This project sets out to understand further the use of the conserved DNA sequence motif CCAAT in the modulation of plant gene transcription, by the isolation and characterisation of CCAAT box binding proteins (CBP's) from the model plant <I>Arabidopsis</I>. A highly conserved CBP complex consisting of CBP2, 3 and 5 has previously been identified in a range of organisms including yeast and vertebrates. Several strategies for the isolation of <I>Arabidopsis</I> CBP homologues were attempted. Functional complementation of the yeast <I>HAP2</I> mutant identified three independent <I>Arabidopsis</I> CBP2 cDNAs. Searching the <I>Arabidopsis</I> expressed sequence tag (est) database revealed two forms each of CBP3 and CBP5. The presence of multiple forms of each CBP homologue is unusual as they are encoded by a single gene in yeast and vertebrates. These seven cDNAs were sequenced on both DNA strands and the putative protein sequence determined. Southern hybridisation gave an indication of the genomic copy number of each clone, while hybridisation of labelled cDNAs to RNA extracted from a range of tissues characterised their expression pattern. While CBP2 and CBP5 homologues were expressed equally in each tissue analysed, the two CBP3 cDNAs were preferentially expressed in aerial parts. Comparison of protein sequences with other CBPs identified conserved amino acid residues that have been shown to be of functional importance. These conserved domain sequences were also used to examine the evolutionary relationship of the multiple <I>Arabidopsis</I> forms and those from other organisms. The identification of multiple, novel forms of CBP from <I>Arabidopsis </I>is discussed in relation to their potential mode of action and role in plant development.

To characterise the circadian transcriptome, the expression profiles of transcripts in whole leaves of mature, soil grown <i>A. thaliana </i>plants were analysed. Circadian-regulated transcripts constituted approximately 9.5% of the transcripts detected, and were found to encode proteins involved both a well-described circadian processes and in pathways that have not been previously identified as having circadian regulation. Transcripts encoding proteins involved in core metabolic processes and stress response pathways were particularly highly representative in the dataset, which suggested a potential basis for the fitness benefits associated with the possession of a functional biological clock. To determine the correlation between rhythms in transcript abundance in whole leaves and in a single cell type, the stomatal guard cell was selected as a model system. However, assessment of two published methods of guard cell isolation, epidermal fragmentation and guard cell protoplasting, revealed that neither method was suitable for analysis of circadian rhythms in transcript abundance. Consequently, single cell analysis was not pursued. Nevertheless, bio-informatic analysis of the whole leaf circadian transcriptome and published microarray data was employed in order to characterise components of the intra-cellular circadian signalling pathway. This analysis revealed a relationship between the circadian oscillator and the regulator of [Ca²⁺]_cyt release, cyclic adenosine disphosphate ribose (cADPR). Evidence is presented suggesting that circadian [Ca²⁺]_cyt oscillations form a component of the oscillator that maintains the periodicity of circadian rhythms in transcript abundance. Collectively the data presented provide an overview of the biological clock in <i>A. thaliana</i>, and form a resource for further analysis of the structure of the clock and its role in integrating diverse cellular and physiological processes into a coherent biological programme.

The aim of the work described in this thesis was to investigate the role and importance of the glucose transporter in the chloroplast envelope. I believed that further characterisation would contribute to our knowledge of transitory starch degradation at night, and to the control of carbon partitioning. I have supplied tracer quantities of [U-<SUP>14</SUP>C]glucose and [U-<SUP>14</SUP>C]glycerol in the dark to leaves selected from a diverse range of species, and demonstrated that the net flux in the cytosol, during starch degradation in the dark, is glycolytic, so that a gluconegoenic flux from triose-phosphates to sucrose does not occur. This provides evidence that a flucose transporter, rather than the phosphate translocator, is active in the chloroplast envelope for the synthesis of sucrose, and suggests that glucose transport across the chloroplast envelope is found throughout the plant kingdom. I have demonstrated, using the technique of silicon oil centrifugation, that chloroplasts of wild-type <I>Arabidopsis thaliana </I>and tobacco take up glucose but do not take up maltose from the external medium, that glucose transport in <I>Arabidopsis </I>is unaffected by ATP. I have also shown, by pre-labelling starch with <SUP>14</SUP>CO<SUB>2</SUB>, that wild-type <I>Arabidoposis </I>chloroplasts export glucose, but not maltose, during starch degradation in the dark. These results imply that maltose is not an important final product of starch degradation in these species. I used transgenic tobacco plants, with altered levels of the triose-phosphate translocater and little impact on carbon fixation, to investigate the role of hexose transport in carbon partitioning. Careful measurements of phosphate and glucose uptake in chloroplasts of antisense and wild-type plants revealed that there was no alteration in glucose uptake, and that the endogenous wild type capacity may be sufficiently high to account for a compensatory flux in glucose transport. I hypothesised that a mutant of <I>Arabidopsis </I>(<I>sex1</I>), with chloroplasts deficient in glucose transport and accumulating starch, did so through increased starch synthesis at night. To support this, I found no differences in the maximal catalytic activities of amylases, starch phosphorylase, phosphoglucomutase, glucokinase and phosphofructokinase (ATP) in chloroplasts isolated from dark-acclimated leaves of <I>sex1</I> and the wild type.

This study combined <i>in vivo </i>leaf gas exchange, chlorophyll fluorescence, and instantaneous carbon isotope discrimination (D) analyses with <i>in vitro </i>measurement of Rubisco and PEPc activity and transcript abundance of key Rubisco and PEPc regulatory enzymes, Rubisco activase (RCA) and PEPc kinase <i>(PPCKI ), </i>and RCA protein abundance, to provide a novel molecular perspective on the co-regulation of carboxylase activity in <i>M. crystallinum.</i> In C₃ <i>M. crystallinum, </i>Rubisco activation state was highest during the photoperiod. C3 Rubisco activity remained uniform diurnally, mirrored by the pattern of stable CO₂ assimilation rate and protein abundance of both RCA isoforms, RCA1 and RCA2. RACE-PCR was used to identify RCA2 as the larger isoform in <i>M. crystallinum. </i>A marked decrease in RCA2 protein occurred during CAM induction, coincident with increased PEPc protein abundance and maximal PEPc activity. Diurnal Rubisco activity was regulated independent of light intensity and electron transport rate (ETR) following CAM induction. Rubisco activation state in CAM <i>M. crystallinum </i>was increased during the latter half of the photoperiod (mid CAM Phase III - Phase IV), coincident with low apparent PEPc activity and the final stages of titratable acidity breakdown. Increased RCA1 protein abundance during mid-CAM Phase III was observed coincident with an increase in Rubisco activation state of approximately one third. Modulation of diurnal Rubisco activity by RCA1 represented a potential regulatory mechanism unique to the CAM mode of <i>M. crystallinum.</i>

Plant growth regulators (PGRs) are thought to play a vital role in the control of plant development. This control is mediated by interplay between the sensitivity of a tissue to a given PGR, and the concentration of the PGR within that tissue. PGR concentration is a function of its biosynthesis and metabolism. Biosynthesis feeds the active pool, but the role of metabolism in PGR homeostasis is more complex. This thesis describes an investigation into the importance of one aspect of metabolism, O-glucosylation, in the control of cytokinin levels in plant tissues. Cytokinins are N<SUP>6</SUP>-substituted adenine derivatives, which occur in many different forms. Glucosylation of "active" forms renders the molecule biologically inactive. It is, however, thought that cytokinins glucosylated at the O-position of the N<SUP>6</SUP> side chain are susceptible to cleavage by β-glucosidases, leading to the release of the active form. Very little work has been carried out on these metabolities, and no accurate technique for their measurement in plant tissues exists. This project therefore involves the development of 3 specific immunoassays for the accurate measurement of (OG)Z, [9R](OG)Z and [9R](OG)DHZ in plant tissues. These assays, were employed, in addition to pre-existing techniques, to assess the importance of cytokinin O-glucosylation in 3 very different systems. Direct ELISAs for 9-riboside and O-glucoside cytokinins were used in conjunction with HPLC to investigate cytokinin metabolism during leaf maturation in <I>Phaseolus vulgaris. </I>Cytokinin O-glucosides were found to increase with leaf age with a concomitant decrease in the ratio of active to conjugated forms. This is consistent with the hypothesis that a decrease in active cytokinins is involved with the onset of foliar senescence. Premature leaf senescence and reduced apical dominance of forest trees are two major symptoms of novel forest decline. Both of these symptoms suggest a perturbation of hormone controlled processes in damaged trees. I therefore investigated the possible inter-relationship between changes in cytokinin physiology and exposure to atmospheric pollution in <I>Picea abies. </I>This was achieved by examining the content of Z-, DHZ- and iP-type cytokinins in a range of metabolic forms, in the needles of trees grown at a polluted and a non-polluted site in central Slovenia. Results indicated that trees exposed to high levels of pollution had elevated needle cytokinin content. This appeared to be due to perturbation of both synthesis and metabolism, as changes were detected in total cytokinin content and the relative proportions of the metabolites measured, in response to pollution stress. The results are discussed with reference to the impact the changes may have on whole plant physiology and the possible relationship between cytokinins and alleviation of environmental stress.

The arabinogalactans are a class of plant cell wall polysaccharides rich in the arabinosyl and galactosyl residues. The synthesis of the arabinogalactans is not well understood at a molecular genetic level. In this work, qualitative techniques were developed for the analysis of both classes of arabinogalactan (type I and II) in Arabidopsis using high-resolution oligosaccharide profiling strategies. An assay for pectic arabinogalactan I was developed using specific enzymatic digestion and oligosaccharide profiling by polysaccharide analysis using carbohydrate electrophoresis (PACE). Arabinogalactan II was studied using a chemical digestion strategy followed by compositional analysis and oligosaccharide profiling with high performance liquid chromatography and tandem mass spectrometry. These techniques were then applied to study the structure and quantity of arabinogalactan I and II in a range of Arabidopsis organs for the first time. This revealed interesting structural features, including the presence of galacturonic acid on arabinogalactan II. The techniques were validated by analysis of previously characterized nucleotide sugar synthesis mutants with arabinogalactan defects. This revealed the channelling of sugars from nucleotide sugar donors into different polysaccharide pools. These and other techniques were then used to study the synthesis of wall polysaccharides, testing the hypothesis that the Cs1A family of plant glycosyltransferases were arabinogalactan synthases. The sequence and expression of selected Cs1A genes was characterized, and mutant Arabidopsis plant lines with genetic lesions in these genes were studied. In summary, this thesis describes both the development and application of tools to learn more about arabinogalactans structure and polysaccharide synthesis in the model organism, Arabidopsis.

The aim of the research described in this thesis was the characterisation of the subcellular localisation and functional properties of two novel matrix attachment region (MAR) DNA-binding proteins, MARBP-1 and MARBP-2, exhibiting strong sequence similarity to each other and to yeast nucleolar box C+D snoRNP proteins Nop56p and Nop58p. MARBP-1 subcellular localisation was examined in onion bulb epidermis and pea leaf cells in a transient expression assay, using fusions of MARBP-1 and the reporters GUS and GFP. These experiments indicated nucleolar localisation of MARBP-1. Deletion analysis demonstrated that nucleolar localisation is conferred within the lysine-rich 98 amino acid C-terminal region. Similarly truncated forms of MARBP-1 were expressed in <I>E. coli</I>, and their ability to bind to a <SUP>32</SUP>P-labelled soybean heat shock (<I>Gmhsp</I>) gene MAR was examined <I>in vitro</I> by filter-based south-western assay. These experiments indicated that MAR-binding capability was also conferred within the 98 amino acid C-terminal region. MAR-binding activity of a soluble form of the 110 amino acid C-terminal region of MARBP-1 was demonstrated by electrophoretic mobility shift analysis, indicating that MAR-binding is not dependent upon prior denaturation of the protein. Disruption by site-directed mutagenesis of seven KKE/D repeats within the C-terminal region did not perturb subcellular localisation or MAR-binding, nor did a 20 amino acid deletion within this region. Disruption of a potential bipartite nuclear localisation sequence also within this region did not perturb subcellular localisation. These experiments suggest that general basic properties of C-terminal residues may be more important than specific sequence motifs for conferring MAR-binding and localisation functions.

With a view to developing inhibitors, two enzymes of the <i>Arabidopsis </i>pantothenate pathway (KPHMT2 and PS) were expressed in <i>E. coli</i>, as MBP fusion proteins purified and used to perform kinetic studies to identify their characteristics. KPHMT2 showed an apparent <i>Km, Vmax</i> and <i>kcat</i> of 0.534 ± 0.089 mM, 0.802 ± 0.032 nmoles/min and 2.79 min/1 respectively for α-KIVA whilst the enzyme appeared to be inhibited by its co-factor, 5,10-methylene tetrahydrofolate, at levels above approximately 25μM. In addition to its potential as a target for herbicides and anti-microbials the pathway is also of commercial interest for the production of pantothenate for use as a supplement in animal feed and for addition to cosmetics. By constitutive expression of the genes of the <i>E. coli </i>pathway, <i>Arabidopsis </i>lines with higher levels of pantothenate than WT were obtained. Expression of <i>E. coli panC</i> resulted in increased levels of the vitamin in some lines, as did the expression of <i>panD</i>, however, expression of <i>panB</i> showed no increase. The β-alanine levels of the transgenic lines ere also assayed and showed hugely elevated levels in the lines expressing <i>panD.  </i>To determine if expression of the <i>E. coli</i> genes or the accumulation of pantothenate effected the expression of the endogenous genes, RT-PCR was performed and showed no significant difference in the expression of endogenous <i>panB2 </i>or <i>panC</i> in lines expressing <i>E. coli panB, panC </i>or <i>panD</i>. Expression of the genes of the pathway was also studied using promoter:GUS fusions, RT-PCR, western blot analysis and enzyme assays. The results showed expression of the genes throughout the plant suggesting <i>de novo</i> pantothenate biosynthesis with <i>panB1 </i>and <i>panB2</i> promoter activity showing up-regulation in flowers and siliques, tissues known to require large amounts of pantothenate.

The aims of this study were to investigate the nature of the interaction between cytochrome <I>f</I> and plastocyanin isolated from higher plants. The reaction kinetics for these redox partners isolated from two homologous systems (spinach and turnip) were investigated. Limited proteolysis of purified spinach cytochrome <I>f </I>with proteinase K generated a form of the protein that was essentially kinetically identical to soluble, monomeric turnip cytochrome <I>f </I>in the kinetics of its reaction with plastocyanin. The second order rate constant for the reaction between oligomeric, nonproteolysed spinach cytochrome <I>f</I> and spinach plastocyanin (7.19 x 10<SUP>7</SUP> M<SUP>-1</SUP> s<SUP>-1</SUP>) was 1.9-fold less than that observed for the reaction between proteinase K-treated spinach cytochrome <I>f </I> and spinach plastocyanin (13.7 x 10<SUP>7</SUP> M<SUP>-1</SUP> s<SUP>-1</SUP>). The reactivity of cytochrome <I>f</I> when present in isolated cytochrome <I>bf</I> complex towards plastocyanin (k<SUB>2</SUB> = 7.78 x 10<SUP>7</SUP> M<SUP>-1</SUP> s<SUP>-1</SUP> for the spinach plastocyanin/spinach <I>bf</I> reaction and 8.68 x 10<SUP>7</SUP> M<SUP>-1</SUP> s<SUP>-1</SUP> for the turnip plastocyanin/turnip <I>bf</I> reaction) was very similar to that of unproteolysed spinach cytochrome <I>f</I>. The difference in rate between proteolysed and non-proteolysed cytochrome <I>f </I>in its reaction with plastocyanin is likely to be due to the occlusion of a region of the cytochrome involved in interacting with plastocyanin, caused by aggregation of the non-proteolysed form of the protein. This may also explain the decrease in rate observed for the reaction between <I>bf</I> complex and plastocyanin compared to the reaction with soluble cytochrome <I>f</I>. The reaction kinetics for homologous (turnip cytochrome <I>f/</I> turnip plastocyanin) and heterologous (turnip cytochrome <I>f</I>/spinach plastocyanin) systems were very similar (k<SUB>2</SUB> = 14.31 x 10<SUP>7</SUP> and 12.9x 10<SUP>7</SUP> M<SUP>-1</SUP> s<SUP>-1</SUP> respectively at pH 6.0. and 100 mM ionic strength). It is concluded that heterologous higher plant systems are valid for examining the reaction between cytochrome <I>f</I> and plastocyanin on the condition that the cytochrome is isolated from a cruciferous source. Mutation of Y83 (a residue suggested to be essential for the electron transfer activity of the protein) to serine, glutamate and tryptophan generated structurally unstable mutants which lost copper during the purification process. Similar instability was observed when the surface exposed copper ligand H87 was mutated to cysteine. Introduction of leucine at position 90 (adjacent to H87), replacing alanine, resulted in a stable mutant.

Recent epidemiological studies have shown that consuming a diet rich in fruits and vegetables is associated with a reduced risk of cardiovascular disease and cancer. The hypothesis of this thesis is that increasing the consumption of fruits and vegetables in the form of soups and drinks will increase the plasma phytochemical concentrations, antioxidant status and improve the oxidative stability of LDL ex vivo. The thesis describes two intervention studies. The first was a chronic study, which investigated the bioavailability of carotenoids and beneficial effects of carotenoid-rich foods in the Carotenoid-Rich Soup and Juice Study (CARS). The second study was an acute intervention, which investigated the effects of fruit and vegetable puree-based drinks (FVPD) in the Flavonoid Kinetics Study (FLaKS). The studies showed that the consumption of fruit and vegetables in the form of soups and puree drink, equivalent to five portions of fruits and vegetables, increased plasma phytochemicals in a chronic intervention, and increased plasma phytochemicals and plasma antioxidant capacity in the acute intervention. However, there was no significant effect of the interventions on the resistance of LDL to copper-induced oxidation. The ability of caffeic acid and rutin to bind to LDL and to affect the susceptibility of LDL to oxidation was studied. The current study showed that the polyphenol compounds did not bind to LDL or increase the oxidative stability, except when added rutin was present. In conclusion, the thesis showed that increasing the consumption of fruits and vegetables in the form of soups and drinks, equivalent to five portions of fruits and vegetables, will increase the plasma phytochemical concentrations, and postprandial antioxidant capacity. Possible effects on vascular reactivity need further study.