<p>Oligosaccharides (carbohydrates) are a large class of biological molecules that are important as energy sources in the human body and have enormously varied biological functions. It is generally believed that biological activities of carbohydrates are related to their internal dynamics. The dynamic properties of some oligosaccharides in solution are studied in this thesis, by NMR relaxation. We have employed relaxation interference effects to investigate the conformational dynamics within oligosaccharides (in-tramolecular dynamics) and paramagnetic relaxation enhancement (PRE) as an experimental tool to study intermolecular dynamics. Most of the thesis concerns the dynamics of the methylene group in the two possibly mobile parts of the oligosaccharide: in the exocyclic hydroxymethyl moiety and in the glycosidic linkage position. To perform conformational dynamic studies, the more traditional auto-relaxation pa-rameters are combined with the relaxation interference terms or the cross-correlated relaxation rates (CCRRs). Some experimental schemes based on the initial-rate technique were developed for measuring CCRRs. The techniques are useful for labelled sugars as well as naturally abundant ones. Furthermore, various dynamical models ranging from the Lipari–Szabo approach to several more informative and complicated models such as the two-site jump model, restricted internal rotation and slowly relaxing local structure (SRLS), have been employed to interpret our experimental data. We have combined and com-pared different models; we have also developed a novel approach to existing models, by scaling dipolar coupling constants (DCC), to extract the dynamic behaviour and structural properties of the system. We found that the auto- and cross-correlated relaxation data analyses yield a consistent picture of the dynam-ics in all cases. Additionally, our investigations show that CCRRs are practically important for verifica-tion of certain dynamical and structural information that is difficult to be determined by other means. Moreover, the anisotropy of the carbon-13 chemical shielding tensor in the methylene group has been estimated, using the interference between dipole-dipole and chemical shift anisotropy.</p><p>This thesis also discusses using the PRE to investigate sugar dynamics relative to a paramagnetic MRI contrast agent in solution, which might be important in medicine. We have studied the intramolecu-lar dynamics of the trisaccharide raffinose in the presence of a gadolinium complex. We also investigated the effect of translational diffusion instead of rotational diffusion, which is normally more important in NMR. The paramagnetically enhanced spin–lattice relaxation rates of aqueous protons over a wide range of magnetic fields and of carbon-13 and protons of the sugar at high fields have been measured. The nuclear magnetic relaxation dispersion of water protons and the PREs of proton and carbon in the sugar are interpreted in terms of the model recently developed in our laboratory, allowing both outer- and inner-sphere PREs for water protons, but allowing only the outer sphere PRE for nuclei in the sugar. We found that the relative diffusion has a stronger effect on the PRE than the electron spin relaxation.</p>

Oligosaccharides (carbohydrates) are a large class of biological molecules that are important as energy sources in the human body and have enormously varied biological functions. It is generally believed that biological activities of carbohydrates are related to their internal dynamics. The dynamic properties of some oligosaccharides in solution are studied in this thesis, by NMR relaxation. We have employed relaxation interference effects to investigate the conformational dynamics within oligosaccharides (in-tramolecular dynamics) and paramagnetic relaxation enhancement (PRE) as an experimental tool to study intermolecular dynamics. Most of the thesis concerns the dynamics of the methylene group in the two possibly mobile parts of the oligosaccharide: in the exocyclic hydroxymethyl moiety and in the glycosidic linkage position. To perform conformational dynamic studies, the more traditional auto-relaxation pa-rameters are combined with the relaxation interference terms or the cross-correlated relaxation rates (CCRRs). Some experimental schemes based on the initial-rate technique were developed for measuring CCRRs. The techniques are useful for labelled sugars as well as naturally abundant ones. Furthermore, various dynamical models ranging from the Lipari–Szabo approach to several more informative and complicated models such as the two-site jump model, restricted internal rotation and slowly relaxing local structure (SRLS), have been employed to interpret our experimental data. We have combined and com-pared different models; we have also developed a novel approach to existing models, by scaling dipolar coupling constants (DCC), to extract the dynamic behaviour and structural properties of the system. We found that the auto- and cross-correlated relaxation data analyses yield a consistent picture of the dynam-ics in all cases. Additionally, our investigations show that CCRRs are practically important for verifica-tion of certain dynamical and structural information that is difficult to be determined by other means. Moreover, the anisotropy of the carbon-13 chemical shielding tensor in the methylene group has been estimated, using the interference between dipole-dipole and chemical shift anisotropy. This thesis also discusses using the PRE to investigate sugar dynamics relative to a paramagnetic MRI contrast agent in solution, which might be important in medicine. We have studied the intramolecu-lar dynamics of the trisaccharide raffinose in the presence of a gadolinium complex. We also investigated the effect of translational diffusion instead of rotational diffusion, which is normally more important in NMR. The paramagnetically enhanced spin–lattice relaxation rates of aqueous protons over a wide range of magnetic fields and of carbon-13 and protons of the sugar at high fields have been measured. The nuclear magnetic relaxation dispersion of water protons and the PREs of proton and carbon in the sugar are interpreted in terms of the model recently developed in our laboratory, allowing both outer- and inner-sphere PREs for water protons, but allowing only the outer sphere PRE for nuclei in the sugar. We found that the relative diffusion has a stronger effect on the PRE than the electron spin relaxation.

Phytophthora infestans (Mont) de Bary is a devastating pathogen in potato producing regions around the world. Populations of the organism in Canada shifted during the mid-1990’s as the US-1 strain (A1, metalaxyl-sensitive) was displaced by the highly aggressive, US-8 strain (A2, metalaxyl-insensitive). An increase in the incidence and severity of late blight has followed. Late blight is controlled by cultural practices aimed at eliminating disease sources and by the application of foliar fungicides. Tubers can become infected at harvest from contact with blighted vines leading to severe losses in storage. In many production areas, growers desiccate vines two to three weeks prior to harvest to reduce late blight tuber rot. However, in Manitoba, because of the loss of potential yield that results from vine killing prior to harvest in a late maturing cultivar such as Russet Burbank, growers are reluctant to adopt this practice. The objective of this study was to develop recommendations for preharvest management practices that reduce storage losses due to late blight. Field trials were conducted in 1997 and 1998 to investigate the effect of vine desiccation with diquat and/or a late season application of chlorothalonil and copper hydroxide on yield, processing quality, and disease development in storage. Desiccating vines with diquat two weeks prior to harvest reduced yield and tuber size. Compared to the untreated control, the largest reductions in marketable yield were observed for the early September harvest. By the late September harvest, however, the effect of vine killing in reducing marketable yield was less apparent. Specific gravity was lower in the vine killed treatment for all harvest dates in 1997 and in the early and mid September harvests in 1998. Vine killing did not contribute to elevated levels of reducing sugars or consistently darker fry colour at harvest or during storage. Skin-set was improved when vines were desiccated for all harvest dates in 1997 and at the early September harvest date in 1998. Vine killing reduced tuber rot in storage caused by Fusarium dry rot and Pythium leak for the early and mid September harvest dates in 1997. The incidence of late blight tuber rot was reduced in storage for the early September harvest in 1998 when vines were desiccated. The late-season application of chlorothalonil and copper hydroxide did not reduce tuber rot in storage in storage either year. Results from this study indicate that vine killing two weeks before an early to mid- September harvest is not recommended in Manitoba because of reductions in yield and specific gravity. Alternative management practices to reduce late blight tuber rot in storage should be investigated. / February 2005

The work embodied in this thesis pertains to an attempt to understand better, the molecular basis of protein-carbohydrate recognition. For this purpose a systematic study was undertaken, not only of the energetics of lectin-sugar interactions, which serve as molecular recognition prototype of protein-carbohydrate interactions, but also of the complex effects of solvent water molecules surrounding both the species in solution state. The systems chosen for investigation include the specific recognition of sugars by lectins from diverse families, leguminosae and moraceae. The following salient aspects of the molecular recognition process constitute the focus of this thesis: • Effect of site specifically modified, deoxy-, fluorodeoxy-, or methoxy- substituted D-galactopyranoside binding to lectins. Isothermal titration calorimetric (ITC) investigations of the binding of these sugars to a model lectin permitted the correct prediction of the architecture of the primary binding site in the absence of x-ray crystal or NMR structure of the combining site (Ref. 7). The study provided the only unambiguous means of a site specific mapping of the hydrogen-bond donor- acceptor relationship of the monosaccharide within the primary combining site of the lectin. • Novel features of lectin-sugar recognition. Molecular interactions and forces contributing to the stabilization of the saccharides in the primary combining site of lectins. Binding of site specifically modified fluoro- substituted D- galactopyranosides to WBA I led to the demonstration of the involvement of C- F««»H-0 hydrogen bonds in stabilizing the saccharide within the combining site of lectin (Ref. 7). Implication of the novel C-H«**O hydrogen bonds at the specificity determining C-4 position in enabling the methoxy- substituted D- galactopyranoside to be stabilized within the primary binding site of galactose specific lectins WBA I and jacalin. • Development of a novel coupled osmotic-thermodynamic approach for investigating the role of water molecules in determining the specificity of lectin- sugar interactions. The results obtained led to the first direct demonstration of a differential uptake of water molecules accompanying the specific process of recognition of sugars by lectins (Ref 2) • On the origin of enthalpy-entropy compensation, a ubiquitous phenomenon accompanying the thermodynamics of several ligand binding reactions in aqueous solutions in general and the molecular recognition involving all known lectin-sugar interactions, in particular. The results provide the first unequivocal solution state proof of water reorganization as the source of enthalpy-entropy compensation (Ref 3). A new diagnostic test of a true osmotic effect in molecular recognition phenomena was proposed (Ref. 2) and validated (Ref. 3). As an introduction, Chapter 1 is a comprehensive review of literature that touches upon the diverse properties of lectins and our present understanding of their multifarious roles and applications, which has led to their christening, perhaps appropriately, as molecules that mediate the 'social' functions of cells and tissues. Although a challenge it is still, to decipher the "glycocode", it is apparent that the fundamental basis of the recognition function of lectin-sugar interactions is the initial specific binding of the saccharide molecule by the globular proteinaceous lectin molecule. It is imperative, therefore, that an incisive investigation of the origin of specificity of the binding reaction as well as the solvent effects influencing both the interacting species be undertaken for a better understanding of the complete molecular recognition process. Towards this end is introduced in Chapter 1 our present understanding of the results on lectin-sugar interactions from two complementary approaches viz structural, including X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, as well as thermodvnamic ones, which have provided important information on the architecture of the combining sites, the dynamic modes of saccharide recognition and forces involved therein. Despite a detailed knowledge available from such methods, a structure-energetics correlation has persisted as a current challenge of the field. Towards achieving this goal, studies on the energetics of the recognition of sugars by lectins were undertaken, with an aim to better understand the origin of specificity of lectin-sugar interactions. This thesis attempts to provide new insights on some of the possible lacunae precluding structure-energetics correlation and suggests ways to overcome them. Chapter 2 deals with ITC investigation of the effect of deoxy-, fluorodeoxy-, and methoxy- substitutions on the binding of monosaccharides to the primary combining site of the lectin WBA I isolated from the mature seeds of the leguminosae family member Psophocarpus tetragonolobus as well as the moraceae lectin jacalin. These studies provide valuable information on the hydrogen-bond donor-acceptor relationships within the combining site of the lectins wherein the sugar molecule is liganded with the amino-acid residues of the lectin. This study is relevant for understanding the origin of specificity of monosaccharide binding within the primary combining site of the lectins. It has recently become apparent that there is a predisposition in three-dimensional space, of the donor-acceptor pairs within the sugar binding site of the lectins. Hence there appears to be a stereochemical basis of distinguishing the recognition of the donor group vis-a-vis that of the acceptor group and that their spatial disposition determines the specificity of the saccharide recognition. Unambiguous assignment of which of the groups within the hydrogen bonded pairs is a donor and which one is the acceptor assumes greater importance. The ITC measurements of the binding of deoxy-, flurodeoxy-and methoxy-derivatives of D-galactopyranoside (oc-D-Gal) to the basic lectin from winged bean Psophocarpus tetragonolobus, WBA I revealed that each of the ligands bind to WBA I with the same stoichiometry of one per subunit (29 kDa) of WBA I. The binding enthalpies for various derivatives were essentially independent of temperature and showed complementary changes with respect to binding entropies. Replacement of the hydroxyl group by fluorine or hydrogen on C3 and C4 of the galactopyranoside eliminated their binding to the lectin, consistent with C3-OH and C4-OH acting as hydrogen bond donors. The affinity for C2 derivatives of galactose decreased in the order: GalNAc>2MeOGal>2FGal=Gal>2HGal which suggests that both polar and non-polar residues surround the C2 locus of galactose, consistent with the observed high affinity of WBA I towards GalNAc, where the acetamido group at C2 position is probably stabilized by both non-polar interactions with the methyl-group and polar interactions with the carbonyl group. The binding of C6 derivatives followed the order: Gal>6FGal>D-Fuc»6MeOGal=L-Ara indicating the presence of favourable polar interactions with a hydrogen bond donor in the vicinity. Based on these results the hydrogen bond donor-acceptor relationship of the complexation of methyl-a-D-galactopyranoside with the primary combining site of WBA I was proposed (Ref. /), which was subsequently validated by the crystal structure of methyl-a-D-galactopyranoside complexed with WBA I. This chapter also describes the results from ITC studies on the binding of monosaccharides and disaccharides to the lectin jacalin isolated from the mature seeds of the moraceae family member Artocarpus integrifolia. The novel observation about the existence of C-F*«*H-0 and C-H**»O hydrogen bonds in lectin-sugar interactions is also discussed in this chapter. Chapter 3 is a description of the detailed investigation on the role of water molecules in influencing the energetics of lectin-sugar recognition. A novel coupled osmotic-thermodynamic approach was developed to dissect the role of water molecules in determining the recognition of the sugars by lectins. For this purpose, the model system of mannotriose-concanavalin A was used because atomic level structural information on these complexes were available. The work described in this chapter, is the first solution state evidence for the role of water molecules in the specific interaction of carbohydrates with a legume lectin, concanavalin A (Con A) (Ref. 2). Sugar binding to Con A was accompanied by linear changes in the logarithm of binding constants as a function of neutral osmolyte strength, and were described by well defined negative slopes characteristic for each sugar. As these changes were independent of the chemical nature of the osmolyte used, the results were rationalized in terms of a true osmotic effect. It was demonstrated that the specific recognition of the branched trimannoside (3,6-di-0-(a-D-mannopyranosyl)~a-D-mannopyranoside), the individual dimannosidic arms (3-<9-(a-D-mannopyranosyl)-a-D-mannopyranoside, and 6-0-(a-D-marmopyranosyl)-a-D-mannopyranoside) and the monomeric unit D-mannopyranoside by Con A was accompanied by differential uptake of water molecules; 1,3 and 5 respectively. We also observed a conservation of the compensatory behaviour of binding enthalpies and entropies in the presence as well as absence of osmolytes. This provided the first definitive evidence that water-reorganization plays a direct role in effecting the phenomenon of enthalpy-entropy compensation in protein-ligand interactions in general and lectin-sugar interactions in particular, and that the specificity of lectin-sugar recognition is characterized by a differential uptake of water molecules. Chapter 3 also describes the first experimental identification of the origin of enthalpy-entropy compensation (EEC), a ubiquitous phenomenon accompanying the thermodynamics of multifarious biomolecular recognition processes. By coupling direct microcalorimetry with osmotic stress technique, an experimental handle was devised to test the hypothesis that solvent reorganization could be the source of EEC. The results provided an unequivocal demonstration that an osmotic change in water activity alone, at the same temperature and pH, is sufficient to result in the conservation of EEC during the molecular recognition of specific ligands by macromolecules belonging to thermodynamically diverse and unrelated systems, a compelling evidence that the primary source of EEC in aqueous solutions is attributable to reorganization of solvent water molecules, thus validating the test for the role of water reorganization as a source of EEC (Ref. 3). This provides the first definitive evidence for the notion that there is a direct involvement of water molecules in originating the EEC effect. Despite the generality of the results it is urged that several systems be subjected to a vigorous application of the coupled osmotic-thermodynamic approach proposed herein before constituting it as a proof. Suffice to say, it is perhaps heartening that at last one has a handle to test the role of water molecules in effecting EEC in the solution state and appreciate the diverse roles played by water molecules in mediating molecular recognition reactions. The proposal presented in Ref 2, that the strong isoequilibrium relationship of enthalpy with entropy during the recognition of saccharides by Con A studied under osmotic stress, be considered as diagnostic of a true osmotic effect was subsequently validated in a thermodynamically diverse and unrelated system of peptide recognition by monoclonal antibody, the results from which are discussed in an Appendix (A) to this thesis (Ref 4). That the stabilities of these lectins are not hampered in the presence of osmolytes was demonstrated using differential scanning calorimetry (DSC) (Ref 2). During the course of these DSC studies, we discovered an unusual feature in an animal galectin. Despite possessing the legume lectin fold, the 14-kDa S- type lectin exhibits multiple oligomeric states that are influenced profoundly by complementary ligands and surprisingly do not dissociate at the denaturation temperature. These results are discussed in an Appendix (B) to this thesis (Ref. 5). The general discussion and conclusions drawn from this work are summarized in chapter 4. Briefly, the following salient conclusions can be drawn from the work presented in this thesis: 1. Unambiguous assignment of hydrogen-bond donor-acceptor relationship at each of the hydroxyl group of the monosaccharide bound to the lectin belonging to different families has been demonstrated (Refs. 1,6). 2. First report of novel hydrogen bonds in lectin-sugar interactions such as C- F«MH-0 (Ref 1) and C-H^*O hydrogen bonds (Ref 6). 3. Unusual structural stabilities in a galectin with a fold similar to that in legume lectins but with starkly different thermodynamic stabilities (Ref 5). 4. We have demonstrated for the first time in solution state, that water molecules are involved in the specific recognition of sugars by concanavalin A (Ref 2). It appears that lectin-sugar recognition reactions are, in general, mediated by a net uptake of water molecules during the binding process (Ref 7). 5. We have provided the first experimental demonstration that reorganization of water molecules is the source of enthalpy-entropy compensation in molecular recognition processes (Ref 3). 6. We provide evidence for another facet in the recognition of antigens by antibodies, viz water release accompanying the binding reaction (Ref 4). The studies reported in this thesis provide the foundation for embarking on a systematic study not only of the origin of specificity of lectin-sugar recognition but also of the complex roles that water molecules play in mediating these molecular recognition processes. These specific binding reactions wherein non-linear thermodynamics predominates and precludes a direct structure-energetics correlation emphasize the need to account for the effect of solvent water molecules in lectin-sugar interactions in particular and, without any overemphasis, in molecular recognition processes in general.

Aggregatibacter actinomycetemcomitans is a Gram negative bacterium found exclusively in the mammalian oral cavity where it resides in the gingival crevice, the space between the tooth and gum tissue. Though it has historically been considered a common commensal organism, it is now appreciated that A. actinomycetemcomitans is an opportunistic pathogen associated with the diseases periodontitis and endocarditis. To cause infection, A. actinomycetemcomitans must interact and compete with neighboring bacteria for space and nutrients, though little is known about the physiology it employs within the gingival crevice. Using A. actinomycetemcomitans grown in a chemically defined medium containing carbon sources found in vivo, I use transcriptome analyses and growth studies to show that A. actinomycetemcomitans preferentially utilizes lactate over the phosphotransferase system (PTS) sugars glucose and fructose. Additionally, the presence of lactate or pyruvate inhibits the transport and metabolism of these sugars in a post-transcriptionally controlled process I have termed PTS substrate exclusion. Since lactate is an energetically inferior carbon source, PTS substrate exclusion appears to be a carbon resource partitioning mechanism that allows A. actinomycetemcomitans to avoid competition for energetically favorable sugars with other species, and I propose a model to describe this phenomenon. To begin to understand the mechanism of PTS substrate exclusion, I examine the first step of the proposed model by purifying and characterizing the L-lactate dehydrogenase (LctD) from A. actinomycetemcomitans. I demonstrate that, unlike other studied lactate dehydrogenases, the LctD from A. actinomycetemcomitans does not exhibit feedback inhibition in the presence of physiologically relevant concentrations of pyruvate, which supports my hypothesis that elevated intracellular pyruvate levels inhibit the PTS. The results of my studies provide insight into a new regulatory mechanism governing carbon utilization in this bacterium. / text

Our understanding of bird pollination systems has changed dramatically in the last few years. A long-standing paradigm was that hummingbirds and passerine birds select for different nectar properties in flowers (phylogenetic hypothesis). However, specialist passerines, such as sunbirds, have similar nectar preferences to hummingbirds and nectar in plants pollinated by these two bird groups is strongly convergent. Thus, as an alternative to the existing paradigm, it has been argued that the most useful distinction that can be drawn is that between specialist and generalist avian nectarivores (feeding niche hypothesis). This was supported by phylogenetically-controlled analyses that show that nectar in plants pollinated by specialist avian nectarivores (whether hummingbird or passerine) tends to have a lower volume, higher concentration and higher sucrose content than that in plants pollinated by generalist avian nectarivores. The aim of this thesis was to determine if these trends can be explained by the nectar preferences of avian nectarivores, and to determine whether the generalist-specialist dichotomy can be applied to Kniphofia, a largely bird-pollinated African plant genus. This thesis consists of two sections. In the first, I present data from equicaloric choice experiments to determine sugar preferences of both specialist and occasional nectar feeding birds across a range of concentrations. In addition, I determine apparent sugar assimilation efficiencies and concentration preferences for a range of occasional nectar feeding birds. Using Malachite Sunbirds (Nectarinia famosa), Dark-capped bulbuls (Pycnonotus tricolor), Speckled Mousebirds (Colius striatus) and Red-winged Starlings (Onychognathus morio) (plus Village Weavers (Ploceus cucullatus) in an appendix) as representatives of the major groups of nectar feeding birds in South Africa, I show that some differences occur between occasional nectar feeding birds and all specialist nectar feeding birds studied so far. Specialists show a switch from hexose preference at low (5%) concentrations, exhibit no preference at medium concentrations (10-20%) and prefer sucrose or show no preference at high concentrations (25%). However, specialists are unable to maintain energy balance at low concentrations, and always select the higher concentration when given choices within this range. Occasional nectar feeding birds prefer hexose solutions at low and medium concentrations (5 and 10%), but show varied preferences at higher concentrations. Occasional nectar feeding birds are able to maintain energy balance at low concentrations, and either show no concentration preference, or a preference for lower concentrations when given a choice. Occasional nectar feeding birds show a range in ability to digest sucrose, although some species, like the Dark-capped Bulbul, are quite proficient at it. Surprisingly, no significant difference is found between specialist and occasional nectarivores sugar preferences when analysed globally, even when phyllogeny is accounted for. Instead, I found a significant relationship between body size and bird sugar preference at different concentrations, from which I build the body size hypothesis, which I suggest is a better predictor to use than bird diet type (specialist or occasional nectarivores – feeding niche hypothesis). In the second section of the thesis, I examine the associations between plant traits and nectarivore nectar preferences. This section focuses on flower morphology and nectar characteristics in Kniphofia species, and ecotypes within species, pollinated by specialist versus occasional avian nectarivores. I show that apart from sugar type, which appears to be phylogenetically constrained, flower morphology and nectar characteristics appear to diverge according to whether plants are pollinated by generalist or specialist nectar feeding birds. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Phytophthora infestans (Mont) de Bary is a devastating pathogen in potato producing regions around the world. Populations of the organism in Canada shifted during the mid-1990’s as the US-1 strain (A1, metalaxyl-sensitive) was displaced by the highly aggressive, US-8 strain (A2, metalaxyl-insensitive). An increase in the incidence and severity of late blight has followed. Late blight is controlled by cultural practices aimed at eliminating disease sources and by the application of foliar fungicides. Tubers can become infected at harvest from contact with blighted vines leading to severe losses in storage. In many production areas, growers desiccate vines two to three weeks prior to harvest to reduce late blight tuber rot. However, in Manitoba, because of the loss of potential yield that results from vine killing prior to harvest in a late maturing cultivar such as Russet Burbank, growers are reluctant to adopt this practice. The objective of this study was to develop recommendations for preharvest management practices that reduce storage losses due to late blight. Field trials were conducted in 1997 and 1998 to investigate the effect of vine desiccation with diquat and/or a late season application of chlorothalonil and copper hydroxide on yield, processing quality, and disease development in storage. Desiccating vines with diquat two weeks prior to harvest reduced yield and tuber size. Compared to the untreated control, the largest reductions in marketable yield were observed for the early September harvest. By the late September harvest, however, the effect of vine killing in reducing marketable yield was less apparent. Specific gravity was lower in the vine killed treatment for all harvest dates in 1997 and in the early and mid September harvests in 1998. Vine killing did not contribute to elevated levels of reducing sugars or consistently darker fry colour at harvest or during storage. Skin-set was improved when vines were desiccated for all harvest dates in 1997 and at the early September harvest date in 1998. Vine killing reduced tuber rot in storage caused by Fusarium dry rot and Pythium leak for the early and mid September harvest dates in 1997. The incidence of late blight tuber rot was reduced in storage for the early September harvest in 1998 when vines were desiccated. The late-season application of chlorothalonil and copper hydroxide did not reduce tuber rot in storage in storage either year. Results from this study indicate that vine killing two weeks before an early to mid- September harvest is not recommended in Manitoba because of reductions in yield and specific gravity. Alternative management practices to reduce late blight tuber rot in storage should be investigated.

Phytophthora infestans (Mont) de Bary is a devastating pathogen in potato producing regions around the world. Populations of the organism in Canada shifted during the mid-1990’s as the US-1 strain (A1, metalaxyl-sensitive) was displaced by the highly aggressive, US-8 strain (A2, metalaxyl-insensitive). An increase in the incidence and severity of late blight has followed. Late blight is controlled by cultural practices aimed at eliminating disease sources and by the application of foliar fungicides. Tubers can become infected at harvest from contact with blighted vines leading to severe losses in storage. In many production areas, growers desiccate vines two to three weeks prior to harvest to reduce late blight tuber rot. However, in Manitoba, because of the loss of potential yield that results from vine killing prior to harvest in a late maturing cultivar such as Russet Burbank, growers are reluctant to adopt this practice. The objective of this study was to develop recommendations for preharvest management practices that reduce storage losses due to late blight. Field trials were conducted in 1997 and 1998 to investigate the effect of vine desiccation with diquat and/or a late season application of chlorothalonil and copper hydroxide on yield, processing quality, and disease development in storage. Desiccating vines with diquat two weeks prior to harvest reduced yield and tuber size. Compared to the untreated control, the largest reductions in marketable yield were observed for the early September harvest. By the late September harvest, however, the effect of vine killing in reducing marketable yield was less apparent. Specific gravity was lower in the vine killed treatment for all harvest dates in 1997 and in the early and mid September harvests in 1998. Vine killing did not contribute to elevated levels of reducing sugars or consistently darker fry colour at harvest or during storage. Skin-set was improved when vines were desiccated for all harvest dates in 1997 and at the early September harvest date in 1998. Vine killing reduced tuber rot in storage caused by Fusarium dry rot and Pythium leak for the early and mid September harvest dates in 1997. The incidence of late blight tuber rot was reduced in storage for the early September harvest in 1998 when vines were desiccated. The late-season application of chlorothalonil and copper hydroxide did not reduce tuber rot in storage in storage either year. Results from this study indicate that vine killing two weeks before an early to mid- September harvest is not recommended in Manitoba because of reductions in yield and specific gravity. Alternative management practices to reduce late blight tuber rot in storage should be investigated.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2010. / Includes bibliography. / ENGLISH ABSTRACT: The study of plants on a sub-cellular level is an important, yet challenging area and its application allows for novel insight into the understanding of metabolism and its regulation. In this study I describe the development of a reverse phase liquid chromatography mass spectrometry (RPLC-MS) technique in which 29 phosphorylated and nucleotide sugars could be detected and quantified. The method was validated with the use of authentic standards and the system displayed very good linearity (Rª > 0.95), while the recovery of the standards added to the plant material before extraction was between 65 and 125%. Further, Arabidopsis thaliana wild type (Col-0) and adenylate kinase (adk1) mutant leaf discs were fed 13C labeled glucose over a period of 24 hours and harvested at defined time intervals. Non aqueous fractionation, and metabolite profiling via the above mentioned rpLC-MS method in conjunction with gas chromatography mass spectrometry (GC-MS) allowed for the detection and quantification of primary metabolites on a sub-cellular level as well as the determination of their relative isotopic label enrichments through primary carbon metabolism. Finally, a yeast complementation system was designed for the identification of tonoplast bound sucrose import proteins. The screening system identified 22 unique sequences from an Arabidopsis thaliana cDNA library. Four unknown sequences were identified, one of which displayed tonoplast membrane association upon in silico analysis. Three ATP-binding proteins were also identified as well as a sub-unit from the exocyst gene family. Further studies will include the functional characterization of the latter, as well as the development of additional cDNA libraries more suited for screening of sequences that encode sucrose importer proteins. / AFRIKAANSE OPSOMMING: Die studie van plante op a sub-sellulere vlak is ‘n belangrike maar uitdagende navorsingsarea en die toepassing daarvan dra by tot unieke insig tot ‘n beter begrip van metabolise regulasie. In die studie bespreek ek die ontwikkeling van ‘n teenoorgestelde fase vloeistof kromatografie massa spektrometrie (RPLC-MS) tegniek waarin 29 gefosforileerde en nukleotied suikers gevind en gekwantifiseer kon word. Geldigverklaring van die metode is bewerkstelling met die gebruik van oorspronklike standaarde and die systeem het baie goeie liniariteit (Rª > 0.95) getoon, terwyl die herstelbaarheid van standaarde wat bygevoeg is by die plant material voor ekstraksie tussen 65% en 125% was. Arabidopsis thaliana wilde type (Col-O) en die adenaliet kinase (adk1) mutant blaar dele is met 13C gemerkte glukose gevoed oor ‘n tydperk van 24 uur en geoes by spesifieke tydstippe. Nie-vloeibare fraksionering en metaboliet uitleg is vermag vanaf die genoemde RPLC-MS metode met behulp van gas kromotografie massa spektrometrie (GC-MS) wat die bepaling en kwantifikasie van primere metaboliete op n sub-sellulere vlak sowel as die bepaling van hul relatiewe isotropiese merker verrykers deur primere metabolisme toelaat. Verder is n gis komplementere systeem ontwerp vir die identifikasie van tonoplas gebinde sukrose invoer proteine. Die verkenningsysteem het 22 unieke volgordes opgelewer vanaf ‘n Arabidopsis thaliana kDNA biblioteek. Vier onbekende volgordes is geidentifiseer, een wat tonoplas membraan assosiasie toon met in silico analise. Drie ATP-bindings proteine is ook geidentifiseer asook ‘n sub-eenheid van die eksosyst geen familie. Verdere studies sal die funksionele karakterisering van die laaste protein insluit, asook die ontwikkeling van additionele kDNA biblioteke meer gepas vir verkenning sodiende identifiseer van volgordes wat sukrose invoer proteine vertaal.