Plants, like most organisms, have developed elaborate mechanisms for anticipating periodic environmental changes. The circadian clock allows an organism to adapt its metabolic, developmental and physiological processes to coincide with favourable environmental conditions. At the centre of the Arabidopsis thaliana clock, linking environmental inputs and driving the overt biological rhythm is a central oscillator that consists of multiple interlocked transcriptional/translational negative feedback loops. What is known about the structure of the central oscillator comes primarily from genetic analysis. Less clear, is how putative oscillator proteins perform their perceived functions in circadian rhythm maintenance. Described are the cloning, expression and purification of clock-associated proteins; TOCI-PRR, ELF4, LUX and LIPl. Purified ELF4 was subjected to unsuccessful crystallisation trials, probably due to its intrinsically unstructured nature. A truncated form of LIP 1 was shown to be an active OTPase, representing the first example of an active OTPase in the plant clock. In addition, a protocol for the production of ssDNA aptamers has been developed (against SRRl), which can be used to replace antibody-based experimentation. The work presented discusses the difficulty in obtaining the novel, plant-specific proteins in quantities required for crystallisation, and suggests alternative methods for structural and biochemical analysis of these proteins. Moreover, this thesis combines experimental data with a range of Bioinformatic tools to aid design for subsequent biochemical expression, purification and crystallisation trials.

Nitrogen enrichment promotes phosphomonoesterase (PME) activity in the common heathland lichen Cladonia portentosa. This is associated with a marked increase in thallus N:P mass ratio and significant up-regulation of inorganic phosphate uptake capacity, evidencing a shift from N-limited to P-limited growth. Phosphomonoesterase activity in C. portentosa responds rapidly to change in N deposition load, with a significant increase in activity recorded within 6 months of transplantation from a low-N to high-N site. The location of PME activity in C. portentosa was revealed using a fluorescent marker and was found to be concentrated on both the outer and inner surfaces of the hollow ‘tube-like’ thallus branches. Activity appeared to be associated exclusively with the mycobiont and was located within the hyphal-lumina, consistent with a membrane bound ecto-enzyme. High PME activity in axenic mycobiont cultures of C. portentosa provided further evidence of a fungal location and confirmed that rates of activity in this lichen are amongst the highest reported for any other plant/fungal system in the literature. Different classes of phosphatases were assayed in a range of N2-fixing and non-N2-fixing lichens in an oligotrophic subarctic environment. No relationship was found between the capacities for nitrogenase and PME activities. Maximum rates of PME activity were recorded in fruticose mat-forming lichens which capture nutrients predominantly from atmospheric deposits. 5´ nucleotide phosphodiesterase activity was readily detected in N2-fixing lichens and was particularly high in rhizine-rich regions of foliose terricolous lichen thalli, consistent with the utilisation of organic phosphates from soil and litter sources.

Remote Sensing (RS) and Geographic Information System (GIS) approaches, combined with ground truthing, are providing new tools for advanced ecosystem management, by providing the ability to monitor change over time at local, regional, and global scales. In this study, remote sensing (Landsat TM and aerial photographs) and GIS, combined with ground truthing work, were used to assess wetland vegetation change over time at two contrasting wetland sites in the UK: freshwater wetland at Wicken Fen between 1984 and 2009, and saltmarsh between 1988 and 2009 in Caerlaverock Reserve. Ground truthing studies were carried out in Wicken Fen (UK National Grid Reference TL 5570) during 14th - 18th June 2010: forty 1 m2 quadrats were taken in total, placed randomly along six transects in different vegetation types. The survey in the second Study Area Caerlaverock Reserve (UK National Grid Reference NY0464) was conducted on 5th - 9th July 2011, with a total of forty-eight 1 m2 quadrats placed randomly along seven transects in different vegetation types within the study area. Two-way indicator species (TWINSPAN) was used for classification the ground truth samples, taking separation on eigenvalues with high value (>0.500), to define end-groups of samples. The samples were classified into four sample-groups based on data from 40 quadrats in Wicken Fen, while the data were from 48 quadrats divided into five sample-groups in Caerlaverock Reserve. The primary analysis was conducted by interpreting vegetation cover from aerial photographs, using GIS combined with ground truth data. Unsupervised and supervised classifications with the same technique for aerial photography interpretation were used to interpret the vegetation cover in the Landsat TM images. In Wicken Fen, Landsat TM images were used from 18th August 1984 and 23rd August 2009; for Caerlaverock Reserve Landsat TM imagery used was taken from 14th May 1988 and 11th July 2009. Aerial photograph imagery for Wicken Fen was from 1985 and 2009; and for Caerlaverock Reserve, from 1988 and 2009. Both the results from analysis of aerial photographs and Landsat TM imagery showed a substantial temporal change in vegetation during the period of study at Wicken Fen, most likely primarily produced by the management programme, rather than being due to natural change. In Cearlaverock Reserve, results from aerial photography interpretation indicated a slight change in the cover of shrubs during the period 1988 to 2009, but little other change over the study period. The results show that the classification accuracy using aerial photography was higher than that of Landsat TM data. The difference of classification accuracy between aerial photography and Landsat TM, especially in Caerlaverock Reserve, was due to the low resolution of Landsat TM images, and the fact that some vegetation classes occupied an area less than that of the pixel size of the TM image. Based on the mapping exercise, the aerial photographs produced better vegetation classes (when compared with ground truthing data) than Landsat TM images, because aerial photos have a higher spatial resolution than the Landsat TM images. Perhaps the most important conclusion of this study is that it provides evidence that the RS/GIS approach can provide useful baseline data about wetland vegetation change over time, and across quite expansive areas, which can therefore provide valuable information to aid the management and conservation of wetland habitats.

The discovery of plastocyanin in 1960 confirmed the long-held belief that copper was an essential component of the electron transfer sequence in photosynthesis (Katoh 1960). Much work has appeared in the literature on the role of plastocyanin and this has been reviewed in recent articles (Vernon and Avron 1965; Hind and Olson 1968; Levine 1969).

In this thesis a range of lattice based spatially explicit models of ecosystems are presented and their applicability to various ecological situations is demonstrated with emphasis on plant communities These mechanistic and individual based models which include coupled map lattices and cellular automata aim to produce ecological insights and testable results Models of both short and long term systems are developed with the former being potentially testable in the eld and the latter promoting understanding where experimentation is not feasible A range of graphical and numerical techniques were developed to investigate both plant and animal model ecosystems The starting point is a short term single species coupled map lattice which investigates popula tion structure arising from local competitive interactions The model concludes that increase of size variation with increasing density indicates the presence of competitive intraspecic asymme try This idea is applied to crop data where considerable asymmetry is identied emphasising the need for balancing crop yield and size consistency Multispecies extensions to this model focus on spatial patterning arising from biotic interac tions and various numerical techniques underline the asymmetrical relationship between long and short lived species Environmental heterogeneity is imposed on the plant species in a third version of the model via the incorporation of an explicit resource base The complex inter dependence of community and environment is highlighted and illustrated by a model of the evolution of seed sizes Through the application of cellular automata to forest and epidemiological systems the concept of memory such as age or stage structuring is shown to be vital in the generation of spatial structure in long term ecological systems Analytical investigations generate further insights and again emphasise the crucial role played by spatial extensiveness in the wide range of ecological situations considered here In conclusion lattice models are ideally suited to the study of ecosystems

During a three year field study (1997-2000) vegetation assemblages, collective vegetation variables, traits of dominant populations and hydrological and hydrochemical variables were repeat-sampled within seven wetland sites across Scotland and northern England. These ranged from the Irish Marshes, Inverness-shire in the north, to Tarn Moss, Cumbria at the southern extreme. Sampling was conducted at a total of fifty-six permanent sample stations located along a total of eleven transects. Vegetation groupings were defined using multivariate analyses, and were classified as various fen, mire, and swamp NVC community types. The various groups were characterised by the values for the range of variables measured, and significant differences were seen between a number of these variables for different groupings. In addition, certain separate groupings with the same community classification were also seen to have significant variations between them in terms of trophic status, and canopy height and biomass values. Collective vegetation variables and dominant population trait values were successfully predicted from physical and chemical variables measured within the groundwater and substrate during 1999. A number of specific models incorporating relatively large numbers of predictor variables were proposed alongside more general models incorporating fewer predictor variables. The greatest predictive power with R2 = 0.67 (p<0.001) for a model predicting stem density (m-2). Conversely, vegetation variables proved useful for predicting characteristics of the groundwater environment, for which specific and general models were against proposed. In this instance, the greatest predictive power was R2 = 0.79 (p<0.001) for a model predicting minimum water table level (i.e. maximum level of drawdown). The models were tested using data collected during 2000 from repeat sites and independent sites. Whilst some of the variables were predicted within noisy limits, predicted values generally corresponded well to observed values.

Oakbank crannog is a Late Bronze/Early Iron Age lake dwelling in Loch Tay, Scotland. The initial free-standing pile construction was rebuilt several times, resulting in an organic mound. The latest remaining consolidation phase incorporated large boulders which sealed the organic material underneath and preserved waterlogged remains of the plants utilised by earlier inhabitants. The base-rich soil of the local environment around the dwelling supported a mixed oak woodland with alder carr in the wetter areas and fringing the loch. Parts of this were cleared for agriculture by the earliest settlers, and woodland trees were utilised to build the dwelling, most especially alder, but also frequently oak, hazel and birch, with others occasionally at low levels. Cereal crops grown included Hordeum vulgare, Triticum dicoccum and T. spelta. Spelt wheat implies trade links with the south of the country or abroad. Linum usitatissiumum was cultivated on a small scale, possibly together with Papaver somniferum, another species which has implications for trade. The abundance of crop contaminant weed species emphasises the soil fertility, and suggests that cereal crops were neither weeded nor hand-picked on the ear. Seeds of low growing weeds indicate harvesting close to ground level. Processing was done for immediate use, at the doorway to the dwelling itself. Crop by-products including chaff and weed seeds were fed to livestock to supplement fodder. Tail grain and some prime barley may have been added or left intentionally during processing to supplement the feed. This generous use of valuable grain and edible weed seeds implies a healthy economy with adequate stores of food. Livestock were housed in the dwelling at least periodically, but were confined to specific areas.

The aim of this project has been to isolate, characterise and investigate the expression patterns of the various members of the castor bean lectin gene family. Genomic Southern blotting experiments showed that the gene family contains approximately eight members, of which two appear to be ricin-like. Castor bean genomic DNA was cloned into a bacteriophage lambda vector and the resulting recombinant clones screened with a ricin cDNA probe. Seventeen positive clones were isolated, amongst which at least five different groups could be recognised, on the basis of Southern blotting data. Four different lambda clones were selected for further analysis. One of the clones was found to contain a functional ricin gene with an identical restriction pattern to that of the ricin cDNA, but none of the clones appears to contain an authentic Riclnus communis agglutinin (RCA I) gene. DNA sequencing and RNAse protection data showed that three of the clones analysed contain lectin pseudogenes. The expression pattern of the functional ricin gene in pCBG3Hl (lambda clone 10) was investigated at the transcriptional level using RNAse protection. The results obtained show that the mRNA transcribed from this gene accumulates during the late (post-testa) stages of seed development. The pCBG3Hl ricin gene appears to use multiple cap and poly(A) sites. The developmental profile of lectin gene transcript levels observed in this study is similar to the patterns previously observed at the protein and translatable mRNA levels. This indicates a close correlation between the accumulation of the lectin proteins and the amounts of steady state transcripts. DNA sequencing enabled the identification of putative transcriptional regulatory elements in the promoter of the pCBG3Hl ricin gene, including an element resembling the RY repeat previously implicated in seed-specific gene expression. Ultimately, it is hoped that studies of this type will make it possible for the network of regulatory processes governing the expression pattern of the castor bean lectin genes to be unravelled.

Biogenesis of the chloroplast involves the activities of both the nuclear and chloroplast genomes. Nuclear-encoded chloroplast proteins are synthesised on free ribosomes in the cytosol and imported into the chloroplast post-translationally. Protein uptake is directed by an N-terminal presequence which is removed after import by specific processing peptidases. Thylakoid lumen proteins must cross three membranes to reach their site of function and have a composite presequence to enable their correct localisation, consisting of an envelope transit domain (ETD) and a thylakoid transfer domain (TTD). The ETD is removed after translocation across the envelope by a stromal processing peptidase (SPP) to produce an intermediate-sized protein and the TTD then allows transport into the thylakoid lumen where it is removed by a thylakoidal processing peptidase (TPP). SPP is a soluble, stromal- located metallopeptidase which also removes the presequences from stromal proteins. Despite its apparently high specificity, there is very little sequence similarity around SPP cleavage sites in stromal and thylakoidal proteins, and little is known about the structural features which SPP recognises. SPP was partially purified from pea chloroplasts and the SPP cleavage site within the presequences of three thylakoid lumen proteins was determined by radiosequencing of the cleavage products with the two-fold aim of identifying the residues around the cleavage site which SPP may recognise and of delineating the ETD and TTD regions of the presequences, allowing their comparison with signal sequences. This information was then used to create four mutant thylakoid lumen precursor proteins by site-directed mutagenesis with altered SPP processing sites. The four mutant proteins all showed a reduced rate and efficiency of processing in an organelle-free time course assay, although all were imported into chloroplasts, correctly localised and processed to the mature size in an in vitro assay. Although SPP has usually been considered to be highly specific for impoited chloroplast precursor proteins, it has recently been suggested that it may also process mitochondrial precursor proteins, which are cleaved in vivo and in vitro to the mature size by the mitochondrial processing peptidase (MPP). These two peptidases were therefore compared in terms of substrate specificity and mechanism by assaying the activity of the two enzymes against chloroplast and mitochondrial precursor proteins. MPP did not process any of the chloroplast precursor proteins available, suggesting that this enzyme is indeed highly specific for mitochondrial precursor proteins. A partially-purified SPP preparation cleaved the mitochondrial precursor proteins tested to a smaller size; however, the site of cleavage in at least some of these proteins was different to the authentic MPP cleavage site, with the stromal preparation cleaving N- terminal to MPP. This stromal processing activity was not due to mitochondrial contamination and evidence from inhibitor sensitivities and column chromatography suggests that SPP cleaves both chloroplast and mitochondrial precursor proteins. Hie SPP processing site within the presequences of two mitochondrial proteins was determined by radiosequencing and compared with the chloroplast cleavage sites, and this data should enable further analysis to determine the features which constitute a site which can be recognised by SPP. An SPP activity was also partially-purified from Chlamydomonas reinhardtii and shown to be located in the stroma. This activity is able to process chloroplast precursor proteins from C. reinhardtii and pea to an intermediate or mature size, emphasising the similarity of the specificity of SPP from these two species.

Studies on aspartate transcarbamoylase from various organisms have been reviewed. The amino acid composition, isoelectric point and peptide maps of purified wheat germ aspartate transcarbamoylase have been elucidated and compared to the E.coli enzyme. Three types of essential amino acid residues have been demonstrated by chemical modification reagents. Essential arginine and lysine residues were studied by reaction with phenylglyoxal and pyridoxal 5'-phosphate respectively. In each case one or more residues were found at the active site, the reaction of which showing many similarities to previous studies on the E.coli catalytic subunit. One exceptionally reactive histidyl residue has been studied in wheat germ and E.coli aspartate transcarbamoylase. Reaction of these histidyl residues with diethylpyrocarbonate again indicated many similarities between the two enzymes. The position of the essential histidyl residue has been located in the published sequence of the E.coli enzyme and found to agree well with X-ray crystallographic and other evidence. The sequences adjacent to the essential histidyl and lysyl residues in the wheat enzyme were elucidated and compared to those of the E.coli enzyme. Attempts to induce the overproduction of aspartate transcarbamoylase in a carrot cell suspension culture by PALA were unsuccessful. These cells were instead found to have an innate mechanism for the detoxification of the drug. The nature of this detoxification mechanism has been partly elucidated.