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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Computational systems biology of sucrose accumulation in sugarcane

Uys, Lafras 03 1900 (has links)
Thesis (MSc (Biochemistry))--University of Stellenbosch, 2006. / This thesis is about mathematical modelling of sucrose accumulation in the storage perenchyma of Saccharum officinarum (sugarcane) In 2001, Rohwer & Botha (76) published a kinetic model that described the defining feature of this process ...
2

Gasification characteristics of sugarcane bagasse

Anukam, Anthony Ike January 2013 (has links)
Sugarcane is a major crop in many countries. It is the most abundant lignocellulosic material in tropical countries such as South Africa. It is one of the plants with the highest bioconversion efficiency. The sugarcane crop is able to efficiently fix solar energy, yielding some 55 tons of dry matter per hectare of land annually. After harvest, the crop produces sugar juice and bagasse. Sugarcane bagasse is a residue that results from the crushing of sugarcane in the sugar industry. It is a renewable feedstock that can be used for power generation and manufacturing cellulosic ethanol. As biomass, sugarcane bagasse holds promise as a fuel source since it can produce more than enough electricity and heat energy to supply the needs of a common sugar factory. However, in the sugarcane industry the bagasse is currently burnt inefficiently in boilers that provide the heating for the industry. This project seeks to investigate the possibility of gasifying sugarcane bagasse as an efficient conversion technology. The investigation is necessary because fuel properties govern the gasifier design and ultimately, the gasification efficiency. Proximate and ultimate analysis of sugarcane bagasse was conducted after which the results were used to conduct a computer simulation of the mass and energy balance during gasification. The kinetic investigation undertaken through the TGA and DTG analyses revealed the activation energy and pre – exponential factor which were obtained by the model – free Kissinger method of kinetic analysis and were found to be 181.51 kJ/mol and 3.1 × 103/min respectively. The heating value of sugarcane bagasse was also measured and found to be 17.8 MJ/kg, which was used in the calculation of the conversion efficiency of the gasification process. Fuel properties, including moisture content and gasifier operating parameters were varied in order to determine optimum gasifier operating conditions that results in maximum conversion efficiency. The highest conversion efficiency was achieved at low moisture content after computer simulation of the gasification process. Moisture content also affected the volume of CO and H2 as the former decreases with increasing moisture content while the latter increases with increasing moisture content, accelerating the water – gas reaction. Scanning electron microscope fitted to an Energy dispersive X – ray spectroscopy was also used in order to view the shape and size distribution as well as determine the elemental composition of sugarcane bagasse. The results obtained established that the fuel properties and gasification conditions affect the conversion efficiency. During computer simulation, it was established that smaller particle size resulted in higher conversion efficiency. The smaller throat diameter also resulted in higher conversion efficiency. The throat angle of 25° also resulted in higher conversion efficiency. The temperature of input air was also found to be one of the major determining factors in terms of conversion efficiency. The dissertation presents the proximate and ultimate analysis results as well as the kinetic analysis results. The SEM/EDX analysis as well as the computer simulation results of the gasification process is also presented. The major contribution of this project was on the investigation of the gasification characteristics of sugarcane bagasse and the utilization of these in the design of a laboratory scale sugarcane bagasse gasifier with enhanced conversion efficiency through computer simulation.
3

Carbon partitioning in sugarcane internodal tissue with special reference to the insoluble fraction

Bindon, Keren (Keren Ann) 12 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: The changes in carbon allocation to sucrose, hexoses, fibre, starch and respiration were investigated in developing internodes of sugarcane. Radiolabelling studies were conducted on internode 3, 6 and 9 tissue, representing three stages of increasing maturity. It was apparent that a high rate of cycling between triose-phosphate and hexose-phosphate occurred. A maximum of 50% of carbon entering triose-phosphates was returned to hexose-phosphate in internode 3 tissue, and this flux decreased with tissue maturity to 30%. Carbon partitioning into sucrose increased from 34% of total 14C uptake in internode 3, to 61% in internodes 6 and 9. In immature tissue, the protein and fibre components were the dominant competing sinks with sucrose for incoming carbon, to which 14 and 16% of carbon were allocated respectively. Increased carbon allocation to sucrose with tissue maturity, coincided with a decrease in partitioning to fibre. This indicated that previous studies had underestimated total carbon allocation to respiration, since the protein component was not considered. In contrast with earlier work, the respiratory pathway was the strongest competitor with sucrose for incoming carbon, even in mature tissue. Between internodes 3 and 6, carbon allocation to total respiration did not change significantly, but decreased 50% in mature tissue. Starch was a weak competitor with sucrose, for incoming carbon, to which a maximum of 2% of 14Cwas allocated in immature tissue. In cane harvested in early spring, radiolabelled maltose was recovered in internode 3 tissue of ripening cane, indicating that concomitant starch synthesis and degradation occurred. The. redistribution of C-1 and C-6 in starch glucose was analysed following feeding of tissue with [1_14C]_and [6_14C]_glucose. Randomization of label in starch indicated that the pathway for carbon movement into sugarcane plastids for starch synthesis is primarily through the triose-phosphate translocator. Finally, this study indicated that radiolabelling of tissue discs is a suitable experimental system to determine carbon flux in sugarcane. During the 3 h labelling period the rate of 14C02 release became linear, indicating that the system approached isotopic steady state between the external and internal glucose pool; and the respiratory processes involved in CO2 release. / AFRIKAANSE OPSOMMING: Die veranderinge in koolstoftoedeling na sukrose, heksoses, vesel, stysel en respirasie is in ontwikkelende internodes van suikerriet ondersoek. Die koolhidraatmetabolisme in internodes 3, 6 en 9, wat drie stadiums van toenemende rypheid verteenwoordig, is met behulp van 14Cmerkingstudies ondersoek. Dit is duidelik dat daar 'n hoë mate van koolstofsirkulering tussen die heksose- en triose-fosfaat poele voorkom. In internode 3 word tot 50% van die koolstofwat in triose-fosfate geïnkorporeer is, weer na heksosefosfaat omgeskakel. Selfs in volwasse weefsel vind daar nog soveel as 30% koolstofsirkukering plaas tussen die twee poele plaas. Koolstoftoedeling vanaf glukose na sukrose het van 34% in internode 3, tot 61% in internodes 6 en 9 toegeneem. Proteïn en selwandkomponente was die belangrikste swelgpunte vir koolstof in onvolwasse weefsel gewees. Namate die weefsel meer volwasse word, word sukrose 'n belangriker swelgpunt. Die koolstoftoedeling aan sukrose is veral ten koste van toedeling aan die selwandkomponente. Die bevinding dat die proteïenpoel 'n sterk swelgpunt is dui aan dat vorige studies die belang van respiratoriese koolstofvloei onderskat het. In teenstelling met vorige aansprake is dit duidelik dat selfs in volwasse weefsel respirasie die grootste swelpunt vir die inkomende organiese koolstof in die internode vorm. Koolstoftoedeling aan respirasie het nie noemenswaardig tussen internodes 3 en 6 verskil nie, maar het met 50% in volwasse weefsel afgeneem. Stysel is deurgaans 'n swak swelgpunt vir koolstof met die hoogste toedeling aan die poel (2%) in die jong weefsel (internode 3) . Na toediening van [U- 14C]-glukose is radioaktief gemerkte maltose gevind in suikerriet wat vroeg in die lente geoes is. Dit dui aan dat gelyktydige afbraak en sintese van stysel plaasgevind het. Die herverdeling van C-l en C-6 in glukose afkomstig van stysel is na toediening van [1_14C]_ en [6-14C]-glukose ontleed. Die ewekansige verspreiding van radioaktiwiteit tussen koolstof 1 en 6 van die glukose in stysel dui aan dat dit hoofsaaklik die triose-fosfaat translokeerder is wat in die plastied verantwoordelik is. Hierdie studie het ook aangetoon dat radioaktiewe merking van weefselsnitte 'n geskikte eksperimentele sisteem is om koolstoftoedeling in suikerriet te ondersoek. Die patroon van 14C02 vrystelling dui daarop dat die weefsel na 'n 3 h inkuberingsperiode isotopiese ewewig tussen die eksterne en interne glukose poele en die respiratoriese prosesse begin bereik het.
4

Effect of alkaline pre-treatments on the synergistic enzymatic hydrolysis of sugarcane (Saccharum officinarum) bagasse by Clostridium cellulovorans XynA, ManA and ArfA

Beukes, Natasha January 2011 (has links)
The continual increase in industrialization and global population has increased the dependency and demand on traditional fossil fuels for energy; however, there are limited amounts of fossil fuels available. The slow depletion of fossil fuels has sparked a fresh interest in renewable sources such as lignocellulose to produce a variety of biofuels, such as biogases (e.g. methane), bioethanol, biodiesel and a variety of other solvents and economically valuable by-products. Agricultural crop wastes produced in surplus are typically lignocellulosic in composition and thus partially recalcitrant to enzymatic degradation. The recalcitrant nature of plant biomass and the inability to obtain complete enzymatic hydrolysis has led to the establishment of various pre-treatment strategies. Alkaline pre-treatments increase the accessibility of the exposed surface to enzymatic hydrolysis through the removal of acetyl and uronic acid substituents on hemicellulose. Unlike the use of steam and acid pre-treatments, alkaline pre-treatments solubilize lignin and a small percentage of the hemicellulose, increasing enzyme accessibility and thus the hydrolysis of lignocellulose. The majority of Clostridium cellulovorans associated enzyme synergy studies have been devoted to an understanding of the cellulolytic and hemi-cellulolytic degradation of plant cell walls. However, little is known about the effect of various physical and chemical pre-treatments on the synergistic enzymatic degradation of plant biomass and possible depolymerization of plant cell walls. This study investigates the use of slake lime, sodium hydroxide and ammonium hydroxide to pre-treat sugarcane bagasse under mild conditions and elucidates potentially important synergistic associations between the C. cellulovorans enzymes for the enhanced degradation of lignocellulose. The primary aims of the study were addressed using of a variety of techniques. This included suitable vector constructs for the expression and purification of recombinant C. cellulovorans enzymes, identification of the effects of various pre-treatments on enzyme synergy, and identification of the resultant reducing sugars and phenolic compounds (released during the pre-treatment of the bagasse). This study also made use of physical and chemical pre-treatment methods, protein purification using affinity, high performance liquid and thin layer chromatography, mass spectrometry, sodium dodecyl sulphate and fluorophore-assisted polyacrylamide gel electrophoresis (FACE) , enzymatic degradation and synergy studies with various substrates indirectly using the 3, 4-dinitrosalicylic acid (DNS) reducing sugar assay. From this investigation, the following conclusions were made: alkaline pre-treatment successfully solublised, redistributed and removed lignin from the bagasse, increasing the digestibility of the substrates. In summary, the most effective pre-treatment employed 0.114 M ammonium hydroxide / gram bagasse at 70°C for 36 hours, followed by hydrolysis with an enzyme cocktail containing 25% ManA and 75% XynA. This increased the production of sugars approximately 13-fold. Analysis of the sugars produced by the synergistic hydrolysis of sugarcane bagasse (SCB) indicated the presence of xylose, indicating that the enzymes are potentially bifunctional under certain conditions. This study indicated that the use of mild pre-treatment conditions sufficiently removed a large portion of lignin without affecting the hemicellulose moiety of the SCB. This facilitated the potential use of the hemicellulose component for the production of valuable products (e.g. xylitol) in addition to the production of bioethanol. Thus, the potential use of additional components of holocellulose may generate an additional biotechnological benefit and allow a certain degree of flexibility in the biofuel industry, depending on consumer and industrial needs.
5

Manipulation of pyrophosphate fructose 6-phosphate 1-phosphotransferase activity in sugarcane

Groenewald, Jan-Hendrik 03 1900 (has links)
Thesis (PhD (Genetics. Plant Biotechnology))--University of Stellenbosch, 2006. / The main aim of the work presented in this thesis was to elucidate the apparent role of pyrophosphate fructose 6-phosphate 1-phosphotransferase (PFP) in sucrose accumulation in sugarcane. PFP activity in sugarcane internodal tissue is inversely correlated to the sucrose content and positively to the water-insoluble component across varieties which differ in their capacities to accumulate sucrose. This apparent well defined and important role of PFP seems to stand in contrast to the ambiguity regarding PFP’s role in the general literature as well as the results of various transgenic studies where neither the downregulation nor the over-expression of PFP activity had a major influence on the phenotype of transgenic potato and tobacco plants. Based on this it was therefore thought that either the kinetic properties of sugarcane PFP is significantly different than that of other plant PFPs or that PFP’s role in sucrose accumulating tissues is different from that in starch accumulating tissues. In the first part of the study sugarcane PFP was therefore purified and its molecular and kinetic properties were determined. It consisted of two subunits which aggregated in dimeric, tetrameric and octameric forms depending on the presence of Fru 2,6-P2. Both the glycolytic and gluconeogenic reactions had broad pH optima and the kinetic parameters for all the substrates were comparable to that of other plant PFPs. The conclusion was therefore that sugarcane PFP’s molecular and kinetic characteristics do not differ significantly from that of other plant PFPs. The only direct way to confirm if PFP is involved in sucrose accumulation in sugarcane is to alter its levels in the same genetic background through genetic engineering. This was therefore the second focus of this study. PFP activity was successfully down-regulated in sugarcane. The transgenic plants showed no visible phenotype under greenhouse and field conditions and sucrose concentrations in their immature internodes were significantly increased. PFP activity was inversely correlated with sucrose content in the immature internodes of the transgenic lines. Both the immature and mature internodes of the transgenic plants had significantly higher fibre contents. This study suggests that PFP plays a significant role in glycolytic carbon flux in immature, metabolically active sugarcane internodal tissues. The data presented here confirm that PFP can indeed have an influence on the rate of glycolysis and carbon partitioning in these tissues. It also implies that there are no differences between the functions of PFP in starch and sucrose storing tissues and it supports the hypothesis that PFP provides additional glycolytic capacity to PFK at times of high metabolic flux in biosynthetically active tissue. This work will serve as a basis to refine future genetic manipulation strategies and could make a valuable contribution to the productivity of South African sugarcane varieties.
6

Marker assisted breeding in sugarcane : a complex polyploid

Butterfield, Michael Keith 03 1900 (has links)
Thesis (PhD (Genetics))—University of Stellenbosch, 2007. / Association analysis was used to improve the efficiency of breeding sugarcane varieties for the negatively correlated traits of resistance to sugarcane smut and the eldana stalk borer. 275 RFLP and 1056 AFLP markers were scored across a population of 77 genotypes representing the genetic variation present within the SASRI breeding programme. Genetic diversity analysis did not detect significant structure within the population. Regression analysis identified 64 markers significantly associated with smut rating and 115 markers associated with eldana rating at r2 > 6.25%. Individual markers with the largest effects explained 15.9% of the phenotypic variation in smut rating and 20.2% of the variation in eldana. Five markers were significantly associated with both smut and eldana. In each case the marker effect was negatively correlated between the two traits, suggesting that they are genetically as well as phenotypically negatively correlated.
7

Bioconversion of sugar cane residues into edible monascus and pleurotus products.

January 1998 (has links)
by Pui-nin Lee. / Thesis submitted in: August 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 141-148). / Abstract also in Chinese. / List of Abbreviations --- p.I / List of Tables --- p.II / List of Figures --- p.III / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Sugar cane --- p.1 / Chapter 1.2 --- Mushroom: Pleurotus pulmonarius --- p.5 / Chapter 1.2.1 --- Fruiting bodies --- p.5 / Chapter 1.2.2 --- Life cycle of the mushroom --- p.6 / Chapter 1.2.3 --- Cultivation --- p.7 / Chapter 1.2.4 --- Nutrition --- p.8 / Chapter 1.2.5 --- Cardiovascular and Renal effects --- p.9 / Chapter 1.2.6 --- Dietary fiber --- p.10 / Chapter 1.2.6.1 --- Physical properties of dietary fiber --- p.10 / Chapter 1.2.6.2 --- Physiological actions --- p.11 / Chapter 1.3 --- Food colorants --- p.13 / Chapter 1.4 --- Quality of food products --- p.14 / Chapter 1.4.1 --- Toxicity studies --- p.15 / Chapter 1.4.2 --- Stability --- p.16 / Chapter 1.4.3 --- Sensory evaluation --- p.18 / Chapter 1.5 --- Secondary metabolism --- p.20 / Chapter 1.6 --- Monascus --- p.24 / Chapter 1.6.1 --- Monascus products --- p.25 / Chapter 1.6.2 --- Monascus products as a functional food --- p.27 / Chapter 1.6.3 --- Monascus pigments --- p.27 / Chapter 1.6.4 --- Factors affect Monascus pigment production --- p.31 / Chapter 1.7 --- Purposes of study --- p.37 / Chapter Chapter 2 --- Materials and Methods --- p.38 / Chapter 2.1 --- Micro-organisms --- p.38 / Chapter 2.2 --- Maintenance of culture --- p.38 / Chapter 2.3 --- Waste Culture medium for production of pigments --- p.38 / Chapter 2.4 --- Chemical analysis of sugar cane residue broth (SCRB) --- p.39 / Chapter 2.5 --- Preparation of inoculum --- p.40 / Chapter 2.6 --- Batch culture for pigment production --- p.41 / Chapter 2.7 --- Fermentor culture for pigment production --- p.44 / Chapter 2.8 --- Cultivation of oyster mushroom --- p.46 / Chapter 2.9 --- Purification and characterization of Monascus pigments --- p.49 / Chapter 2.9.1 --- Extracellular pigments --- p.49 / Chapter 2.9.2 --- Intracellular pigments --- p.50 / Chapter 2.10 --- Toxicity test --- p.56 / Chapter 2.11 --- Sensory evaluation --- p.61 / Chapter 2.12 --- Statistical analysis --- p.62 / Chapter Chapter 3 --- Results --- p.62 / Chapter 3.1 --- Extracellular pigment of screening test --- p.63 / Chapter 3.2 --- Batch culture for pigment production by Monascus purpureus --- p.69 / Chapter 3.2.1 --- Consumption of glucose --- p.69 / Chapter 3.2.2 --- Consumption of sucrose --- p.59 / Chapter 3.2.3 --- Biomass production --- p.69 / Chapter 3.2.4 --- Residual protein content --- p.73 / Chapter 3.2.5 --- pH value of the fermented medium --- p.73 / Chapter 3.2.6 --- Production of crude extracellular pigments --- p.73 / Chapter 3.2.7 --- Production of crude intracellualr pigments --- p.75 / Chapter 3.2.8 --- Total crude pigment yield --- p.75 / Chapter 3.3 --- Fermentor --- p.77 / Chapter 3.3.1 --- "Pigmentation of control group, treatment A and B" --- p.77 / Chapter 3.3.2 --- Nutrition of the biomass from control group and treatment B of fermentor products --- p.80 / Chapter 3.4 --- Production of oyster mushroom --- p.80 / Chapter 3.4.1 --- Biological efficiency of mushroom --- p.80 / Chapter 3.4.2 --- Amino acid profile and total amino acid content of the fruitbidies --- p.87 / Chapter 3.4.3 --- Total dietary fiber content --- p.87 / Chapter 3.4.4 --- "The contents of carbon, hydrogen and nitrogen" --- p.91 / Chapter 3.4.5 --- Sensory evaluation of the mushroom --- p.91 / Chapter 3.5 --- Toxicity --- p.91 / Chapter 3.5.1 --- Acute toxicity --- p.91 / Chapter 3.5.1.1 --- Body weight --- p.95 / Chapter 3.5.1.2 --- Food consumption --- p.95 / Chapter 3.5.2 --- Subacute toxicity --- p.99 / Chapter 3.5.2.1 --- Food consumption --- p.99 / Chapter 3.5.2.2 --- Body weight --- p.99 / Chapter 3.5.2.3 --- The organ weight to body weight ratios --- p.99 / Chapter 3.5.2.4 --- The glutamate-pyruvate transaminase (GPT) and glutamate- oxaloacetate transaminase (GOT) level in blood serum --- p.103 / Chapter 3.6 --- Analysis of Monascus pigments from fermentor system by HPLC and spectrophotometry --- p.108 / Chapter 3.6.1 --- Extracellular pigment from control group --- p.108 / Chapter 3.6.2 --- Extracellular pigment from the group supplemented with 1 % MSG --- p.108 / Chapter 3.6.3 --- Intracellular pigment --- p.111 / Chapter 3.6.3.1 --- Standard of intracellular pigments (conventional pigments) --- p.111 / Chapter 3.6.4 --- Intracellular pigments extracted from SCRB group --- p.111 / Chapter 3.6.5 --- Intracellular pigment extracted from SCRB with 1 % MSG --- p.115 / Chapter 3.7 --- Qualitative and quantitative of the extracellular and intracellular pigments --- p.118 / Chapter 3.8 --- Detection of citrinin by HPLC --- p.123 / Chapter Chapter 4 --- Discussion --- p.128 / Chapter 4.1 --- Screening test --- p.128 / Chapter 4.1.1 --- Batch culture system --- p.128 / Chapter 4.2 --- Toxicity test of crude extracellular pigment --- p.129 / Chapter 4.2.1 --- Acute toxicity --- p.129 / Chapter 4.2.2 --- Subacute toxicity test --- p.130 / Chapter 4.2.3 --- Organ to body weight ratios and cytotoxicity --- p.131 / Chapter 4.3 --- The two new water soluble pigment --- p.132 / Chapter 4.4 --- Extraction and purification of Monascus pigments from fermentor system --- p.133 / Chapter 4.4.1 --- Qualitative and quantitative of the intracellular and extracellular pigments --- p.133 / Chapter 4.5 --- Nephrotoxic-citrinin --- p.134 / Chapter 4.6 --- Oyster mushroom cultivation --- p.135 / Chapter 4.6.1 --- Fruiting yield (biological efficiency) --- p.136 / Chapter 4.6.2 --- Amino acid content --- p.136 / Chapter 4.6.3 --- Total dietary fiber content --- p.137 / Conclusion --- p.139 / References --- p.141 / Appendix
8

Manipulation of neutral invertase activity in sugarcane

Joubert, Debra 12 1900 (has links)
Thesis (MSc (Genetics. Institute for Plant Biotechnology))--University of Stellenbosch, 2006. / The main goal of this project was to elucidate the apparent role of neutral invertase (NI) in sucrose accumulation in sugarcane. In the first part of the study putative transgenic cell lines (transformed with antisense NI constructs) were characterised to confirm the stable integration and expression of the transgene. Batch suspension cultures were used to initiate replicate cultures of several of these transgenic lines as well as a control, and the metabolism of the cultures during a 14 day growth cycle was examined. The transgenic lines had substantially reduced levels of NI activity. While the activities of the other invertases remained unchanged, the activity of sucrose synthase (SuSy) was significantly higher in the transgenic suspension cultures relative to the control. Throughout the growth cycle, sucrose concentrations in the transgenic lines were consistently higher, and glucose and fructose concentrations lower, than the control. The transgenic cultures also exhibited a decreased growth rate in comparison to the control. Labelling studies confirmed a decrease in the in vivo rate of invertase-mediated sucrose hydrolysis in the transgenic lines, as well as indicating a decline in the partitioning of carbon to respiratory pathways in these cultures. In the second part of the study, which focussed on greenhouse-grown transgenic plants, similar results were reported. NI activity was significantly decreased, and SuSy activity increased in all of the tissues sampled. The sucrose concentration and purity were also higher in the transgenic tissues, while the in vivo sucrose hydrolysis rate was lower. Allocation of carbon to respiration was lower in the transgenic plants, suggesting that a decrease in sucrose breakdown reduces the availability of hexoses for growth and respiration. Overall, the results suggest that NI plays a key role in the control of sucrose metabolism, and that changes in the activity of this enzyme have far-reaching effects on cellular metabolism. The fact that the trends reported in the whole-plant studies parallel those of the suspension cultures confirms that suspension cultures can be used as a model system in metabolic engineering research in sugarcane. Thus the possibility now exists to analyse large numbers of transgenic lines in a quicker time frame and at a reduced cost in comparison to conventional methods.
9

Influence of hexose-phosphates and carbon cycling on sucrose accumulation in sugarcane spp.

Van der Merwe, Margaretha Johanna 12 1900 (has links)
Thesis (MSc (Genetics. Plant Biotechnology))--University of Stellenbosch, 2005. / Sucrose accumulation, marked by a continuous cycle of synthesis and degradation, is characterised by a shift of carbon away from the insoluble matter and respiratory intermediates into sucrose. Despite this shift, a significant proportion of carbon is returned to these pools by hexose-phosphate: triose-phosphate cycling and/or sucrose cycling. Little is known about the magnitude and behaviour of these cycles in sugarcane. Contradictory reports on the relationship between these two cycles have led to the evaluation of the link between the hexose-phosphate: triose-phosphate- and sucrose cycle. In addition, it still needs to be tested whether these cycles could significantly influence carbon partitioning within sugarcane internodal tissue. In this work, a comprehensive metabolic profile was constructed for sugarcane internodal tissue by gas chromatography-mass spectrometry (GC-MS) in order to determine the steady state levels of a broad range of primary metabolites that are involved in these cycles. The power of GC-MS was illustrated by the detection of raffinose, maltose, ribose, xylitol, inositol, galactose, arabinose and quinic acid, which was quantified for the first time in sugarcane internodal tissue. Analyses were not solely based on the prevailing metabolite levels, but also on the interactions between these metabolites. Thus, in a complementary approach the metabolic flux between the two substrate cycles was assessed by 13C nuclear magnetic resonance (NMR). Analyses of transgenic sugarcane clones with 45-95% reduced cytosolic pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) activity displayed no visual phenotypic change, but significant changes were evident in in vivo metabolite levels. Sucrose concentrations increased six and three-fold in young and maturing internodal tissue, respectively. Reduced PFP activity also resulted in an eight-fold increase in the hexose-phosphate: triose-phosphate ratio in the transgenic immature internodes. In addition, the hexose-phosphate: triose-phosphate cycling decreased in the immature internodes of the transgenic lines if compared to the immature control internode. However, there was no significant difference between the hexose-phosphate: triose-phosphate cycling in the mature internodal tissue of the transgenic and the control lines. This illustrated that PFP mediates hexose-phosphate: triose-phosphate cycling in immature sugarcane internodal tissue. Unpredictably, reduced PFP activity led to a ten-fold increase in sucrose cycling in the transgenic immature internodes. The combination of metabolite profiling and flux distribution measurements demonstrated that the fluxes through the sucrose and the hexose-phosphate pools were not co-regulated in sugarcane internodal tissue. From these observations a model was constructed that implicates higher sucrose cycling as a consequence of increased sucrose concentrations.
10

The manipulation of fructose 2,6-bisphosphate levels in sugarcane

Hiten, Nicholas Fletcher 03 1900 (has links)
Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2006. / Fructose 2,6-bisphosphate (Fru 2,6-P2) is an important regulatory molecule in plant carbohydrate metabolism. There were three main objectives in this study. Firstly, to determine whether the recombinant rat 6-phosphofructo 2-kinase (6PF2K, EC 2.7.1.105) and fructose 2,6-bisphosphatase (FBPase2, EC 3.1.3.11) enzymes, which catalyse the synthesis and degradation of Fru 2,6-P2 respectively, showed any catalytic activity as fusion proteins. Secondly, to alter the levels of Fru 2,6-P2 in sugarcane, an important agricultural crop due to its ability to store large quantities of sucrose, by expressing the recombinant genes. Thirdly, to investigate whether sugar metabolism in photosynthetic- (leaves) and non-photosynthetic tissue (internodes) were subsequently influenced. Activity tests performed on the bacterially expressed glutathione-S-transferase (GST) fusion 6PF2K and FBPase2 enzymes showed that they were catalytically active. In addition antibodies were raised against the bacterially expressed proteins. Methods for extracting and measuring Fru 2,6-P2 from sugarcane tissues had to be optimised because it is known that the extraction efficiencies of Fru 2,6-P2 could vary significantly between different plant species and also within tissues from the same species. A chloroform/methanol extraction method was established that provided Fru 2,6-P2 recoveries of 93% and 85% from sugarcane leaves and internodes respectively. Diurnal changes in the levels of Fru 2,6-P2, sucrose and starch were measured and the results suggested a role for Fru 2,6-P2 in photosynthetic sucrose metabolism and in the partitioning of carbon between sucrose and starch in sugarcane leaves. Transgenic sugarcane plants expressing either a recombinant rat FBPase2 (ODe lines) or 6PF2K (OCe lines) were generated. The ODe lines contained decreased leaf Fru 2,6-P2 levels but increased internodal Fru 2,6-P2 levels compared to the control plants. Higher leaf sucrose and reducing sugars (glucose and fructose) were measured in the transgenic plants than the control plants. The transgenic lines contained decreased internodal sucrose and increased reducing sugars compared to the control plants. Opposite trends were observed for Fru 2,6-P2 and sucrose when leaves, internodes 3+4 or internodes 7+8 of the different plant lines were compared. In contrast, no consistent trends between Fru 2,6-P2 and sucrose were evident in the OCe transgenic lines.

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