This thesis describes the discovery of a new class of related proteins which has been named the chaperonins (Hemmingsen, Woolford, van der Vies, Tilly, Dennis, Georgopoulos, Hendrix & Ellis, Nature 333, 330-334, 1988). The proteins in this highly conserved class are structurally and immunologically related and ubiquitous in their occurrence in plastids, mitochondria and bacteria. The chaperonins comprise one class of the larger family of molecular chaperones since their function of assisting in the folding and assembly of other polypeptides without being components of the final structure meets the criteria suggested for molecular chaperones (Ellis, Nature 328, 378-379, 1987). The chaperonin class of proteins was discovered during studies on the assembly of the hexadecameric enzyme ribulose-1.5-bisphosphate carboxylase-oxygenase (Rubisco); this enzyme is found in the chloroplasts of plants where it catalyzes the first step in the pathways of both photosynthesis and photorespiration. The assembly of Rubisco in vivo had been proposed to require the activity of another chloroplast protein, originally known as the Rubisco large subunit binding protein because it binds to Rubisco large subunits newly- synthesized in isolated chloroplasts of Pisum sativum (Barraclough & Ellis, Biochim. Biophys, Acta 608, 19-31, 1980). Newly-imported Rubisco small subunits have since been shown to bind to the same chloroplast protein, which has therefore been renamed the Rubisco subunit binding protein (abbreviated to binding protein), or the plastid chaperonin. Antibodies raised against the plastid chaperonin purified from P.sativum recognize two subunit polypeptides with an apparent Mr of 61 500 and 60 500 (termed alpha and beta respectively) in extracts of Triticum aestivum leaves. With the aid of these antibodies a cDNA fragment has been isolated and sequenced from a lambda gtl 1 expression library of cDNA from leaves of Triticum aestivum. The cDNA fragment of 1834 bp encodes the entire mature plastid chaperonin alpha subunit plus two amino acids of the presequence. The amino acid sequence of the T.aestivum alpha chaperonin shows 46% identity to a protein from Escherichia coli known as the groEL protein; this protein had previously been shown to be essential for cell viability and is required for the assembly of bacteriophage capsids. An identity of 59% is found between the wheat alpha chaperonin and a groEL-like protein present in Mycobacterium leprae and M. tuberculosis. Immunologically related proteins were also detected in a variety of prokaryotes including cyanobacteria and Prochlorothrix hollandica, the eukaryote Chlamydomonas reinhardii and in mitochondrial fractions from P.sativum leaves and Solarum tuberosum tubers. All these related proteins comprise the new class of chaperonins. Amino acid sequences of all the known chaperonins from plastids, mitochondria and bacteria were compared and show 41%-58% amino acid identity. The chloroplast alpha chaperonin is as closely related to the chloroplast beta chaperonin (amino acid identity is 50%), as it is to the bacterial and mitochondrial chaperonins. The most interesting finding to emerge from the analysis of the deduced amino acid sequences is the presence of a possible dinucleotide binding site. A highly conserved region of 36 amino acids shows 9 out of 11 matches reported for the dinucleotide binding sit fingerprint (Wierenga, Terpstra & Holl, J. Mol. Biol. 187, 101-107, 1986), but only when the chaperonin sequence is read from the carboxy to the aminoterminus and two additional amino acids are allowed. Other proteins such as the Ca2+-ATPase of the sarcoplasmic reticulum and the ecdysone-induced protein Eip 28129 of Drosophila melanogaster also contain this reversed dinucleotide binding site sequence. This finding raises the novel possibility that a given binding site can be constructed from a set of amino acids running in either direction along the polypeptide chain; this possibility should be tested for other consensus sequences. When the T.aestivum chloroplast alpha chaperonin is synthesized in E.coli cells, it forms a hybrid oligomeric complex with the host chaperonin. The T.aestivum Rubisco large subunits that are synthesized in E.coli are found associated with either the E.coli chaperonin or with the hybrid chaperonin complex, whereas co-synthesized Rubisco small subunits bind neither to the large subunits nor to the chaperonin complexes. The T.aestivum Rubisco subunits fail to assemble into an enzymically active oligomer when synthesized in the presence of the T.aestivum chloroplast alpha chaperonin. This work is discussed in light of the conclusion emerging from studies in several laboratories that chaperonins function in many processes within the cell, the common feature of which is the requirement to prevent folding and assembly occurring between transiently exposed interactive protein surfaces.

The aim of this study was to formulate an integrated disease management (IDM) strategy for winter sports turf. (Winter sports turf, or coarse turf, consists primarily of perennial rye grass, Lolium perenne, which is used for football, rugby and hockey pitches because of its wear tolerant characteristics). IDM involves the use of a number of control strategies to suppress disease economically and efficiently. Such strategies incorporate cultural, biological, genetic, legislative and chemical control. In order to formulate a successful disease management strategy, all the significant diseases affecting winter sports turf and the effects of different management strategies on these target pathogens needed to be identified and collated. This was achieved by a comprehensive questionnaire survey to professional football clubs (who require a high level of turf maintenance) and local authorities (moderate/low maintenance). The questionnaire sought information regarding disease, pest and weed incidence, control measures employed and general problems, e. g. drainage, wear and routine management practices. Red thread, Laetisariafiiciformis, and Fusarium patch, Microdochium nivale appeared to be the most ubiquitous diseases on winter sports turf. Some important management practices that suppress red thread can, however, encourage Fusarium patch, e. g. the application of nitrogenous fertiliser. A series of experiments and field trials have been initiated to identify a number of specific factors which manage to effectively suppress both diseases. A field trial involving the use of species mixtures, perennial rye grass and smooth stalked meadow grass (Poa pratensis), illustrated that genetic diversity can help to reduce both red thread and Fusarium patch compared to turf grown in monoculture. The amount Fusarium patch and red thread cover indicated that disease severity was significantly lower in dual species stands as compared to monoculture. A mixture comprising 50% perennial rye grass and 50% smooth stalked meadow grass appeared the most effective at suppressing disease incidence. Similarly, mixtures of three perennial rye grass cultivars appeared more successful at suppressing slight outbreaks of red thread as compared to bi-blends and monoculture. In addition, individual perennial rye grass cultivars also vary in tolerance to red thread. One hundred and ten cultivars, under three different nitrogen regimes were assessedto determine which were the most disease resistant. The cultivars, received artificial football type wear treatment throughout the winter, to determine if red thread incidence predisposes rye grass to be less wear tolerant. The results indicate that a number of cultivars tolerant to red thread throughout the summer were also more resistant to wear. These cultivars included Quickstart, DelDwarf and Brightstar. Wear tolerance was also increased under a moderate nitrogen level (150 kg/ha/yr). Finally, a field trial investigating the effect of nitrogen rate on red thread and Fusarium patch incidence on five different constructions for football pitches was set down. Both diseases appeared to be efficiently suppressed under a moderate/high nitrogen level (N=225 kg/ha/yr). The 'pipe/slit' construction type also appeared to contain both diseases effectively, whilst sustaining a healthy, vigorous sward throughout the Winter when subjected to artificial football-type wear treatment. In addition to the field studies, an investigation to isolate potential microbial antagonists for use as biocontrol agents against Fusarium patch was undertaken; Fusarium patch was identified as the most economically important disease on winter sports turf from the original survey. A number of known antagonists and indigenous fungi and bacteria isolated from the phylloplane and rhizosphere of Lolium perenne were screened in vitro on turfgrass extract agar against Fusarium patch. This in vitro assay identified which species effectively suppressed disease growth. These potential antagonists were further tested in vivo to determine efficacy under field conditions. Fungi from the genus Trichoderma and bacteria from the genera Bacillus and Pseudornonos appeared the most effective antagonists against Fusarium patch in the in vivo study. In all cases where an antagonist was present, Fusarium patch severity was significantly lower than the untreated control, e. g. the indigenous Bacillus sp. reduced disease severity by 76.1 %. The results obtained from the field trials are encouraging and suggest that the use of species/cultivar mixtures, disease tolerant cultivars and a balanced fertiliser regime on a freely-draining construction type can successfully be incorporated into an IDM plan. An IDM strategy will help to effectively suppress both red thread and Fusariurn patch on winter sports turf. Biological control of Fusariurn patch was successful on an experimental basis, although further research is required to identify an appropriate formulation and optimum application technique for successful commercial use. The use of IDM on winter sports turf will help reduce reliance on chemical control, may delay the onset of fungicide resistance and reduce non-target impacts of fungicides. IDM will also help limit the need for potentially hazardous chemicals in recreational areas open to the public.

Bacterial lignin-degrading strain Sphingobacterium sp. T2 is reported as most active strain among 12 isolates and proteomic analysis of extracellular fractions gave hits for two superoxide dismutase enzymes. Two novel lignin-oxidising enzymes from Sphingobacterium sp. T2 were identified. Experimental and bioinformatic evidence revealed that these enzymes are extracellular superoxide dismutases, namely SOD1 and SOD2. Both enzyme genes have been cloned and overexpressed in Escherichia coli. The metal specificity of both enzymes was investigated using ICP-OES, UV-vis spectra and superoxide dismutation activity of different metal-containing enzyme preparations, the results indicate that both enzymes (SOD1 and SOD2) are manganese-containing SOD. MnSOD1 and MnSOD2 from Sphingobacterium sp. T2 (SpMnSODs) exhibit time-dependent lignin-oxidising ability, they can depolymerise wheat straw organosolv lignin, modify Kraft lignin, and lignocellulose materials (pine and miscanthus). Analysis of reaction components of SpMnSOD enzymes with organosolv lignin by HPLC, LC/MS and GC/MS show that SOD1 and SOD2 generate 10 new products. These metabolites were identified by comparison of their retention times and mass fragmentation pattern produced by LC/MS and GC/MS with their authentic analogue. The mechanism of reaction and reactive species generated by SpMnSODs were elucidated from their identified reaction products. Results indicate that SpMnSOD enzymes produce highly reactive species (hydroxyl radical) and catalyse several types of reactions which include: Ca-Cß cleavage, arylC-Ca Cleavage, -OCH3 group replacement with -OH group, hydroxylation of aromatic rings, Ipso-substitution and decarboxylation. The X-ray crystal structure of MnSOD1 was determined to 1.35 Å resolution, showing that MnSOD1 has a typical homodimer with a Mn(II) ion in each active site ligated by three His, one Asp, and a water/hydroxide forming trigonal bipyramidal geometry. The reaction of SpMnSOD enzymes with lignin model compounds confirms that these enzymes can catalyse Ca-Cß cleavage, arylC-Ca Cleavage, ipso-substitution, decarboxylation, demethoxylation, hydroxylation of aromatic rings and fragmentation of propenoic acid side chains which in turn are evidence of generation of highly oxidising species (hydroxyl radical).

The Arbuscular Mycorrhizal (AM) fungi are a group of obligate plant root endosymbionts, which form associations with an estimated two thirds of terrestrial plant species. Their extra-radical mycelium extends throughout the soil and absorbs nutrients that are transferred to their host plant in exchange for a purely phytogenic carbon supply. Due to their ubiquity and their functional importance, they are the subject of much research into their community ecology, yet much is still unknown. For instance: whether or not AM fungi display preference for certain host plant species; how environmental heterogeneity and energy availability affect communities; and the relative influence of niche and neutral processes. This thesis describes experiments which profile AM fungal communities and environmental properties of their habitat at different spatial scales in different plant species. Network analysis revealed patterns indicative of niche-based processes structuring AM fungal communities more than neutral processes, phenotypic trade-offs between AM fungi, and of priority effects influencing diversity and unevenness. Difference between plant species in the heterogeneity of surrounding soil was dependent on spatial scale. The effect of decreased carbon allocation on AM fungal communities is greater in more heterogeneous habitats. These results suggest that the detection of host plant preference in AM fungi is dependent on spatial scale of sampling, driven by interspecific variation in plant root architecture, soil physical properties and AM fungal vital rates.

With popular interest in Linnaean botany thriving at the turn of the century, the Proserpina myth and its central focus on flowers and the feminine support nineteenth-century approaches to nature as an object of both scientific study and a source of spiritual or moral contemplation and guidance. The mythological figure of Proserpina with her dual nature of innocence and sexuality, is easily transposed into or appropriated as a flower-woman who can be identified with the moral typology or teaching of a mother’s botany—whether it be the maternal ideology of the “Linnaean years” or the Wordsworthian nature philosophy of Victorian Romantics—or the scientific knowledge of the “sexual system” and its link to industrial, technological science. Drawing upon historicist myth criticism, I trace the nineteenth-century evolution of the Proserpina myth into botanical discourse within contemporary views of myth’s organic quality and enduring aesthetic significance as a product of the imagination. Like modern critics of myth, nineteenth-century writers valued myth as literature or art and as adaptable and evolving. I follow the botanical evolution of the Proserpina myth, as a historical, literary construct, from its reception in the late eighteenth-century botanical poetry of Catherine Maria Fanshawe and Erasmus Darwin through the Romantic poetry of William Wordsworth and into its Victorian evolution as a narrative of change in the fiction of George Eliot and Elizabeth Gaskell and the prose of John Ruskin. Language, form and structure, morality and science, are concerns which literature, botany and myth all share in the nineteenth century, as the Victorians attempt to articulate their relationship to a changing natural world. The myth’s reception by my nominated writers reveals three readings of female sexuality as passive, active or ambivalent, based upon the identification of girl and flower as a contested site between conflicting sides of a maternal or sexual nature. Proserpina’s coming-of-age highlights the tension within nature and indicates predominant attitudes toward or preferences for moral nature, scientific nature or ambivalence, which ultimately signify corresponding perceptions of social change. Nature is sacred, violated by industrialism and in need of preservation and protection, or nature is ripe and ready for scientific exploration and industrial development. The Victorian preoccupation with myth, flowers and the feminine is evident in the appropriation and interpretation of the popular myth of Proserpina as a narrative of change capturing an ambivalence toward industrial society: a fractured consciousness caught between nostalgia and progress that is in keeping with the narrative’s double cast, looking backward to childhood and forward to romance or marriage. An innocent female protagonist and daughter figure, nurtured by a rural, maternal nature, is threatened by the entrance or intrusion of a male seducer/suitor figure associated with the industrial, scientific world. The heroine exists as a contested site of innocence, threatened like the landscape itself.

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.

Aspergillus niger is a black-spored filamentous fungus that forms asexual spores called conidospores (‘conidia’). Germination of conidia, leading to the formation of hyphae, is initiated by conidial swelling and mobilisation of endogenous carbon and energy stores, followed by polarisation and emergence of a hyphal germ tube. These morphological and biochemical changes which define the model of germination have been studied with the aim of understanding how conidia sense and utilise different soluble carbon sources for germination. Microscopy and flow cytometry were used to track the morphological changes and results showed that the germination of A. niger conidia was quicker and more homogenous in rich media than in minimal media. The germination of conidia was also shown to be quicker in the presence of D-glucose than D-xylose. In the absence of a carbohydrate, no visual indicators of germination were evident. Added to this, the metabolism of internal storage compounds was shown to only occur in the presence of a suitable carbon source. Specific environmental carbon sources may therefore serve as triggers of germination, i.e. to initiate the catabolism of stores such as D-trehalose and the swelling of conidia. Studies carried out using D-glucose analogues identified the structural features of sugars that trigger or support conidial germination. These studies showed that the arrangement of atoms on carbons 3 and 4, on the pyranose ring structure of D-glucose, are essential to serve as a trigger of germination. The trigger step preceeds, and is separate from, the energy generation step that supports the continued outgrowth. Transcriptomic studies found that the most significant changes were associated with the breaking of dormancy. The data also revealed that fermentative metabolism present at the early stages of spore germination is rapidly replaced by respiratory metabolism.

Atmospheric CO2 levels have been increasing at an accelerated rate for the last 250 years, much of which is absorbed by the oceans, resulting in a process called ocean acidification (OA). This phenomenon has the capacity to disrupt many marine biological processes that utilise carbon, in particular photosynthesis and calcification, and as such phytoplankton have been a main topic of OA studies. While research has accelerated over the last decade, establishing general trends still remains confounded by methodological inconsistencies. Coccolithophores, particularly the species Emiliania huxleyi, are both ecologically and biogeochemically important phytoplankton; however, one strain (NZEH) has produced highly varied results. Here, we present a multivariate analysis that suggests previous inconsistencies between past studies of NZEH may be driven by variance of the light:dark (L:D) cycle used for growth. Experimental analysis on NZEH showed that under a 14:10h L:D cycle, CO2 induces significantly slower growth rates and higher PIC and POC cell-1, but this effect is dampened under 24h of light. This was widened to encompass more taxa, including more isolates of E. huxleyi (PLY70-3, PLY124-3, RCC962), and two other species of coccolithophore; Gephyrocapsa oceanica and Coccolithus pelagicus. L:D cycle changed the observed OA response, with two main responses divided by biogeographical origin. In tropical taxa, 24h light enhanced the effects of increased photosynthesis, but dampened the decrease in calcification in response to CO2. For temperate taxa, 24h dampened both the increases in photosynthesis and calcification with CO2. Evaluation of photobiology reveals that both CO2 and longer photoperiods induce a “high light” acclimation response, and changes in coccosphere thickness suggest it has a photoprotective role. Finally, results from bioassay experiments on natural phytoplankton populations in the polar regions show that CO2 response is hard to predict and based on community composition and ambient starting conditions. This work serves to further highlight the importance of environmental variables that moderate the OA response in accurately understanding future biogeochemical cycles. Future models attempting to predict the impact of OA upon marine systems must critically account for interactive role of light availability.

Stomata open in response to light allowing CO2 uptake for photosynthesis and they close in response to abiotic stress, such as drought, to prevent transpirational water loss from the plant. A pair of guard cells surrounds each stoma and stomatal movements depend on K+ fluxes across the guard cell plasma membrane. These fluxes are mediated by inward and outward rectifying K+ channels (K+in and K+out). The SNARE SYP121 was originally identified in association with ion channel regulation in guard cells. SNARE proteins mediate vesicle fusion and facilitate delivery of membrane proteins to target membranes. They are also linked to a variety of physiological responses. In particular, the plasma membrane SNARE SYP121 has been attributed a role in immune response and K+ nutrition. I have used the Arabdopsis loss-of-function mutant syp121 and uncovered a set of mutant phenotypes associated with impaired stomatal opening. In the syp121 mutant stomatal reopening was delayed and incomplete following Ca2+-induced closure, and increase in stomatal transpiration was slowed in the light. Incomplete reopening was rescued by complementation with wild-type SYP121 and was not observed in the syp122 mutant, lacking the homologous gene product. Guard cell K+ in current, necessary for K+ uptake during stomatal reopening, was reduced in syp121 mutant guard cells. Analysis of current gating characteristics suggested an impaired delivery of K+in channels to the plasma membrane, which was consistent with inhibition of stomatal reopening by the trafficking inhibitor Brefeldin A in wild-type plants. Impaired stomatal reopening in the syp121 mutant was phenomenologically similar to a Ca2+-encoded form of ‘programmed closure’ and my results suggest that endocytosis and delayed recycling of K+in channels may underly this phenomenon. Impaired stomatal function manifested in a conditional syp121 mutant growth phenotype dependent on high light and low humidity, characterised by reduced stomatal conductance and photosynthetic CO2 assimilation. My results suggested the necessity for SYP121-dependent K+in channel traffic during stomatal reopening. My results revealed a novel syp121 stomatal phenotype that was linked to K+in channel recycling in guard cells and had consequences for whole-plant water use and biomass production.

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.