Xyloglucan is thought to form cross-links between cellulose microfibrils within the plant cell wall. The hydrolysis of these 'tethers' may be involved in wall loosening, and a control of cell expansion. Transglycosylation reactions between xyloglucan 'tethers' has for some years been suggested as an alternative mechanism to hydrolysis by which the plant cell wall may be loosened. The work described in this thesis is an investigation of this hypothesis. Spinach (<i>Spinacia oleracea</i> L.) cell suspension cultures were incubated with [<i>xylosyl</i>-<SUP>3</SUP>H]xyloglucan nonasaccharide (XG9; Glc<SUB>4</SUB>.Xyl<SUB>3</SUB>.Gal.Fuc) and [<i>reducing terminus</i>-<SUP>3</SUP>H]XG9. The majority of <SUP>3</SUP>H-labelled material remained soluble and extracellular (69-98% ), and 53-57% of it underwent an apparent increase in molecular weight as shown by gel permeation chromatography. This result could suggest the occurrence of a transglycosylation reaction in the apoplast. A proportion of XG9 was hydrolysed to low molecular weight <SUP>3</SUP>H-labelled products. [<SUP>3</SUP>H]-XG9 was found to have undergone no appreciable re-arrangementon incorporation into the high molecular weight product as complete acid hydrolysis and Driselase digestion released similar <SUP>3</SUP>H-labelled hydrolysis products from the putative transglycosylation product, as from [<SUP>3</SUP>H]-XG9. The reducing terminus of the oligosaccharide remained as a reducing terminus of the high molecular weight product. This was shown by sodium borohydride reduction of the product formed during incubation of cells with [<i>reducing terminus</i>-<SUP>3</SUP>H]XG9. This would suggest that XG9 acts as the acceptor substrate, an apoplastic donor being cleaved and the newly formed potentially reducing terminus of this polymer being transferred onto XG9.

The aim of this project was to examine the process by which cultured cells of <i>Papaver somniferum</i> and related species, biotransform codeinone to codeine. It was confirmed in this study that plants of <i>P.somniferum</i> produce the morphinan alkaloids thebaine, codeine and morphine and therefore, possess the enzyme involved in the biotransformation of codeinone to codeine. However plants of <i>P.bracteatum</i> and <i>P.orientale</i> only accumulate thebaine apparently lacking one or more of the enzymes necessary to convert thebaine to morphine. Suspension cultures derived from <i>P.somniferum</i>, <i>P.bracteatum</i> and <i>P.orientale</i> all produced the morphinan alkaloid(s) common to the parent plant but in significantly lower amounts. <i>P.somniferum</i> suspension cultures converted a maximum of 35.3% of added codeinone to codeine. However, 'meristemoid' cultures of <i>P.somniferum</i>, which displayed a high state of differentiation and consisted of densely cytoplasmic cells organised in recognisable morphological structure, converted up to 60.0% of added codeinone to codeine. By contrast, suspended cells of <i>P.bracteatum</i> and <i>P.orientale</i> biotransformed less than 5% of added codeinone to codene. This strongly suggests the existence of a relationship between biotransformation capacity (amount of product produced per specified volume or unit of biomass) and culture origin. Immobilisation increased the biotransformation capacity of <i>P.somniferum</i> suspension cultures but not of <i>P.somniferum</i> 'meristemoid' cultures. Suspension cultures of <i>P.somniferum</i>, at different stages of development, converted approximately equal quantities of codeinone to codeine over a 72h period. However, the rate at which codeine was produced in these cultures varied considerably and was fastest in cultures entering the stationary phase. This also corresponded to the stage at which the concentration of endogenous alkaloids in the cells was highest.

This thesis describes the analysis of the AtSTP1 protein of <i>Arabidopsis thaliana</i> with regard to its physiological role. The AtSTP1 protein is a high affinity proton monosaccharide symporter which is located within the plasmamembrane. The AtSTP1 gene is expressed in various tissues of<i> Arabidopsis</i> including stems, roots, flowers, sepals, ovaries and leaves where the AtSTP1 mRNA is most abundant. The AtSTP1 protein belongs to a gene family with at least 16 members in <i>Arabidopsis</i>. The physiological function which has been proposed for individual members of this family includes the import of monosaccharides into symplastically isolated sink cells like pollen grains, embryos and guard cells or into cells with an increased carbohydrate demand due to wounding, pathogen infection or developmental processes. A role for monosaccharide transporters as part of one sugar sensing system which controls the carbohydrate metabolism of the plant has also been proposed. In order to elucidate the biological function of the AtSTP1 protein an AtSTP1 knock-out line, a line which overexpresses the AtSTP1 gene under control of the 35S-CaMV promotor and wild type plants were analysed with regard to their ability to transport monosaccharides and phenotypic effects after feeding with monosaccharides. Feeding whole seedlings with μM concentrations of glucose showed that in the knock-out line glucose uptake is decreased by 40% whereas in the overexpressing line in a two fold increase glucose uptake is observed compared to wild type line under these conditions. When the seedlings are fed with 200 mM glucose there is no difference in the glucose concentration accumulated in the knock-out line and wild type line whereas the overexpressing line shows a significantly higher glucose content. When the plants are grown in the absence of any added monosaccharides no phenotypic differences between the knock-out line, the overexpressing line and the wild type line can be observed.

No description available.

This work has investigated the permeability of amyloplasts isolated from developing wheat endosperm to a range of metabolites that could be taken lip by the amyloplasts for starch synthesis and carbohydrate oxidation. A rapid method for the isolation and purification of amyloplasts from the endosperm of spring wheat, Triticum aestivum L.cv Axona, has been developed. Amyloplasts were mechanically released from cells of plasmolysed endosperm tissue and purified by low speed centrifugation through 2% Nycodenz onto an agar cushion. Amyloplasts prepared by this method were routinely 55-65% intact and the recovery was greater than 20% as determined by the plastid marker enzyme, alkaline inorganic pyrophosphatase (APPase). Contamination by the cytosol determined by UDP-glucose pyrophosphorylase and alcohol dehydrogenase was less than 1% whilst the contamination attributable to mitochondria, the endomembrane system and microbodies was 0.2%, 0.5% and 0% respectively. Amyloplast integrity was dependent upon the sorbitol concentration of the extraction buffer and at the optimal concentration of O.8M sorbitol, intactness was maintained for up to 2 hours. Proteolytic protection experiments demonstrated that amyloplast marker enzymes were protected from digestion with trypsin. Preparations incubated with a range of [U_14C] labelled substrates, in the presence and absence of ATP, were able to support starch synthesis at physiological rates similar to rates obtained in whole endosperm when supplied with 5 mM GlclP and 1 mM ATP or 5 mM ADP-glucose. The Km for GlclP was calculated as 0.46 mM. The ability of amyloplast preparations to support starch synthesis from GlclP and ATP in the presence of known translocator inhibitor compounds was examined. DIDS, P5P and CAT, inhibitors of the phosphate and adenylate trans locators, reduced the amount label incorporation from [U_14C] GlclP, supplied in the presence of ATP, into starch by up to 80%. Stimulation" of the OPPP by the addition of substrates for the glutamate synthase reaction, 2-oxoglutarate and glutamine in the presence of either Gle 1P or Glc6P and ATP resulted in an asymmetric distribution of carbon utilisation between starch synthesis and carbohydrate oxidation. 14C from supplied [U_I4C] GlclP and [U_14C] Glc6P was measured in synthesized starch and evolved CO2. Whilst GlclP was clearly the preferred substrate for starch synthesis, Glc6P was readily oxidised when 2-oxoglutarate and glutamine were supplied to isolated plastid preparations. Metabolite levels inside the amyloplast were quantified under different conditions of starch synthesis and carbohydrate oxidation. Under conditions in which the OPPP was not stimulated, GlclP demonstrated no interconversion to Glc6P, suggesting that the enzyme which interconverts the two compounds is not catalysing an equilibrium reaction and may be the regulatory branchpoint between the pathways of starch biosynthesis and carbohydrate oxidation.

It is thought that structural features of eukaryotic mRNA, including S' terminal caps, repeated sequence transcripts and 3' poly A tracts, may be involved in post-transcriptional regulation of gene expression. This thesis considers the possible role of the poly A on artichoke mRNA.

Phenolic acid residues e.g. feruloyl groups are found esterified to specific sugars of primary cell wall polysaccharides of plants and phenylpropanoid derivatives are incorporated into lignin. These phenolic moieties probably have profound effect on cell wall properties such as extensibility (and thus growth) and digestibility. The work described in this thesis is an investigation into some aspects of the biosynthesis of cell wall phenolic residues. The site and mechanism of the feruloylation of primary wall polysaccharides has been a matter of some debate, although it is likely to be enzyme mediated. Fescue and maize cell cultures were supplied with radiolabelled precursors in order to ascertain the kinetics of this process and thus its likely sub-cellular site. [<SUP>3</SUP>H]Arabinose uptake was negligible in maize cell cultures and labelling with [<SUP>14</SUP>C]glucose resulted in very high background levels of radioactivity making analysis of feruloylated fragments difficult. However, radiolabelled [<SUP>3</SUP>H]arabinose was rapidly taken up and incorporated into nascent polysaccharides of fescue cells and polymer bound [<SUP>3</SUP>H]arabinose residues were beginning to be feruloylated within 5 minutes of the radiolabelled precursor being supplied. However, radiolabelled polymers were not secreted into the culture medium until 15-30 minutes after this point. Assuming that the onset of secretion of radiolabelled polymers is the time before which essentially all of the radiolabel was internal to the plasma membrane, the data presented show that feruloylation of polymer bound [<SUP>3</SUP>H]arabinose residues must occur intraprotoplasmically. As feruloylation was shown to occur intraprotoplasmically and polysaccharide synthesis occurs mainly in the Golgi bodies, endomembrane preparations from maize cell cultures were assayed to ascertain the presence of feruloyl-CoA : polysaccharide feruloytransferase activity.

We analysed immediate-early local and systemic responses to the perception of Pi in Pi-deplete plants or suspension cells. In Pi-deplete plants or cells, RNA abundance of Pi starvation-induced (PSI) genes is very rapidly downregulated in response to Pi or phosphonate, a non-metabolic analogue of Pi. This indicates that Pi perception, not its metabolism is the crucial event in Pi-signalling. We proposed a model for local Pi signalling in which the perception of Pi controls the stability of negative regulators of downstream responses.  Systemic signalling was examined in a split-root system. Pi perception also initiates systemic Pi signalling. The slow systemic translocation of Pi demonstrates that Pi itself is not the systemic signal. To access on the relationship between different nutrient metabolic and signalling pathways, the effects of varying carbon (C) and nitrogen (N) availability on phosphate (Pi) starvation responses and on plant growth were examined. Increased levels of C enhance PSI gene expression responses in roots and shoots, while elevated nitrogen or cytokinin (CK) treatments suppress these responses strongly in roots only. Elevated nitrogen or CK availability shifts shoot-root mass ratios in favour of shoots and alters cellular Pi concentration in both tissues, while increased carbon specifically promotes root growth. Our data indicate that the carbon-nitrogen balance informs growth control networks controlling shoot-root mass ratios. Altered allocation of growth potential by these two nutrients is not dependent on Pi nutritional status and is dominant over Pi starvation-induced growth responses. To identify genes involved in Pi signalling, a genetic screen with Pi starvation responsive reporter (luciferase) line was also performed. A candidate mutant was identified and initial analysis showed it is likely to be a signalling mutant.

I have developed a method which allows the existence of an intermolecular diferuloyl linkage to be tested. Plant cell-suspension cultures were fed a radiolabelled ferulic acid precursor, (E)-[U-^l4C]cinnamic acid, and maize was shown to have the ¹⁴C-incorporation of into polymer-esterified groups. More [¹⁴C]diferulate formed in the cell wall of older cultures than in young cultures, where intraprotoplasmic coupling was prevalent. I was also able to detect a putative [¹⁴C]hydroxycinnamoyl-CoA, a donor molecule for feruloylation, in 2-d-old maize cell-suspension cultures up to 8 min after [¹⁴C]cinnamic acid feeding. Iodide diminished dimerisation taking both intraprotoplasmically and within the cell wall. The effect of KI or NaI could be reversed and dimerisation increased with its removal and addition of H₂O_2.¹⁴C-Feruloylated polysaccharides secreted in the presence of NaI or KI passed through the cell wall directly into the culture medium, where they accumulated as soluble extracellular polysaccharides. Inhibition of diferulate formation may prevent the integration of newly synthesised arabinoxylans into the cell wall as diferulate cross-links cannot form to integrate the polysaccharides into the wall, suggesting that an intermolecular diferuloyl linkage may act as an anchorage point. Feeding cells with [U-¹³C]glucose in the presence of NaI or KI for 24 h allows the formation of a ¹²C/¹³C interface within the cell wall. Removal of the inhibitor and addition of H₂O₂ would allow the formation of diferulate groups across the ¹²C/¹³ interface, if intermolecular dimerisation is possible <i>in vivo.</i> I have developed a method of extraction and purification of fragments of the isotopically labelled cell wall, namely feruloyl-arabinose and diferulic acids, which allow the existence of an intermolecular diferuloyl linkage to be tested. To monitor the incorporation of carbon from exogenous glucose, I fed [U-¹⁴C]glucose to cell-suspension cultures. Autoradiography and scintillation counting of the isolated cell wall fragments have shown the incorporation of radioactivity into the groups of interest, namely feruloyl-arabinose and diferulate, confirming that glucose was a suitable compound in which to deliver the ¹³C. Preliminary results from this method with both [¹⁴C]glucose and [¹³C]glucose gave some evidence, such as the secretion of ^l4C-feruloylated polysaccharides in the presence of an H₂O₂scavenger and the detection of cell wall fragments into which ¹³Chad been incorporated, for an intermolecular diferuloyl linkage to exist and that my approach is a suitable method to detect such linkages.

The leaves of <i>Antirrhinum </i>provide a good model system to study the mechanisms that specify formation of axes during plant development because mutation of the <i>PHANTASTICA </i>gene (<i>PHAN</i>) causes more complete disruption of these features than does mutation of its homologues in <i>Arabidopsis </i>or maize. <i>PHAN</i> encodes a Myb homologue and is expressed in the whole of the developing lateral organs yet the mutant phenotype suggests that it has functions restricted to the dorsal domain as well as in meristem maintenance. Mutants of <i>PHAN </i>in <i>Antirrhinum, Arabidopsis</i> and maize all missexpress <i>knox</i> genes so a related function of <i>PHAN </i>homologues is repression of meristem genes in lateral organs. The yeast 2-hybrid assay was used here to isolate PULP a novel potential modifier of PHAN protein function which, based on expression pattern in plant tissue has potential to interact with PHAN in vivo. The yeast 2-hybrid assay suggested that the interaction between PHAN and PULP is conserved because <i>Arabidopsis</i> PHAN and PULP homologues, AS1 and T20403, interact in the same way. Mutagenesis indicated that the interaction is biologically relevant because a putative T20403 mutant enhanced the <i>as1</i>-<i>1</i> phenotype causing it to resemble that of <i>phan</i>. PULP expression pattern suggested that it may function in collaboration with PHAN in lateral organs but independently of PHAN in stem tissues to repress <i>knox</i> genes. The yeast 2-hybrid assay also revealed potential homotypic and heterotypic interaction between PHAN, AS1 and PHANL1 which could provide a mechanism for PHAN and AS1 repression of <i>knox</i> expression, but taken together with the function of <i>knox</i> gene and Myb homologues in animals and the nature of the genetic pathways important to regulation of growth of the tissue where they are expressed, a degree of functional homology between PHAN and cMyb or aMyb in animals is also suggested.