Thesisa (MSc (Genetics. Plant Biotechnology))--University of Stellenbosch, 2005. / The study had two main objectives: 1) to investigate specific enzyme activity profiles at various developmental stages and to determine possible implications for sucrose metabolism, 2) to incorporate enzyme activity data of different internodes to obtain a detailed model of every stage in the tissue maturation process. The most significant findings of the regulation of sucrose accumulation in this study are centred on three main point controls in sucrose metabolism pathway. Firstly, the maturation of sugarcane internodes coincided with an increase of SPS in most genotypes, and this underlines the key role of this enzyme in sucrose accumulation. Secondly, SuSy activity (cleavage reaction) correlated negatively with sucrose concentration and hence with tissue maturation process, in most of the varieties. This finding indicates that SuSy could well be implicated in sucrose metabolism. Thirdly, in vitro PFP activity was found to be negatively correlated to sucrose content in sugarcane varieties differing in amount of sucrose.

Thesis (MSc (Genetics. Plant Biotechnology)--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Glycogen was isolated from E. coli and analysed for the amount of phosphate present within it. It was confirmed that a significant proportion of the glucose residues were phosphorylated at the C6 position. This glycogen phosphate was found also in both glgb- (glycogen branching enzyme) and glgp- (glycogen phosphorylase enzyme) mutants, demonstrating that a mechanism for phosphate incorporation that does not involve GlgP alone, and which is capable of incorporating phosphate into linear glucans could exist. The degree of phosphorylation depended on the amount of phosphate present in the media, which less being incorporated in media where phosphate was reduced. Screening for glycogen phosphorylating genes using a E. coli genomic library in a functional expression system identified the malP gene as a possible candidate for incorporation of the phosphate at the C6 position. There was no difference, however, between the glycogen phosphate content of the mutant and wild type. Efforts were made to construct a malp-/glgp- double mutant, but these were unsuccessful. In addition the influence of plants and human proteins on yeast glycogen metabolism was also investigated. These proteins have been demonstrated to have an effect on starch or glycogen in humans, plant and E. coli, but the data from this study indicated that this was not the case in yeast. / AFRIKAANSE OPSOMMING: Glikogeen, wat geisoleer was uit E.coli was geanaliseer vir fosfaat inhoud daarin. Daar was gevind dat `n beduidende proporsie van die glukose residue gefosforileerd was op die C6 posisie. Hierdie gefosforileerde glikogeen was ook gevind in glg- (glikogeen vertakkingsensieme) en glgp- (glikogeen fosforileringsensieme) mutante wat daarop dui dat `n meganisme vir fosforilering bestaan was nie slegs aangewese is op die aktiwiteit van GlgP nie, en om fosfaat te inkorporeer in linêre glukane. Die graad van fosforilering was ook afhanklik van die hoeveelheid fosfaat teenwoordig in die medium, met gevolglik minder wat geinkorporeer kan word in medium waar fosfaat verminderd was. Seleksie-gebaseerde ondersoeking vir fosforileringsensieme van glikogeen deur gebruik te maak van E. coli genomiese biblioteke in `n funksionele uitdrukkingssisteem het die malP geen geidentifiseer as een van die moontlike kandidate wat verantwoordelik kan wees vir inkorporering van fosfaat in the C6 posisie. Daar was egter geen verskil in die fosfaat inhoud van glikogeen tussen die wilde tipe en die mutante. Pogings wat aangewend is om `n malp-/glgpdubbel mutant te konstrueer was onsuksesvol. Verder is die invloed van plant en mens proteine op gis glikogeen ook bestudeer. Vroeër is aangetoon dat hierdie proteine `n invloed op stysel en glikogeen het in mense, plante en E. coli, maar data van hierdie studie toon aan dat dit nie die geval in gis is nie.

Thesis (MSc (Genetics. Plant Biotechnology)) --University of Stellenbosch, 2009. / ENGLISH ABSTRACT: This project examined the role of proteins in starch phosphate metabolism. The first part was aimed at the functional characterization of the SEX4, LSF1 and LSF2 genes in both plants and bacteria. Constructs were produced to allow for expression of the three proteins in E. coli with the SEX4 and LSF2 proteins being successfully purified and used to produce antibodies. Immunoblot analysis indicated that the antibodies recognised the repective proteins in extracts, but it was not clear if they actually recognised the proteins or the GST tags they were fused to. Virus induced gene silencing constructs were also produced to allow repression of these three genes in Nicotiana benthamiana. This resulted in a starch excess phenotype being observed in the leaves of silenced plants which is consistent with the known or presumed roles for the genes. The antibodies produced were not specific enough to confirm that the respective protein were actually repressed, but it is likely that this was the case as plants infiltrated at the same time with a VIGS vector designed to repress phytoene desaturase exhibited a chlorophyll bleaching phenotype. These data confirm that SEX4 and LSF1 probable play the same role in N. benthamiana as in Arabidopsis, and provide evidence that LSF2 is also necessary for starch degradation. It was also attempted to characterise these proteins with respect to their substrate utilization by setting up a glyco-array experiment. Various potato starches from genetically modified plants were subjected to hydrolytic attack by starch degrading enzymes and fractionated by anion exchange chromatography to produce a multitude of glucans. These will be spotted onto glass filters and probed with the purified proteins to see if they bind to specific starch breakdown products preferentially. iv The project also involved investigating the effect the SEX4 protein has on E. coli glycogen contents. SEX4 was expressed in wild type and glgX mutant E. coli strains as it has been shown that this stops glycogen accumulation in the wild type, but not the glgX mutant. The cells were grown in liquid culture and glycogen contents measured. In liquid cultures SEX4 had no effect on glycogen contents in the wild type, possible because of problems with plasmid stability in the strain used. This final part of the project investigated the effect that a gwd mutation has on carbohydrate metabolism in leaves and fruits of the Micro-tom tomato cultivar. Starch and soluble sugar contents were measured in leaves and ripening fruits. A starch excess phenotype was found in the leaves, but no change in starch contents was determined in either the placenta or pericarp of the fruit. Soluble sugar contents were reduced in the fruit tissues, although the reason for this in unclear. / AFRIKAANSE OPSOMMING: Hierdie projek het die rol van proteine in stysel-fosfaat metabolisme ondersoek. Die eerste deel handel oor die funksionele karaktiseering van die SEX4, LSF1 en LSF2 gene in beide plante en bakteriee. Vektore is gekonstrueer om die uitdrukking van die drie proteine in E.coli toe te laat terwyl die SEX4 en LSF2 proteine suksesvol gesuiwer is vir die gebruik vir teenliggaam produksie. Immunoklad analises het getoon dat die teenligame die spesifieke proteine in die ekstrak herken het, maar dit was nie duidelik of dit die onderskeie proteine was of die GST-verklikker waaraan die onderskeie proteine verbind was nie. Virus geindiseerde geen onderdrukking konstrukte is ook geproduseer om toe te laat vir die onderdrukking van hierdie drie gene in Nicotiana benthamiana. Dit het ‘n stysel oorskot fenotipe tot gevolg gehad in die blare van onderdrukte plante wat konstant is met die bekende of voorgestelde rolle van die gene. Die teenliggame wat geproduseer is was nie spesifiek genoeg om te bewys dat die onderskeie proteine wel onderdrukis nie. Dit kon wel die geval gewees het want plante geinfiltreer op dieselfde tyd met ‘n VIGS vektor wat ontwerp is om phytoene desaturase te onderdruk het ‘n chlorofil bleikings fenotipe getoon. Hierdie data bevestig dus dat SEX4 en LSF1 moontlik dieselfde rol speel in N. benthamiana as in Arabidopsis, en toon bewyse dat LSF2 ook nodig is vir stysel afbreek. Karakterisasie van die onderskeie proteine met respek tot hul substraat gebruik is ondersoek deur ‘n gliko-array eksperiment. Verskillende aartappel stysels van genetiese gemodifiseerde plante was geonderwerp aan hydrolitiese afbreek deur stysel afbrekende ensieme en geskei deur anioon uitruilings chromotografie om veelvuldige glukans te vi vervaardig. Dit is geplaas op glas filters en is ondersoek saam met die gesuiwerde proteine om te sien of dit mag bind aan spesifieke stysel afbreek produkte. ‘n Verdere ondersoek is onderneem na die effek van die SEX4 protein op E. coli glikogeen inhoud. SEX4 was uitgedruk in die E .coli wildetipe en glgX mutant omdat dit reeds bewys is dat SEX4 glikogeen ophoping veroorsaak in die wildetipe maar nie in die glgX mutant. Die selle is opgegroei in vloeibare media en glikogeen inhoud is gemeet. In vloeibare media het SEX4 geen effek op die wildetipe se glikogeen inhoud nie wat moontlik kan wees as gevolg van plasmied stabiliteit in die E. coli ras wat gebruik is. Die finale deel van die projek was om die effek van ‘n gwd mutasie op koolhidraat metabolisme in blare en vrugte van die Micro-tom tamatie kultivar te ondersoek. Stysel en oplosbare suikers is gemeet in blare en rypwordende vrugte. ‘n Oortollige stysel fenotipe is in die blare gevind maar geen verandering in stysel inhoud is waargeneem in die plasenta of perikarp van die vrug nie. Oplosbare suiker inhoud het afgeneem in die vrugweefsel dog is die rede hiervoor nie te verstane.

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 (Genetics. Institute for Plant Biotechnology)--Stellenbosch University, 2008. / Strawberry (Fragaria x ananassa) fruit quality is largely determined by the relative amounts of sugars and organic acids present, as well as soluble solid content. This study had three components: 1) Characterisation of cytosolic carbohydrate metabolism and carbon partitioning to sugars and organic acids in two commercial varieties, 2) analysis of transgenic strawberry fruit with increased pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase (PFP) activity and 3) analysis of transgenic strawberry fruit with increased ß-fructosidase (invertase) activity in either cytosol or apoplast. Analyses of transgenic strawberry may inform similar attempts in grape berries. Festival and Ventana, two popular commercial strawberry cultivars in South Africa, were fairly similar with respect to sugar and organic acid content. Twelve cytosolic enzymes were investigated. Temporal differences in maximum catalytic activity were observed for invertase, PFP, pyruvate kinase and ADP-glucose pyrophosphorylase (AGPase). Invertase, PFP and AGPase activity also differed between the cultivars. One enzyme, SuSy, could not be analysed effectively, due to the purification method employed. These analyses established methodology for the analysis of transgenic berries. Constructs were designed to constituitively express Giardia lamblia PFP (GL-PFP), or to express Saccharomyces cerevisiae invertase (SCI) in a fruit-specific manner. A second invertase construct was designed to target SCI to the apoplast. Strawberry (cv. Selekta) was transformed and the presence of each transgene confirmed by PCR. Untransformed Selekta was used as control in both transgenic studies. Transgenic lines were selected based on GL-PFP activity in leaves and total PFP activity in ripe fruit. Sugar and organic acid content of ripe berries with high PFP activity was determined. Although berries displayed marked changes in sugar composition, the total sugar content was similar to controls, in all except one line. Organic acid content was decreased, leading to a clear reduction in organic acid-to-sugar ratio. This points to a gluconeogenic role for PFP in strawberry fruit. Transgenic berries were screened for SCI activity. Berries containing untargeted SCI exhibited total invertase activity similar to controls and were not analysed further. Berries with apoplasttargeted SCI displayed three-fold increases in invertase activity compared to controls. Total sugar content was reduced and exhibited reduced sucrose content relative to hexoses. Despite the effect of increased invertase activity on metabolites, maximum catalytic activity of enzymes involved in cytosolic sucrose, hexose and organic acid metabolism were unchanged. Transgenic plants selected in these studies were subsequently vegetatively replicated and future work will include immature fruit.

Thesis (MSc (Genetics. Institute of Plant Biotechnology))--Stellenbosch University, 2008. / Traditional methods used for the discovery of novel genes have previously relied upon the ability to culture the relevant microbes and then demonstrate the activity of a specific enzyme. Although these methods have proved successful in the past, they severely limit our access to the genomes of organisms which are not able to be cultured under laboratory conditions. It was therefore the aim of this project to use metagenomic strategies for the identification of novel polymer-producing genes with the prospect of commercial exploitation. In this study, soil-derived metagenomic libraries were functionally screened for potential -glucan producing clones using aniline blue staining. Positive reacting clones were selected and sequenced. Initial sequencing revealed a gene with high homology to previously described glucan synthases, the products of these genes all having significant industrial value. The clone was transformed into a suitable bacterial host, cultured and allowed to produce the polymer of interest. The polysaccharide was purified and subjected to various chemical analyses so as to confirm its monosaccharide composition. Data suggests that this polymer is composed mainly of glucose units and that it may be secreted out of the cell. Purification of the active enzyme was attempted using classical protein purification methods with faint activity being detected using Native polyacrylamide gel electrophoresis (PAGE). Further attempts to demonstrate activity were made through the construction of a GST (glutathione S-transferase) tagged fusion protein. The second part of this study focuses on the construction and screening of a metagenomic DNA library from whey, a by-product of the cheese manufacturing process. It was envisaged that this could provide a resource for the identification of high value polymers when lactose is provided as a sole carbon source. The library was screened for function using Congo Red for the detection of extra-cellular polysaccharides.

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.