Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The deterioration of harvested sugarcane as a result of bacterial growth causes major losses of sucrose and a build-up of exopolysaccharides (EPS). Polysaccharides present during production increase the massecuite viscosity, which negatively influences evaporation and crystallisation. In this study 38 culturable EPSproducing bacteria were isolated from milled sugarcane. Analysis of the EPS showed the ubiquitous presence of glucose, however, 14 polysaccharides also contained mannose, fructose or galactose. In vitro treatment using Chaetomium erraticum dextranase to evaluate is effectiveness indicated that 37 of the EPS were hydrolysed to some extent. There were 21 polysaccharides that were only partially digested. The capacity of the isolates to produce EPS on different sugars indicated a correlation between sucrose and polysaccharide formation in 37 isolates. The results indicate there are more species involved in EPS production than previously thought as well as the presence of non-dextran polysaccharides. / AFRIKAANSE OPSOMMING: Bakteriële groei veroorsaak ‘n afname in gehalte, sukrose en ‘n verhoging in die hoeveelheid van eksternepolisakkeriede (EPS). Die verhoogde konsentrasie van polysakkariede gedurende die verwerkingsprosses veroorsaak ‘n verhoging in “massecuite” viskositeit. Hierdie verskynsel het ‘n nadelige uitwerking op die verdamping en kristalvorming van die produk. In gemaalde skuikerriet was 38 groeibare EPS-produserende bakterieë geisoleer. Die geanaliseerde EPS van hierdie bogenoemde bakterieë was daar in almal glukose teenwoordig. In 14 van hulle was mannose, fruktose en galaktose ook gevind. Die in vitro effektiwieteit van Chaetomium erraticum dekstranase op die EPS het gewys dat 37 het tot ‘n mate gehidroliseer maar 21 was net gedeeltelik verteer. As gevolg van die bo-genoemde resultate was daar gevind dat sukrose was ‘n noodsaaklike subtraat vir EPS produksie in die geisoleerde bakterieë. In hierdie studie was bevestig ‘n groter verskiedenheid EPS-produserende bakterieë gevind was en dat hulle assosiasie aan sukierriet prossering meer kompleks is as wat vooreen gedink was.

Thesis (MSc (Biochemistry))--Stellenbosch University, 2008. / The aim of this study is the theoretical investigation of carbon fixation in sugarcane leaves. Sugarcane has a well known reputation for accumulating sucrose in the stalk to levels as high as 650 mM, almost a fifth of the plant’s fresh weight. Although this is an efficient accumulating mechanism, there is an even more efficient ‘carbon pump’ found in C4 plants. This is a well documented carbon concentrating mechanism and one of the first to be studied. However scientists are still trying to understand the carboxylating mechanism and the regulation thereof. It has been speculated that this mechanism is at its saturation level and elevating carbon dioxide will have little or no effect on further carbon fixation. Futher, studies suggest that the sucrose accumulating sink is able to regulate photosynthesis. Therefore a regulatory mechanism should exist from the sink to carbon fixation in order for such regulation to occur. Thework in this thesis therefore lays the foundation for investigating regulation of photosynthesis. The field of systems biology is the study of cellular networks by assemblingmodels. Pathways are considered as systems and notmerely collections of single components. This allows the interaction of pathway metabolites and the regulation that they have on one another to be studied. The questions asked pertaining to a pathway, will determine the types of model analysis. Structural analysis is useful for studying stoichiometric models, determining characteristics like energy consumption, futile cycles and valid pathways through a system at steady-state. Kinetic analysis on the other hand, gives insight into system dynamics and the control exerted by the system components, predicting time-course and steady states. In this thesis we begin to investigate photosynthesis in sugarcane leaves and the role it has in accumulating sucrose in the plant. Firstly, a structural model was developed incorporating carbon fixation, sucrose production in the leaf and subsequent transport of sucrose to the storage parenchyma and accumulation. The model was analysed using elementary mode analysis, showing that there are twelve routes for producing sucrose with no pathway beingmore energy efficient than any other. Further, it highlighted a futile cycle transporting triose phosphates and phosphoglycerate between the two photosynthetic compartments in the leaf. In the storage parenchyma, manymore futile cycleswere revealed,many of them energetically wasteful. Three other sets of elementary modes describe sucrose’s destination in either the vacuole or use in glycolysis or fibre formation, each with a different amount of required energy equivalents. The fourth set describes how sucrose cannot be converted to fibre precursors without also being used for glycolyis building blocks. Secondly, a kinetic model of carbon fixation in the leaf was assembled with the primary goal of characterising thismoiety-conserved cycle. This included the collation of kinetic data, incorporating volumes of the compartments and the areas of the location of the transporters into the model. This model was then analysed using metabolic control analysis. The model was able to predict metabolite concentration in the pathway at steady-state which were compared to those found experimentally. However, modifications need to be made to the model before further analysis is done so that the model predicted values match the experimental values more accurately. Time course analysis and response coefficients were also calculated for the carbon fixation cycle. Thework in this thesis therefore paves the way for understanding photosynthesis and its regulation in sugarcane leaves. Such work has the potential to pinpoint genetic engineering target points, allowing for better hybrid selection and propagation.

Thesis (MSc (Genetics. Plant Biotechnology))--University of Stellenbosch, 2007. / Carbon partitioning was investigated in sugarcane (Saccharum spp. hybrids) that was genetically modified with sucrose: sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99) from Cynara scolymus. This enzyme catalyses the transfer of a fructosyl moiety from one sucrose molecule to another to produce the trisaccharide 1-kestose. Molecular characterisation of four sugarcane lines, regenerated after transformation, confirmed that two lines (2153 and 2121) were transgenic, with at least one intact copy of 1-SST present in line 2153, and a minimum of five copies (or portions thereof) present in line 2121. The novel gene was successfully transcribed and translated in both lines, as confirmed by cDNA gel blot hybridisation and HPLC analysis respectively. Kestose production was stable under field resembling conditions and levels of this trisaccharide progressively increased with increasing internodal maturity from 7.94 ± 2.96 nmol.g-1 fresh mass (fm) in internode 6 to 112.01 ± 17.42 nmol.g-1 fm in internode 16 of 2153, and by 1.05 ± 0.93 nmol.g-1 fm from the youngest to the oldest internode in line 2121. Sugarcane line 2153 contained 100 times more 1-kestose than 2121 in the oldest sampled internode hence the lines were referred to as high- and low-1-kestose producers. The production of 1-kestose did not reduce sucrose levels in the transgenics, instead they contained significantly higher levels of sucrose than the control line NCo310 (p<0.01, N=72). The production of this alternative sugar in addition to elevated sucrose levels significantly increased the total sugar content in the transgenic lines (p<0.01, N=72). Moreover, the high-1-kestose producer had statistically more total sugar than the low-1-kestose producer (p<0.01, N=72). Soluble acid invertase (SAI) and neutral invertase (NI, β-fructofuranosidase EC 3.2.1.26) from non-transgenic sugarcane internodal tissues were separated and partially purified. Kinetic analysis of the purified invertases revealed two isoforms of SAI eluting at approximately 100 mM KCl in a linear gradient while NI eluted at approximately 500 mM KCl. The final specific activities of SAI and NI were 88.57 pkat.mg-1 protein and 92.31 pkat.mg-1 protein, respectively. This implied a 16- fold purification of SAI, and 4- fold purification of NI. The pH optimum for NI was 7.0 and that for soluble acid invertase less than 5.0. Due to the broad pH activities of the invertases, activities significantly overlapped between pH 4.5 and 7.0. The affinity of these invertases for 1-kestose hydrolysis was tested. The invertases displayed hyperbolic saturation kinetics for sucrose, and had low affinities for 1-kestose with Km values ranging from 50 - 247 mM. Furthermore, the presence of 200 mM 1-kestose had an inhibitory effect on SAI-mediated sucrose hydrolysis reducing activity to 51 % and 54 % for isoform 1 and 2 respectively. To determine whether carbon allocation had been altered by the expression and activity of 1-SST, 14C whole-plant radiolabelling experiments were conducted. Radiolabelled CO2 was fed to the leaf subtending internode 5 and the allocation of carbon to different parts of the culm was assessed. There was no significant difference in the distribution of total radiolabel down the culm of the three sugarcane lines (p>0.05, N=72). However, the percentage of total radiolabel in the water-soluble fraction per internode in the high-1-kestose producer was significantly higher than the other two lines (p<0.01, N=72). As a result, the percentage radiolabel in the waterinsoluble fraction in this transgenic was concomitantly lower than in the other lines. Carbon was therefore redirected from the water-insoluble fraction to the water-soluble fraction to account for the additive production of 1-kestose. The expression of 1-SST in sugarcane therefore established an additional carbohydrate sink by the flow of carbon from the sucrose pool into 1-kestose. This did not lead to a depletion of the sucrose pool, but rather stimulated carbon channelling into this pathway, thereby increasing the non-structural carbohydrate content of the plant in one of the transgenics. The work described in this study is the first to report on carbon partitioning in 1- kestose-producing sugarcane grown under field resembling conditions. It contributes significantly to an improved understanding of carbon partitioning in the culm, and demonstrates that an alternative sugar can be produced in sugarcane under field resembling conditions.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2007. / Sugarcane is cultivated because of the high levels of sucrose it stores in its internodes. Starch metabolism has been a neglected aspect of sugarcane research despite the problems caused by it during sugarcane processing. Currently there is no information available on the starch content in different South African commercial sugarcane varieties. This project had two main aims of which the first was to determine the starch content in the internodal tissues of six commercial sugarcane varieties. The activities of ADP-Glucose Pyrophosphorylase (AGPase) and β- amylase were also determined. The second aim of the project was to manipulate starch metabolism in sugarcane using transgenesis. To achieve this, transformation vectors for the down-regulation of AGPase activity and over-expression of β-amylase activity were designed. These vectors were then used to transform sugarcane calli and the results were analysed in suspension cultures. Starch levels in sugarcane internodal tissue increased more than 4 times from young to mature internodes. There were also large differences between varieties. When mature tissues of different varieties were compared, their starch concentration varied between 0.18 and 0.51 mg g-1 FW, with the majority of the varieties having a starch concentration between 0.26 and 0.32 mg g-1 FW. NCo376’s starch concentration was much lower than the rest at 0.18 mg g-1 FW and N19’s was much higher at 0.51 mg. g-1 FW. There was also a very strong correlation between starch and sucrose concentration (R2 = 0.53, p ≤ 0.01) which could be due to the fact that these metabolites are synthesized from the same hexose-phosphate pool. No correlation was evident between starch concentration and AGPase activity. This was true for correlations based on either tissue maturity or variety. β-amylase activity expressed on a protein basis was almost 5 times higher in the young internodes compared to mature internodes, suggesting that carbon might be cycled through starch in these internodes. AGPase activity in the transgenic suspension cultures was reduced by between 0.14 and 0.54 of the activity of the wild type control. This reduction led to a reduction in starch concentration of between 0.38 and 0.47 times that of the wild type control. There was a significant correlation between the reduction in AGPase activity and the reduction in starch (R2 = 0.58, p ≤ 0.05). β-amylase activity in the transgenic suspension cultures was increased to 1.5-2 times that of the wild type control. This led to a reduction in starch concentration of between 0.1 and 0.4 times that of the wild type control. Once again the increase in β-amylase activity could be correlated to the reduction in starch concentration of the transgenic suspension cultures (R2 = 0.68, p ≤ 0.01). In both experiments there was no significant effect on sucrose concentration.

Thesis (MSc (Genetics. Plant Biotechnology)) --Stellenbosch University, 2008. / The main aim of the work presented in this thesis was to further our understanding of the role of Pyrrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) in sugarcane, by specifically investigating its potential contribution to phloem metabolism. PFP activity in sugarcane internodal tissue is inversely correlated to sucrose content across varieties that differ in their sucrose accumulation abilities. This apparent correlation is in contrast to previous studies that suggest PFP plays an insignificant role in metabolism. In the first part of this study an immunological characterisation of the two subunits of sugarcane PFP was conducted to establish whether it differ significantly from other plant species in terms of size and distribution. Both the alpha and beta subunit appears to be approximately sixty kilo Daltons in size and uniform in their relative distribution to each other in the various plant organs of sugarcane. Although the observed alpha subunit size is less than that predicted this could be explained at the hand of post translational modification, in essence the sugarcane PFP subunits appear similar than that described for other plants especially that of tobacco which was employed as a model system later on in this study. The only direct way to investigate PFP’s contribution to phloem metabolism is to alter its activity by recombinant DNA technologies. Therefore, in the second part of the study transformation systems were designed for both the constitutive and phloem specific downand up-regulation of PFP activity. For the down-regulation of activity a post transcriptional gene silencing system, i.e. a complementary strand intron hairpin RNA (ihpRNA) silencing system, was employed. A partial sequence of the PFP-beta subunit was isolated and used in vector construction. For the over-expression the Giardia lamblia PFP gene was used. The model plant tobacco was employed to investigate PFP’s effect on phloem metabolism and transport of assimilate. Transgene insertion was accomplished by means of Agobacterium mediated transformation and tissue specific manipulation of PFP activity was confirmed by in situ activity staining.