration traits, and the markers linked to the QTL can be used for marker assisted selection. Significant but weak correlations were recorded between discolouration and important agronomic traits, however as these were not highly correlated this means that post harvest discolouration and agronomic traits can generally be independently selected for by breeders without having to compromise on other traits. Research was also initiated to understand the metabolic changes underlying the phenotype change. Significant variation in levels of metabolites related to post harvest discolouration including phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and total phenolic content (TPC) was observed in RILs with extreme phenotypes. The differences in metabolite levels were significantly correlated with the discolouration phenotypes. Work was also initiated to identify candidate genes associated with the biosynthetic pathway responsible for discolouration (phenylpropanoid pathway) in an attempt to identify genes underlying QTL. Twenty-three genes have been placed on the Sal x Ice map using comparative genomic approaches. Some of these co-locate within the region of a discolouration QTL and are therefore candidate genes for the QTL effect. Mutants with altered post harvest discolouration phenotypes were also confirmed in this study; however the point of mutation could not be identified although it is thought to be downstream of PPO. This indicates that a desired phenotype with reduced levels of post harvest discolouration can be achieved by two approaches to breeding; using natural and induced variation. This study has provided the tools and knowledge to do this.