Browsing Masters Degrees (Genetics) by Title
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- ItemThe analysis and reduction of starch in sugarcane by silencing ADP-glucose pyrophosphorylase and over-expressing β-amylase(Stellenbosch : University of Stellenbosch, 2007-12) Ferreira, Stephanus Johannes; Groenewald, J-H.; Lloyd, James RichardSugarcane 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.
- ItemAnalysis of aspects of the starch metabolic pathway in lower plants(Stellenbosch : Stellenbosch University, 2018-12) Jacobs, Ingrid; Lloyd, James Richard; Stellenbosch University. Genetics & Institute of Plant Biotechnology.ENGLISH ABSTRACT: Starch is an important plant storage polysaccharide that has been demonstrated to have a major influence on plant growth. Transitory starch is synthesized in the leaves of plants during the day as a product of photosynthesis and degraded at night to allow continued carbon allocation for growth and cellular processes. It is also produced and stored for longer periods of time in non-photosynthetic organs such as stems, tubers and seeds. The study of starch is important for several reasons – not only is it a vital part of the human diet, it is also utilised in many non-food applications such as the paper, textile, oil and pharmaceutical industries. The pathway of starch metabolism in higher plants has been studied for decades and of late, Arabidopsis has become the workhorse plant for many starch researchers due to the plethora of insertion mutants that are readily available. However, the predominant use of Arabidopsis as a model system has led to a narrow understanding of starch metabolism restricted to that of synthesis and degradation of leaf starch. There are ongoing attempts to translate the knowledge gleaned from Arabidopsis into studies on the storage organs of crop plants (e.g. rice and maize endosperm, potato tubers), as well as starch metabolism in lower plants (e.g. algae and mosses) to aid in elucidating the evolutionary development of starch metabolism in land plants. This study investigated two aspects of starch metabolism in lower plants to determine whether the pathway of starch metabolism observed in higher plants is conserved. Firstly, a previously uncharacterised starch synthase from the red alga Chondrus crispus was examined due to the reported differences in substrate preference between red algal and green plant starch synthases, and the deviation in compartmentalisation of starch synthesis and storage in members of the red algae. The C. crispus starch synthase was analysed by means of multiple sequence alignment, site-directed mutagenesis and recombinant protein expression and purification. Features unique to red algal starch synthases were identified, including a C-terminal glycogen binding domain and sequence variations in important residues involved with substrate binding. During recombinant expression, the C. crispus protein was insoluble and accumulated in inclusion bodies. Attempts to recover active protein through optimisation of expression, the use of alternative expression systems and protein refolding were unsuccessful and biochemical characterisation of the starch synthase could not be performed. Secondly, four putative orthologues of the Arabidopsis maltose excess (MEX) transporter were identified in the moss Physcomitrella patens and their functions examined through the generation of knockout mutant lines and complementation of Escherichia coli mutants defective for sugar transporters. Knockout mutants were successfully generated for the P. patens MEX1a gene, while complementation studies failed to produce active protein. Expression profiling in wild type P. patens suggest that the four PpMEX genes are differentially expressed depending on the developmental stage of the culture and may have specialised functions in various growth structures.
- ItemAnalysis of dextrin dextranase from Gluconobacter oxydans(Stellenbosch : Stellenbosch University, 2008-12) Van Wyk, Nathan; Lloyd, James Richard; Kossmann, J. M.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.Dextran is a high value glucose polymer used in medicine and an array of laboratory techniques. It is synthesised by lactic-acid bacteria from sucrose but has also reportedly been produced by Gluconobacter oxydans (G. oxydans) from a range of maltooligosaccharides (MOS) via the action of dextrin dextranase (DDase). In this study the presence of DDase is investigated in two G. oxydans strains (ATCC 621H and ATCC 19357) and shown to be present in the ATCC 19357 strain, but not in the ATCC 621H strain. The enzyme was partially purified from the ATCC 19357 strain, and its kinetic properties investigated. The partially purified protein was also digested with trypsin, and de novo peptide sequences obtained from it. Several attempts were made to obtain the gene coding for the DDase. These include amplifying an open reading frame from the G. oxydans genome coding for a glycosyltransferase with the approximate molecular weight of the DDase, using the peptide sequences obtained from the partially purified protein to design degenerate PCR primers and the production of a genomic DNA library for functional screening in E. coli. None of these approaches led to the successful isolation of the extracellular DDase sequence.
- ItemAnalysis of enzymes involved in starch phosphate metabolism(Stellenbosch : University of Stellenbosch, 2009-12) Samodien, Mugammad Ebrahim; Lloyd, James Richard; Kossmann, J. M.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB)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.
- ItemAnalysis of genes implicated in iron regulation in individuals presenting with primary iron overload in the South African population(Stellenbosch : University of Stellenbosch, 2007-03) Booley, Fadwah; Zaahl, M. G.; Warnich, L.; Robson, K. J. H.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics.Hereditary haemochromatosis (HH), a common autosomal recessive disease, is characterized by increased iron absorption leading to progressive iron accumulation in organs such as the liver, heart and pancreas. In the South African population the disease is prevalent in individuals of Caucasian origin, with a carrier frequency of one in six for the C282Y mutation in the HFE gene. We investigated the role of genes implicated in iron metabolism, including the high-iron gene (HFE), haem oxgenase-1 gene (HMOX1), solute carrier family 40 (iron-regulated transporter) member 1 gene (SLC40A1), cytochrome b reductase gene (CYBRD1), hepcidin antimicrobial peptide gene (HAMP) and the hemojuvelin gene (HJV) in a patient cohort with non-HFE iron overload. DNA analysis was performed on samples from 36 unrelated South African Caucasian patients presenting with primary iron overload, who tested either negative or heterozygous for C282Y. In this study, mutation screening was performed by PCR amplification and HEX-SSCP analysis. Sixteen previously described and two novel variants were identified by semi-automated DNA sequencing. Common variants identified in the HFE gene included C282Y, H63D, IVS2+4T→C, IVS4-44T→C, IVS4+48G→A and IVS5-47G→A. The Q127H mutation in exon 3 of the HFE gene was identified in one patient, who tested negative for both C282Y and H63D. Mutation S65C was identified only in the population-matched controls and was absent in the patient group. Other previously described polymorphisms identified included the IVS5+51delTGGCTGTCTGACT deletion in HMOX1, I109 and V221 in SLC40A1, IVS1-4C→G, IVS2+8T→C and S266N, in the CYBRD1 gene and, S264 and A310G in the HJV gene. The novel variants, -89C→T, in the promoter region of the CYBRD1 gene, was detected in only one patient, while S333 in exon 4 of the HJV gene was present in three patients. These variants were not identified in any of the population-matched controls screened and could explain the non-HFE iron overload presented by these patients. This study clearly demonstrates the importance of modifier genes in patients with iron overload that cannot be explained by the common C282Y mutation. Studies on iron-related genes and the identification of mutations in these genes in non-HFE patients could lead to improved diagnosis and counselling of South African patients presenting with primary iron overload.
- ItemAnalysis of genetic variants in the 5’ regulatory region of the ALAS1 gene in South African patients with Variegate Porphyria (VP)(Stellenbosch : University of Stellenbosch, 2007-03) Du Plessis, Nelita; Warnich, L.; Zaahl, M. G.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics.The porphyrias are a group of genetic disorders arising from mutations in either one of the final seven genes encoding the haeme synthesis enzymes. These disease-causing mutations lead to an enzyme deficiency that disrupts normal haeme production, resulting in clinical features due to the subsequent accumulation of porphyrin precursors. Like most of the porphyrias, variegate porphyria (VP) is characterized by high inter- and intra- familial clinical variability, with no apparent genotype-phenotype correlation. The delta-aminolevulinate synthase-1 gene (ALAS1) is an apparent candidate gene to explain the variable clinical expression observed in VP, since it encodes the first and rate-determining enzyme of haeme synthesis. Several studies have defined important regulatory elements for the human-, rat- and chicken ALAS1 gene that regulate expression patterns of this gene. It was hypothesized that in VP individuals, variants within/near critical regulatory sites might alter the transcription rate of this gene, and consequently increase/decrease the amount of haeme precursors accumulating as a result of the defective haeme synthesis enzyme. The aim of this study was to identify genetic variants that could influence gene expression in the proximal promoter area of the ALAS1 gene, as well as the two ALAS1-drug responsive enhancer sequences (ADRES) located further upstream. DNA (2133 bp per patient) of 19 clinically defined VP patients was analysed by polymerase chain reaction (PCR) and semiautomated DNA sequencing. Subsequently, in silico analyses using appropriate software programs, and in vitro studies using the luciferase reporter system, were performed to investigate the functionality of the identified variants on ALAS1 gene transcription...
- ItemThe analysis of glycogen phosphate and glucose-1,6-bisphosphate metabolism in escherichia coli(Stellenbosch : Stellenbosch University, 2015-03) Jewell, Jonathan Frederick; Lloyd, James Richard; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.ENGLISH ABSTRACT: This thesis examined two aspects of E. coli carbon metabolism, the incorporation of covalently bound phosphate into glycogen as well as the manufacture of glucose-1,6-bisphosphate (GBP). In vitro analysis using recombinant maltodextrin phosphorylase (MalP) incubated together with maltodextrin, glucose-1-phosphate (Glc-1-P) and GBP resulted in the incorporation of phosphate into manufactured polymer at levels of 15 nmol Glc-6-P/mg polymer. No phosphate could be detected in the same incubation lacking only GBP. Moreover, higher amounts of polymer were also present in incubations where GBP was present with Glc-1-P, compared with Glc-1-P alone. Attempts were made to purify glycogen phosphorylase (GlgP), but these were unsuccessful. To examine if MalP and/or GlgP carry out this reaction in vivo, strains lacking them were produced. However, analysis revealed no significant difference in the phosphate content of glycogen extracted from wild type, single and double mutants lacking glgP and malP. A protein responsible for the synthesis of a phosphoglucomutase (PGM) stimulatory compound was purified to apparent homogeneity. This was identified, through tryptic fingerprinting, as the acid glucose-1-phosphate phosphatase (AGP) protein. Using recombinant AGP protein it was demonstrated that it was able to produce GBP from Glc-1-P in a phosphotransferase reaction, where one phosphate from Glc-1-P phosphorylates the C6 position of another. However, agp mutant cells were unchanged in the amounts of GBP they accumulate and crude protein extracts from them were still capable of synthesizing GBP from Glc-1-P. A mutant strain lacking both agp and pgm could no longer produce a PGM stimulatory compound, indicating that PGM most likely also synthesises GBP.
- ItemAnalysis of intermediate carbon metabolism in strawberry plants(Stellenbosch : Stellenbosch University, 2008-12) Basson, Carin Elizabeth; Groenewald, J.-H.; Bauer, R.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.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.
- ItemAnalysis of phosphoglucomutase isoforms from physcomitrium patens(Stellenbosch : Stellenbosch University, 2023-03) De Stadler, Jessica Amy; Lloyd, James R.; Stellenbosch University. Faculty of AgriSciences. Department of Genetics & Institute of Plant Biotechnology.ENGLISH ABSTRACT: Starch is the main storage polymer found in most plants and plays a significant role in plant fitness. It is a polyglucan and is composed of two separate fractions named amylose and amylopectin. In photosynthetic tissues starch is synthesized in chloroplasts during the day and degraded at night to provide energy which supports metabolism. The pathways of starch synthesis and degradation have been extensively characterised in Angiosperms, however, in non-vascular plants these are less well understood. Physcomitrium patens is a Bryophyte that is an excellent non-vascular model plant due to its fully sequenced genome and the ease at which mutants can be created using homologous recombination. As Bryophytes have been classified as a transitionary species between water-based algae and land plants, research using them sheds light on how biochemical processes (such as starch metabolism) have changed during land colonisation. The first committed step of the starch biosynthetic pathway is the creation of ADP-glucose from glucose 1-phosphate (G1P). The formation of G1P is catalysed by plastidial isoforms of phosphoglucomutase (PGM) and these enzymes are encoded by a highly conserved family of genes. Previous studies in Angiosperms have demonstrated that mutating the plastidial isoform of PGM results in plants that accumulate almost no starch in all organs (Hanson & McHale, 1988; Harrison, et al., 2000; Vriet, et al., 2010). Cytosolic phosphoglucomutase isoforms are also present and the protein sequences of these are similar to those of plastidial isoforms. The first aspect of this project was to identify PGM genes in P. patens that demonstrate high similarity to PGM genes from Arabidopsis thaliana. Four were identified (named PpPGM1-4), and the amino acid sequences of the translated P. patens PGM polypeptides have high similarity to other phosphoglucomutases. The predicted intron-exon boundaries showed that PpPGM1 and PpPGM2 genes contain no introns whereas PpPGM3 and PpPGM4 contain seventeen introns each. Phylogenetic analysis of PGM sequences from red algae, Cyanobacteria and Viridiplantae demonstrated that sequences could be divided into three clades. One contained red algal and cyanobacterial sequences while the other two contained only Viridiplantae PGM’s. One of the Viridiplantae clades contained all isoforms that have been demonstrated experimentally to be plastidial (and PpPGM1 & 2), and the other all isoforms that have been demonstrated experimentally to be localised in the cytosol (and PpPGM3 & 4). The sub-cellular localization of the PGM protein was examined by transiently expressing PGM genes, which had been fused to a gene encoding the green fluorescent protein, in P. patens protoplasts. This demonstrated that PpPGM1 and PpPGM2 were imported into plastids and PpPGM3 and PpPGM4 were localised in the cytosol. All four genes were shown to encode active proteins and their expression restored the wildtype phenotype in an Escherichia coli pgm mutant. Furthermore, analysis of the PGM amino acid sequence showed that PpPGM1 and PpPGM2 both contain a SASHNP active site motif whereas in PpPGM3 and PpPGM4 this is TASHNP. Similarly, a metal binding motif differed between the four polypeptides, being DGDGD in PpPGM1 and PpPGM2 and DGDAD in PpPGM3 and PGM4. The known sugar binding sequence CGEESF was found in all four proteins. These versions of the active sites were conserved across the two Viridiplantae clades. The final aspect of this project was to create knockout mutants of the two plastidial PGM genes in P. patens to identify their effect on phenotype, this was attempted using PEG mediated homologous transformations, several plants survived selection but were demonstrated to be untransformed.
- ItemAnalysis of schizophrenia susceptibility variants identified by GWAS : a bioinformatics and molecular genetics approach(Stellenbosch : Stellenbosch University, 2014-12) Coffee, Michelle; Warnich, Louise; Niehaus, D. J. H.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.ENGLISH ABSTRACT: Described as one of the costliest and most debilitating disorders, schizophrenia has proven to be among the greatest challenges for medical researchers. The disorder poses difficulties on all levels: from genotype to phenotype. Even though it is known that there is a substantial genetic contribution to schizophrenia susceptibility (~80%), it is unknown whether this is due to common variants, rare variants, epigenetic factors, polymorphisms in regulatory regions of the genome or a combination of all these factors. Over the past few decades, many approaches have been employed to elucidate the genetic architecture of schizophrenia, with the latest and most promising being genome wide association studies (GWAS). However, nearly a decade after the first GWAS, the limitations are increasingly being recognised and new avenues need to be explored. Studies have recently started to focus on the analysis of non-coding regions of the genome since these regions harbour the majority of variants identified in GWAS thus far. This study aimed to use recently developed programs that utilize data from large scale studies such as previous GWAS, the Encyclopaedia of DNA Elements (ENCODE), 1000 Genomes, HapMap and Functional Annotation of the Mammalian Genome (FANTOM) to establish a simple, yet effective bioinformatics pipeline for the identification and assessment of variants in regulatory regions. Using the established workflow, 149 single nucleotide polymorphisms (SNPs) in regulatory regions were implicated in schizophrenia susceptibility, with the most significant SNP being rs200981. Pathway and network analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID) and GeneMANIA respectively indicated that the most frequently affected genes were involved in immune responses or neurodevelopmental processes, which support previous findings. Yet, novel findings of this study implicated processes crucial for DNA packaging (from DNA level to chromatin level). The second part of the study used restriction fragment length polymorphism analysis of polymerase chain reaction-amplified fragments (PCR-RFLP) to genotype ten of the most significant SNPs (identified by bioinformatic analyses in the first part of the study) in a South African Xhosa cohort of 100 cases and 100 controls, while bi-directional Sanger sequencing was used to confirm the presence of these SNPs. Statistical analyses revealed two haplotypes of regulatory variants, rs200483-rs200485-rs2517611 (p = 0.0385; OR = 1.71; 95% CI = 1.01-2.91) and rs200981-rs2517611-rs3129701 (p = 0.041; OR = 0.51; 95% CI = 0.27-0.98) associated with schizophrenia susceptibility. Bioinformatic analysis indicated that these haplotypes affect DNA packaging, which supported the findings of the first part of the study and could implicate epigenetic processes. The findings of this study support the importance of regulatory variants in schizophrenia susceptibility. This study also showed the importance of combining GWAS data with additional analyses in order to better understand complex diseases. It is hoped that these findings could fuel future research, specifically in genetically unique populations.
- ItemAnalysis of starch metabolism in South African pigeon pea (Cajanus cajan) varieties(Stellenbosch : Stellenbosch University, 2023-03) Kulu, Nokwanda; Lloyd, James R. ; Peters, Shaun Wayne; Stellenbosch University. Faculty of AgriSciences. Department of Genetics & Institute of Plant Biotechnology.ENGLISH ABSTRACT: Starch is a major storage polyglucan in plants that is composed of two fractions, amylose and amylopectin. The biosynthesis and degradation pathways of starch are well documented, with phosphoglucomutase (PGM) and ADP-glucose pyrophosphorylase (AGPase) catalysing the first two steps in its biosynthesis. This project examined starch in five pigeon pea (Cajanus cajan) varieties: uDhali, SEFA, Nondolo, Lari and India by measuring both total and resistant starches in the seeds and leaves, activities of PGM and AGPase as well as expression of the genes encoding these enzymes. The findings demonstrated that the seeds from these South African pigeon pea varieties are rich in starch, containing an average of 47% starch on a dry weight basis; however, one variety (SEFA) contained only 0.3% starch. The starch in the high- starch varieties contained a minimum of 50% resistant starch, with the India variety reaching 70%. Assessment of soluble sugars in seeds revealed sucrose to be the only sugar present in abundance in all varieties while amounts of galacto-oligosaccharides were low in all seeds. Starch in leaves was observed to be 10 fold less than that found in seeds and the amount of resistant starch in leaves was less than 2 mg/g fresh weight (7.6% of the total). The AGPase gDNA nucleotide sequence from one variety was identical to an already sequenced pigeon pea variety, whereas amplification PGM gDNA was unsuccessful. Amplification of coding sequences (CDSs) for both AGPase and PGM were also identified to be the same as the already sequenced AGPase and PGM genes from the pigeon pea genome resource database. Gene expression for both genes varied throughout a 24 h period and was at its peak during the day (light period). Activities of both AGPase and PGM were determined in seeds from all varieties whereas the AGPase enzyme activity was the same in leaves throughout the day while PGM activity varied between the day (light) and night (dark).
- ItemAnalysis of sugar accumulation under cold stress in Physcomitrium patens(Stellenbosch : Stellenbosch University, 2021-12) Jacobs, Desren Brando; Lloyd, James Richard; Peters, Shaun; Kossmann, Jens; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.ENGLISH ABSTRACT: Plants adapt to cold temperatures through the process of cold acclimation. Key experimental observations indicate that one of the common elements in cold acclimation are the accumulation of various sugars. In the moss Physcomitrium patens, it has been reported that a trisaccharide, theanderose, accumulates in response to cold acclimation and is induced by abscisic acid (ABA) – the major stress-linked hormone in plants. Due to its rarity in nature, little is known about the biochemistry of theanderose production. Some sugars accumulating under cold acclimation may be the result of pathways leading from starch degradation. For example, the accumulation of maltose pools which further leads to glucose and sucrose synthesis. While the starch degradation pathway is well characterised in angiosperms, little is known of its importance in bryophytes such as P. patens. Recently, the effects of specific mutations in starch metabolism have been examined in P. patens. In that study mutations in glucan-water dikinase (Ppgwd1) and a disproportionating enzyme (Ppdpe2) were targeted, as mutations in homologs of these gene are known to affect starch degradation in other angiosperms. This study investigated two aspects of soluble sugar accumulation in P. patens. Firstly, we examined if a combination of ABA and exposure to low temperatures led to starch degradation alongside increased soluble sugar accumulation. Wild type plants alongside Ppgwd1 and Ppdpe2 mutants were grown, in vitro, at 25°C for 8 weeks on synthetic growth media and sub-cultured onto growth media supplemented with 10μM ABA at 4°C for 2 days. The starch and sugar contents in each line were measured and no significant increases were seen in any of the wild type or the mutant lines across the two-day incubation. A second experiment over 6 days was attempted on wild type material, but again no significant trend was noticed. The use of LC-MS/MS allowed to examine the presence of other possible sugars accumulating in P. patens during cold exposure. Interestingly we did not observe theanderose as being present but detected raffinose along with two other unknown sugars. Secondly, we examined various enzymes that may be involved in theanderose or raffinose synthesis in P. patens. Expression of three α-glucosidase enzymes were performed in an E. coli malq mutants, however, none appeared to catalyse glucosyltransferase reactions when that would lead to the formation of theanderose. Putative P. patens raffinose synthase enzymes were also assessed in a bacterial system. Gene sequences encoding two putative PpRAFS protein were amplified and heterologously expressed to produce recombinant protein. Unfortunately, after affinity purifications bands of the predicted size were identified in the negative control, indicating that PpRAFS was not present in the samples. Gene expression of the two putative PpRAFS was examined within wild type tissue. Both genes showed high expression at 25 °C when incubated with ABA.
- ItemAnalysis of the effects of the plant growth promoting substances GR24 and smoke water on abiotically stressed Nicotiana benthamiana seedlings(Stellenbosch : Stellenbosch University, 2012-03) Steenkamp, Letitia Elizabeth; Hills, Paul N.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.ENGLISH ABSTRACT: Almost all processes during the life of a plant are affected by the environment. Changes in phytohormone, metabolite and protein levels follow in response to changes in the environment. Plant growth promoting substances can stimulate changes at these levels to facilitate increased plant growth and yields above what the plant would normally establish. In this study, the effects of two growth promoting substances, smoke water (SW) derived from bubbling smoke from the burning of plant material through water, and a synthetic strigolactone analogue, GR24, on plant growth and architecture, as well as the proteome and metabalome of salt stressed Nicotiana benthamiana seedlings were investigated. Physiological studies were conducted to identify the effects of the growth substances on salt stressed seedlings in a tissue culture system. Under non-stress conditions, SW treatment increased seedling fresh mass, root length and leaf area. Under salt stress conditions (100 mM and 150 mM NaCl), SW increased fresh mass, root length, leaf number and lateral root number significantly. Under non-stress conditions, GR24-treated seedlings showed increased fresh mass, leaf number and area and root length. When GR24-treated seedlings were placed under salt stress, the seedlings showed significant increases in fresh mass, leaf number and lateral root number, but only marginal increases in root length and leaf area. Despite these similarities, slight differences were observed in the metabolomes and proteomes of smoke water and GR24-treated seedlings, both with and without the addition of salt stress. Relatively few of the differentially expressed proteins could be identified with the instruments available. Changes in the metabolome indicated that photoassimilation and photosynthesis could be affected in response to smoke water and GR24 treatment. Our results suggest that smoke water and GR24 both promote growth under salt stress conditions in seedlings and we furthermore conclude that, although there are distinct overlaps between treatments, this is accomplished via slightly different mechanisms.
- ItemAnalysis of the relationship between glycogen turnover and cell size in Escherichia coli(Stellenbosch : Stellenbosch University, 2020-03) Van der Walt, Felix; Lloyd, James Richard; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB).ENGLISH ABSTRACT: Glycogen represents an important carbon energy store in organisms across all domains of life. Under permissible conditions, excess environmental glucose is incorporated into glycogen by the Gram-negative bacterium Escherichia coli to provide the cell with an endogenous carbon store. This can rapidly be mobilized to provide the cell with energy for sustained viability when nutritional conditions deteriorate. Extracellular nutrient availability positively impacts cell size and growth rate in a variety of organisms. Bacteria cultured in nutrient-rich media display significant increases in growth rate and cell size, compared to their slow-growing counterparts in nutrient-deprived conditions. Such nutrient-dependent increases in size and growth are accompanied by equally dramatic elevations in the rates of macromolecular biosynthesis (DNA/RNA/protein). How bacteria respond to environmental cues through their ability to sense size and correct random fluctuations that would deviate it from ‘normal’ has been the subject of substantial investigations over the last few decades. This is unsurprising as cell size control and homeostasis are fundamental to cell biology and, of course, to the survival of unicellular bacteria like E. coli. Research has historically focused on cell size and progression of the bacterial cell cycle within the context of extracellular nutrient availability, yet little is known about how endogenous metabolism affects these aspects of bacterial physiology. This investigation aimed to elucidate how glycogen turnover impacts cell size and progression of cell cycle events using E. coli mutants affecting three glycogen catabolic enzymes, glycogen phosphorylase (GlgP), glycogen debranching enzyme (GlgX) and maltodextrin phosphorylase (MalP). Disruption of malP resulted in a profound effect on cell size as ΔmalP mutants are unable to properly coordinate cell cycle progression during exponential growth, leading to substantial heterogeneity in size. This manifests as subpopulations of elongated and filamentous cells. Whilst such mutants do not necessarily form fewer Z-rings per cell, they clearly delay division and grow into filaments and the underlying reason for this appears to be a malfunction of DNA replication. Mutations in either glgP or glgX differently impact DNA replication and cell size and mutants with a lesion in the latter allele contain coinciding glycogen and protein inclusion bodies, particularly noticeable during exponential growth. The nature of the flaws to cell size control and DNA replication observed in ΔmalP mutant strains, specifically the ΔmalP/ΔglgP/ΔglgX triple mutant, was further scrutinized by introducing lesions to genes involved in several interacting processes. Mutating genes associated with glycogen accumulation, pyruvate kinase activity, and SOS-mediated or UDP-glucose-dependent division inhibition led to the formation of mutant cells either smaller or equal in size to the wild type. Partial suppressions to the size defects of the triple mutant were observed in quadruple mutant strains with disruptions to genes involved in amino acid metabolism, ppGpp biosynthesis, UDP-glucose generation, DNA replication and nucleoid structuring. DNA replication is clearly coordinated with diverse physiological processes acting in concert to link duplication of the genome with cell size, growth rate and environmental conditions.
- ItemAnalysis of the role of transcription factors in enhancing drought tolerance in sugarcane (Saccharum spp.)(Stellenbosch : Stellenbosch University, 2020-12) Mbambalala, Nelisa; Van der Vyver, Christell; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.ENGLISH ABSTRACT: Sugarcane is a large perennial grass of the genus Saccharum. Economically, this grass species is an important source of sugar for food purposes and biomass for biofuel production. However, the sustainability of production is greatly constrained by drought, which directly affects crop yield. Drought can lead to modification of metabolic processes in the plant, membrane disorder, disruptions and instabilities of many physiological and biochemical process, including photosynthesis and increased production of reactive oxygen species (ROS), which causes oxidative stress and can ultimately lead to plant death. It is therefore essential to continue developing cultivars with improved drought tolerance, which can possibly be achieved through the identification and introduction of genes that confer tolerance in crops. Transcription factors (TF) are gene regulators that control gene expression and consequently stress responses in plants. A single TF can regulate the expression of many target genes. This study aims at analysing the role of two TFs namely, BBX (B-Box Zinc Finger) and NAC2 (NAM, ATAF and CUC) in enhancing drought tolerance in sugarcane. For this, sugarcane was independently genetically transformed via particle bombardment with a BBX TF from Arabidopsis thaliana and a NAC2 TF from tomato. Attempts were also made to determine the subcellular localization of the AtBBX29 and SINAC2 genes but results were inconclusive due to poor microscopic imaging and faint GFP reporter signals. Transgene insertion was confirmed in putative transformed sugarcane through PCR analysis and transgene expression through semi-quantitative reverse transcriptase PCR. Transgenic sugarcane plantlets were planted ex vitro and drought pot trials were setup in the glasshouse. Once plants were deprived of water, phenotypic changes in transgenic sugarcane lines were compared to non-transgenic control sugarcane plants. Under drought conditions, both AtBBX29 and SINAC2 overexpression in sugarcane enhanced drought tolerance. All transgenic plants exhibited higher survival and recovery rates than wild-type (WT) plants. Transgenic plants overexpressing AtBBX29 maintained relative water content (RWC) at levels not significant different from the WT plants. However, these plants maintained significantly higher chlorophyll fluorescent rates and stomatal conductance under mild and severe drought conditions. Under severe water-deficit stress, oxidative damage was reduced in BBX transgenic plants which exhibited low malondialdehyde (MDA) levels and less accumulation of reactive oxygen species (ROS) throughout the water-deficit stress period. The scavenging activity of antioxidants, which was present at significantly higher levels in the transgenic plants under severe water-deficit stress, most likely played a role in reducing the ROS levels. Transgenic plants also accumulated significantly more proline under mild and severe stress conditions compared to the WT plants. Abscisic acid levels varied between WT and transgenic plants exposed to drought. In addition, BBX transgenic sugarcane was grown to maturity (8 months) under a normal watering regime in the glasshouse where these plants displayed normal phenotypes and no significant difference in carbohydrate content compared to non-transgenic control plants. A preliminary drought trial was conducted with the SINAC2 transgenic sugarcane plants. Overexpression of SINAC2 enhanced drought tolerance in transgenic sugarcane plants exposed to water-deficit stress with higher survival rates seen in the transgenic lines compared to the WT plants. Transgenic plants overexpressing SINAC2 maintained significantly higher RWC levels and displayed less visual damaged such as leaf wilting and yellowing than the WT plants. Over the course of water-deficit period the root biomass increased in all genotypes, but less so in the transgenic plants, SINAC2 overexpression however enhanced root elongation. Transgenic plants also upheld photosynthesis, with high chlorophyll fluorescence and stomatal conductance seen in most transgenic plants under severe water-deficit stress.
- ItemApplication of plant growth promoting substances and arbuscular mycorrhizal fungi for phytostabilisation of mine tailings(Stellenbosch : Stellenbosch University, 2016-03) Rossouw, Marthinus Jacob; Hills, Paul N.; Thompson, David I.; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB).ENGLISH ABSTRACT: This study focused on investigating methods of phytostabilisation of mine tailings operated by Palabora Copper in South Africa. Capping material and mine tailing at various sites of the mine were collected and used in pot trials to investigate the effect of a number of plant growth promoting substances (PGPS) on several of grass species currently used in effort to stabilise the areas in question. Lumichrome, strigolactones (GR24), flavonoids (CropbioLife™), smoke-water (karrikins) and arbuscular mycorrhizal fungi (Mycoroot™) were used as PGPS to investigate growth-promoting effects on i) Anthephora pubescens, Cenchrus ciliaris, Chloris gayana, Cynodon dactylon, and Panicum maximum which are species currently used by Palabora Copper for rehabilitation of mine tailings, and ii) Additional grass species theoretically suited to surviving the environment. Treatments were applied on 2-week old transplanted grass seedlings in pot trials containing mine capping material as the substrate, to infer treatment responses. Trypan-Blue staining procedures were used to ascertain which grass species formed symbiotic relationship with arbuscular mycorrhizal fungi (AMF), which would potentially aid in their survival in deleterious areas. Germination rates were measured to determine the fastest germinating species of the selected grasses with Eragrostis teff and Melinis repens germinating the quickest in the mine capping material. Capping material and mine tailing samples were collected at sites under revegetation by Palabora Copper. This included samples of the rhizosphere of locally abundant plants at two sites: a recently (two years) capped mine tailing, and a rock dump site (capped 10-12 years previously). Five rhizosphere samples were collected from individuals of Cenchrus ciliaris, Enneapogon cenchroides, and Tephorisia polystachya (a locally abundant forb species) at site 1 and Cenchrus ciliaris, Stipagrostis hirtigluma, Tephrosia polystachya, and Pennisetum setaceum at site 2. At both sites the soil of open areas devoid of plants was also sampled. Metagenomic DNA was extracted from the collected samples, often following enrichment techniques. Dilution series spread plates to determine culturable bacteria present in the tailing samples were also utilised. Polymerase Chain Reactions were implemented to produce amplicons of conserved regions within AMF and bacteria present in the mine tailing site. The predominant genera of bacteria detected in the collected tailing samples belonged to Bacillus. However, due to the use of enrichment techniques it was not possible to comment on the relative abundance of different bacteria in the environment where the samples were collected. Due to the small-scale ex situ nature of the experiments the results gained from the PGPS treatment trials and microbial DNA isolation are not necessarily representative of the ecological environment present in situ. However, PGPS treatment of the selected grasses did not elicit any clear beneficial responses in the measured growth parameters, making application thereof of limited benefit for phytostabilisation purposes. Trypan staining revealed most of the grass species are capable of forming symbiotic relationships with mycorrhizal fungi, with trials indicating that AMF might benefit plants present in the mine tailings.
- ItemApproaches for the study of leaf carbohydrate metabolic compartmentation in arabidopsis thaliana(Stellenbosch : University of Stellenbosch, 2010-12) Fly, Richard Derek; Lloyd, James Richard; Van der Merwe, M. J.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB).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.
- ItemThe Arabidopsis GolS1 promotor as a potential biosensor for heat stress and fungal infection?(Stellenbosch : Stellenbosch University, 2016-12) Janse van Rensburg, Henry Christopher; Peters, Shaun; Loedolff, Bianke; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.ENGLISH ABSTRACT: Galactinol (Gol) has classically been considered to serve as a galactose donor during the biosynthesis of raffinose family oligosaccharides (RFOs). These sucrosyl oligosaccharides have been well characterised in their roles in carbon translocation and storage and, abiotic stress protection in plants. However, recent findings have demonstrated Gol to be an efficient free radical scavenger and it has also been suggested to act as signalling molecule during induced systemic resistance (ISR), upon pathogen infection. Collectively, these findings centres to the involvement of only a single galactinol synthase gene (GolS, synthesising Gol) in Arabidopsis (AtGolS1, At2g47180). The AtGolS1 isoform has been shown to be transcriptionally up-regulated during heat stress and Botrytis cinerea infection. Further, it is also responsive to jasmonic acid, a key component of the ISR pathway. Here we targeted the AtGolS1 promotor containing well defined heat shock transcription factor elements and a single putative jasmonate binding element, to develop a dual-functional biosensor with the ability to detect both heat stress and Botrytis cinerea infection. We created transgenic Arabidopsis lines where the reporter genes β-glucuronidase (GUS) and the green florescent protein (GFP) were under the control of the AtGolS1 promotor. Using the native AtGolS1 gene as a point of reference, we confirmed that the reporter genes were transcriptionally responsive to both heat stress and methyl jasmonate treatment in transgenic Arabidopsis. Under the same experimental conditions, both GUS assays and GFP imaging correlated with these transcriptional responses. Finally, we infected the transgenic lines with Botrytis cinerea infections to analyse reporter activity. Transcript analysis of transgenic lines clearly showed an increase in transcript abundance for both the native AtGolS1 and the reporter genes in reponse to B. cinerea infection. Similarly, reporter assays revealed a distinct difference in activity between infected and uninfected plants from 24h to 96h after Botrytis cinerea infection. These results provide sufficient proof-of-concept for the AtGolS1 promotor to be used as a dual functional biosensor for both heat stress and fungal infection.
- ItemAssessing the cyto-genotoxic impacts of un-neutralised and pH-neutralised acid mine drainage on the human breast cancer cell line, MCF-7(Stellenbosch : Stellenbosch University, 2015-12) Botha, Shirmone; Botha-Oberholster, Anna-Maria; Genthe, B.; Oberholster, P.; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.ENGLISH ABSTRACT: The use of toxicity tests to evaluate the quality of streams affected by mixtures such as acid mine drainage (AMD), adds value to assessments whereby site-specific toxicological data may identify toxicants that pose a threat to humans. To successfully evaluate the risk of combined mixtures, an improved understanding of the individual components, their uptake, metabolism, excretion and mode of action is required. This study aimed to identify the extent of AMD toxicity in a dose dependant manner on the MCF-7 cell line. The first study site associated with gold mining was chosen as the Tweelopies Stream situated in the Gauteng province of South Africa. The AMD effluent (un-neutralised) contaminating the Tweelopies Stream had undergone pH-neutralisation using a reactor-bed limestone technology incorporating the use of both calcium carbonate (CaCO3) powder and limestone beds. The second study site, the Kromdraai River, is situated in the eMalahleni region of South Africa where a predominance of coal mining exists. The pH -neutralisation of the AMD (un-neutralised) contaminated Kromdraai River was performed using a caustic soda (NaOH) precipitation technique. This study demonstrated the rapid and effective application of the comet assay as a screening tool for AMD-associated DNA breakages in the human cell line, MCF-7. Moreover, the study analysed parameters of cellular survival, DNA fragmentation and variations in morphologies indicative of cellular death. Collectively, the cyto-genetic aberrations observed in the MCF-7 cells as a result of exposure to gold and coal mining associated AMD, confirms the urgency of incorporating high-throughput screening in ecological toxicity assessment to evaluate cellular damage at genetic levels in low dose exposures where detection might be missed.
- ItemAssessment of yield traits between family groups of the cultured abalone (Haliotis midae) in South Africa(Stellenbosch : Stellenbosch University, 2012-03) Van Schalkwyk, Hester Josina; Brink, Danie; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.ENGLISH ABSTRACT: The abalone Haliotis midae is the most important aquaculture species in South Africa. The industry is dependent on export to Far Eastern markets in a variety of forms, including live, frozen, canned or dried. The species is considered undomesticated in the sense that the current commercial broodstock has been obtained from natural populations through a process of random collection. Global competition has necessitated the South African industry to introduce a genetic improvement program to increase biological productivity and financial profitability. The objective of this study was to assess the genetic variation and to estimate key parameters in terms of growth and yield related traits, between family groups that form part of the breeding program. The study reports on heritability estimates of growth rate (0.14 ± 0.05), canning yield (0.08 ± 0.03), and drip loss during live export (0.03 ± 0.02). The high genetic correlation (0.94 ± 0.34) between shell length and live weight enables industry to utilise either weight or shell length as a criteria during operational practices such as sorting, grading and harvesting. The correlation of 0.85 ± 0.01 between live weight and canning loss indicates that animals that weigh more have a lower dressing percentage. Based on these low heritability values obtained for yield related traits it is recommended not to include these traits in the selection program at this stage. The findings of the study were however, compromised by the availability of a limited number of family groups, the age differences between families and the effect of different locations on the variance in phenotypes. Further investigation is needed to confirm the credibility of the results.