Department of Genetics

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Abiotic stress-induced accumulation of raffinose in Arabidopsis leaves is mediated by a single raffinose synthase (RS5, At5g40390)
(BioMed Central, 2013) Egert, Aurelie; Keller, Felix; Peters, Shaun
Background: The sucrosylgalactoside oligosaccharide raffinose (Raf, Suc-Gal1) accumulates in Arabidopsis leaves in response to a myriad of abiotic stresses. Whilst galactinol synthases (GolS), the first committed enzyme in Raf biosynthesis are well characterised in Arabidopsis, little is known of the second biosynthetic gene/enzyme raffinose synthase (RS). Conflicting reports suggest the existence of either one or six abiotic stress-inducible RSs (RS-1 to -6) occurring in Arabidopsis. Indirect evidence points to At5g40390 being responsible for low temperature-induced Raf accumulation in Arabidopsis leaves. Results By heterologously expressing At5g40390 in E.coli, we demonstrate that crude extracts synthesise Raf in vitro, contrary to empty vector controls. Using two independent loss-of-function mutants for At5g40390 (rs 5–1 and 5–2), we confirm that this RS is indeed responsible for Raf accumulation during low temperature-acclimation (4°C), as previously reported. Surprisingly, leaves of mutant plants also fail to accumulate any Raf under diverse abiotic stresses including water-deficit, high salinity, heat shock, and methyl viologen-induced oxidative stress. Correlated to the lack of Raf under these abiotic stress conditions, both mutant plants lack the typical stress-induced RafS activity increase observed in the leaves of wild-type plants. Conclusions Collectively our findings point to a single abiotic stress-induced RS isoform (RS5, At5g40390) being responsible for Raf biosynthesis in Arabidopsis leaves. However, they do not support a single RS hypothesis since the seeds of both mutant plants still contained Raf, albeit at 0.5-fold lower concentration than seeds from wild-type plants, suggesting the existence of at least one other seed-specific RS. These results also unambiguously discount the existence of six stress-inducible RS isoforms suggested by recent reports.
Accomplishments in wheat rust research in South Africa
(ASSAf, 2020-11-26) Pretorius, Zacharias A.; Prins, Renee; Wessels, Elsabet; Bender, Cornel M.; Visser, Botma; Boshoff, Willem H. P.
Rust diseases, although seasonal, have been severe constraints in wheat production in South Africa for almost 300 years. Rust research gained momentum with the institution of annual surveys in the 1980s, followed by race identification, an understanding of rust epidemiology, and eventually a focused collaboration amongst pathologists, breeders and geneticists. Diversity in South African populations of Puccinia triticina, P. graminis f. sp. tritici and P. striiformis f. sp. tritici has been described and isolates are available to accurately phenotype wheat germplasm and study pathogen populations at national, regional and global levels. Sources of resistance have been, and still are, methodically analysed and molecular marker systems were developed to incorporate, stack and verify complex resistance gene combinations in breeding lines and cultivars. Vigilance, capacity, new technologies, collaboration and sustained funding are critical for maintaining and improving the current research impetus for future management of these important diseases.
AFLP and PCR markers for the Ht1, Ht2, Ht3 and Htn1 resistance genes in maize
(Stellenbosch : Stellenbosch University, 2001-12) Van Staden, Derick; Retief, A. E.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Maize is undoubtedly South Africa's most important field crop. The identification of markers and genes for traits of interest is important to sustain the improvement of maize cultivation. Northern corn leaf blight (NClB) is a disease that occurs worldwide and can dramatically reduce yield. A number of single dominant resistance genes have been identified for NClB and some have been mapped. Currently there are no simple PCR markers for any of these resistance genes, making markerassisted selection (MAS) difficult. The aim of this study was to develop PCR markers for the NClB resistance genes Ht1, Ht2, Ht3 and Htn1 in maize. To accomplish this, the AFlP (amplified fragment length polymorphism) technique was first optimised. The results indicated that the Mlul/Msel restriction enzyme combination produces a higher percentage of polymorph isms when compared to the PstllMsel enzyme combination. It was also shown that the enzyme combination plays an important role in the percentage of polymorphic fragments observed, whereas the number of restriction enzymes used in AFlP analysis only significantly affects the total number of fragments scored. Populations segregating for the different resistance genes were not available for this study. Nearly-isogenic lines (Nils) were used in combination with AFlP technology to identify markers that map close to the genes. AFlP markers common in at least two resistant or susceptible lines were cloned and converted to PCR markers. Two commercially available recombinant inbred line (Ril) populations were then used to map the identified markers. For Htn1 fifteen polymorphic fragments were present in both resistant lines. They were selected for sequence specific marker conversion. Seven of the fifteen sequence characterized amplified region (SCAR) markers were polymorphic on the Nil pairs and five mapped to one region of maize chromosome 8.05/06. Twenty-one AFlP markers were identified for Ht1 and four SCAR markers were polymorphic In the Ht1 Nils. Three of these were mapped to chromosome 2.07. Three AFlP markers were identified for Ht2 of which two were converted to SCAR markers. Both SCAR markers were polymorphic on the Ht2 Nils and mapped to chromosome 8.05/06. On the Ht3 NILs, four AFLP markers were identified and two converted SCAR markers and one microsatellite marker (bnlg1666) were polymorphic. One of the SCAR markers and the microsatellite marker were mapped to chromosome 7.04 using a RIL population. This reports the first tentative mapping position for the Ht3 locus. The next step was to determine if a set of marker alleles could be used in a number of Htn 1 resistance lines to identify a common donor region selected by the breeders. Nine markers consisting of five SCAR markers, three converted RFLP markers and one microsatellite marker were used on 16 Htn1 resistant lines. The marker allele of us3 was in 12 of the 16 lines in coupling with Htn1 resistance. Second was the marker us5 in 11 of the 16 lines. Using this data 14 of the 16 lines shared a common introgressed region between the markers us3 and us5. A further common introgressed region between 11 of the inbred lines was found between the markers us14 and asg17. The last aim of this study was to propose a new marker technique that might be more successful than the AFLP technique in the identification of markers closely linked to genes. A new marker approach was identified where a MITE (Hbr) primer was used as an anchor primer in combination with resistance gene analog primers. This was found to be a highly polymorphic marker technique that could be used to identify markers and possibly candidate genes. It is a robust technique, which is affordable since amplifications occur from undigested genomic DNA and the primers mainly amplify fragments from genic regions.
African origin for Madagascan dogs revealed by mtDNA analysis
(Royal Society, 2016) Ardalan, Arman; Oskarsson, Mattias C. R.; Van Asch, Barbara; Rabakonandriania, Elisabeth; Savolainen, Peter
Madagascar was one of the last major land masses to be inhabited by humans. It was initially colonized by Austronesian speaking Indonesians 1500–2000 years ago, but subsequent migration from Africa has resulted in approximately equal genetic contributions from Indonesia and Africa, and the material culture has mainly African influences. The dog, along with the pig and the chicken, was part of the Austronesian Neolithic culture, and was furthermore the only domestic animal to accompany humans to every continent in ancient times. To illuminate Madagascan cultural origins and track the initial worldwide dispersal of dogs, we here investigated the ancestry of Madagascan dogs. We analysed mtDNA control region sequences in dogs from Madagascar (n=145) and compared it with that from potential ancestral populations in Island Southeast Asia (n=219) and sub-Saharan Africa (n=493). We found that 90% of the Madagascan dogs carried a haplotype that was also present in sub-Saharan Africa and that the remaining lineages could all be attributed to a likely origin in Africa. By contrast, only 26% of Madagascan dogs shared haplotypes with Indonesian dogs, and one haplotype typical for Austronesian dogs, carried by more than 40% of Indonesian and Polynesian dogs, was absent among the Madagascan dogs. Thus, in contrast to the human population, Madagascan dogs seem to trace their origin entirely from Africa. These results suggest that dogs were not brought to Madagascar by the initial Austronesian speaking colonizers on their transoceanic voyage, but were introduced at a later stage, together with human migration and cultural influence from Africa.
The 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 Richard
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.
Analysis of aspects of starch metabolism in Physcomitrella patens
(Stellenbosch : Stellenbosch University, 2021-12) Mdodana, Ntombizanele Thobela; Lloyd, James Richard; Kossmann, Jens; Stellenbosch University. Faculty of AgriSciences. Department of Genetics. Institute of Plant Biotechnology.
ENGLISH ABSTRACT: Starch is an important polysaccharide produced by plants and is widely used in industry mainly as a food thickener, but also in other important processes, such as the development of textiles and p aper manufacture. This polyglucan consists of two glucose polymers, amylose and amylopectin. As the major storage carbohydrate, starch is synthesised during the day before being catabolised at night to sustain plant growth and metabolism. Starch metabolism is well studied in vascular plants such as Arabidopsis thaliana and Solanum tuberosum, however, information about these processes are less well understood in non-vascular plants. The bryophyte Physcomitrella patens is an excellent plant model system for many reasons; for example its fully sequenced genome and the ability to produce knockout mutants using homologous recombination. Attempts to gain a better understanding of the function and regulation of some of the key metabolic enzymes involved in starch metabolism in non-vascular plants have recently emerged (Stander, 2015; Jacobs, 2018; Mdodana et., al 2019). This project focused on two aspects of starch metabolism to determine whether some of the pathways and mechanisms involved during these processes are conserved between Physcomitrella patens and vascular plants. The first part of this dissertation examines the roles of glucan water dikinase enzymes (GWD) in P. patens. In angiosperms these polypeptides are involved in starch degradation through catalysing starch phosphorylation. Five isoforms, PpGWDa-e, were identified in and phylogenetic analysis demonstrated the two (PpGWDa and PpGWDb) were most similar to Arabidopsis GWD1 or GWD2, while another two (PpGWDd and PpGWDe) were most similar to GWD3/PWD. The final isoform (PpGWDc) was likely to be inactive as it lacks the essential catalytic histidine. Both PpGWDa and PpGWDb targeted to chloroplasts. Using homologous recombination, knockout mutant lines were successfully generated for PpGWDa and PpGWDb each isoform both as individually and together in double mutants. Inserts in either gene resulted in reduced amounts of starch phosphate compared to the control, with Ppgwda mutant lines interestingly containing less glucose 6-phosphate in starch than Ppgwdb lines. Double mutant (Ppgwda/Ppgwdb; DM) lines contained even less glucose 6-phosphate in starch than Ppgwda single mutants. When plants were grown over a diurnal cycle it was shown that, like vascular plants, starch accumulated in the light period and was degraded at night. Both Ppgwd1a and DM lines accumulated significantly higher amounts of starch compared to Ppgwd1b and the control lines at almost all time points. Soluble sugars on the other hand were significantly reduced in Ppgwd1a and the DM lines compared with Ppgwd1b and the control lines. The Ppgwd1a and DM mutant lines also demonstrated a n observable morphological phenotypic alteration characterized by lack of gametophore development which could be reversed by growing the plant on media supplemented with glucose. In the second experimental chapter, two isoamylase (ISA) like starch debranching enzymes, ISA1 and ISA2, were examined. Mutations in these genes in vascular plants can result in the production of the water-soluble polysaccharide (WSP), phytoglycogen at the expense of starch. Both single and double mutants showed loss of a debranching enzyme activity band in activity gels indicating that ISA1 and ISA2 exist as a heterocomplex in Physcomitrella patens. Analysis of purified recombinant protein demonstrated, surprisingly that both PpISA1 and PpISA2 peptides were catalytically active. Analysis of single and double mutant plants demonstrated that all contained increased amounts of WSP.
Analysis 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.
Analysis 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.
Analysis of eight genes modulating interferon gamma and human genetic susceptibility to tuberculosis : a case-control association study
(BioMed Central, 2010-06) Moller, Marlo; Nebel, Almut; Van Helden, Paul D.; Schreiber, Stefan; Hoal, Eileen G.
Background: Interferon gamma is a major macrophage-activating cytokine during infection with Mycobacterium tuberculosis, the causative pathogen of tuberculosis, and its role has been well established in animal models and in humans. This cytokine is produced by activated T helper 1 cells, which can best deal with intracellular pathogens such as M. tuberculosis. Based on the hypothesis that genes which regulate interferon gamma may influence tuberculosis susceptibility, we investigated polymorphisms in eight candidate genes. Methods: Fifty-four polymorphisms in eight candidate genes were genotyped in over 800 tuberculosis cases and healthy controls in a population-based case-control association study in a South African population. Genotyping methods used included the SNPlex Genotyping System™, capillary electrophoresis of fluorescently labelled PCR products, TaqMan® SNP genotyping assays or the amplification mutation refraction system. Single polymorphisms as well as haplotypes of the variants were tested for association with TB using statistical analyses. Results: A haplotype in interleukin 12B was nominally associated with tuberculosis (p = 0.02), but after permutation testing, done to assess the significance for the entire analysis, this was not globally significant. In addition a novel allele was found for the interleukin 12B D5S2941 microsatellite. Conclusions: This study highlights the importance of using larger sample sizes when attempting validation of previously reported genetic associations. Initial studies may be false positives or may propose a stronger genetic effect than subsequently found to be the case.
Analysis 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.
Analysis 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.
Analysis 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...
The 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.
Analysis of interactions between glucan, water dikinase and either isoamylase or starch branching enzymes in detrmination of starch structure
(Stellenbosch : Stellenbosch University, 2022-12) Adegbaju, Muyiwa Seyi; Lloyd, James Richard; Van der Vyver, Christell; Stellenbosch University. Faculty of AgriSciences. Department of Genetics. Institute of Plant Biotechnology.
ENGLISH ABSTRACT: Starch is the main form in which carbon is stored in plants and it is used in a wide array of applications, from its role as a source of calories in human diets, to livestock feed and as an industrial feedstock. It is composed of two types of glucan polymer, amylose which is com- posed mainly of α-1,4 linked chains and highly branched amylopectin which contains α1,4 chains which are connected together with α1,6 branchpoints. Synthesis of the starch polymer involves co-ordinated activities of starch synthases, starch branching enzymes (SBE) and de- branching enzymes (DBE). In addition, the enzyme glucan, water dikinase (GWD) can intro- duce phosphate into amylopectin. The critical roles these enzymes play during starch metabo- lism have been investigated by knockout or silencing of genes encoding them in some plants, mainly Arabidopsis thaliana. Interactions between some of these enzymes have also been demonstrated, but there are still many questions about how these interactions occur and how they influence starch metabolism. In this study two sets of experiments were performed. The first involved analyses of potato (Solanum tuberosum L.) plants where the expression of SBEI, SBEII and/or Glucan, Water Dikinase 1 (GWD1) were repressed using RNAi technology. Individual or joint repression of the two SBE isoforms resulted in a significant increase in starch phosphate, whereas repression of GWD1 led to synthesis of low phosphate starch. Starch phosphate decreased in SBEI/GWD1 and SBEII/GWD1 lines but not to the levels found in lines where only GWD1 was repressed. The apparent amylose content increased in starch from SBEI/GWD1 lines was greater than either the SBEI or GWD1 lines. These alterations in starch composition influenced its granule morphology, swelling power and freeze-thaw stability. Silencing of GWD1 reduced starch deg- radation in cold-stored tubers, but this was not the case in lines repressed in either starch branching enzyme. The second set of experiments examined polyglucan metabolism in potato lines repressed in isoamylase 2 (ISA2) and/or GWD1. Transgenic potato lines were produced by RNAi gene silencing and tuber starch metabolism analysed. The water-soluble glucan content in ISA2 and ISA2/GWD1 lines tuber was higher than GWD1 lines. Analysis of starch structure indicated that there was an increase in the apparent amylose content of starches from GWD1 lines whereas it was unchanged ISA2/GWD1 lines. There was a reduction in the starch phosphate in both GWD1 and ISA2/GWD1 lines, and this reduction was greatest in the GWD1 lines. The ISA2 lines contained a proliferation of small starch granules, but this was not observed in ISA2/GWD1 lines which indicates that ISA2 and GWD1 interact during granule initiation. The effect of both enzymes on cold-induced sweetening (CIS) was also investigated. As expected, CIS was reduced in lines where GWD1 was silenced. Interestingly CIS was also inhibited in the ISA2 lines indicating that the pathway of starch degradation may differ between cold-stored potato tubers and Arabidopsis leaves.
Analysis 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.
Analysis 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.
Analysis of schizophrenia susceptibility variants identified by GWAS : a bioinformatics and molecular genetics approach
(Stellenbosch : Stellenbosch University, 2014-12) Coffee, Michelle; Warnich, L. ; 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.
The analysis of starch degradation in Solanaceae species
(Stellenbosch : Stellenbosch University, 2014-04) Samodien, Mugammad Ebrahim; Lloyd, James Richard; Kossmann, J. M.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics and Institute of Plant Biotechnology.
ENGLISH ABSTRACT: This project involved the analysis of genes in Solanaceae species that have previously been shown to be involved in the phosphorylation of starch or its subsequent dephosphorylation. Both these processes are essential for normal starch mobilization. A tomato conditional mutant lacking the starch phosphorylating enzyme glucan water dikinase was analyzed. It is known that starch accumulates transiently in tomato fruit and is degraded throughout the ripening process. The study aimed to determine the effect of inhibited starch degradation on fruit development. Unfortunately no effect on starch mobilisation was found in the fruit of the mutant. Immunoblot analysis revealed expression of Glucan Water Dikinase (GWD) within the fruit of the tomato mutant indicating that the conditionality of the mutation was compromised. The second set of experiments analyzed the roles of Starch Excess4 (SEX4), Like Sex Four-1 and Like Sex Four-2 (LSF1 and LSF2) in starch degradation in potato and Nicotiana benthamiana. These enzymes have, thus far, only been studied in Arabidopsis, with the proposed role for SEX4 and LSF2 being that they are involved in dephosphorylation of the C-6 and C-3 positions of starch breakdown products. The role of LSF1 is unclear, although it is not thought to be a phosphatase. SEX4, LSF1 and LSF2 were repressed individually while the expression of SEX4 and LSF2 were also inhibited simultaneously. Using a transient repression system in N. benthamiana it was shown that all of the genes play a role in leaf starch degradation. The SEX4 and LSF2 enzymes were shown to influence the proportion of phosphate located on the starch which contained an altered ratio of C-3/C-6 phosphate. Stably transformed potato plants were produced where SEX4 and LSF2 were successfully repressed in potato leaves and tubers. Although AtLSF2 had been shown not to be essential for normal starch degradation on its own, in potato plants when LSF2 was repressed, the plants developed a starch-excess phenotype. Taken together with the N. benthamiana data this indicates that LSF2 plays a bigger role in leaf starch degradation in Solanaceae than in Arabidopsis. The ratio of C-3/C-6 phosphate was also altered in tuber starch from some of the silenced plants. Starch from SEX4 repressed potato plants contained increased amounts of glucose-6-phosphate and increased glucose-3-phosphate in the tuber when compared to the WT. An increase in the proportion of C-6 or C-3 phosphate is not surprising with SEX4 being characterized as a phosphatase specific for C-6 position and LSF2 for the C-3 position in Arabidopsis, however the combined increase in C-3 and C-6 amounts in StSEX4 silenced plants is interesting. The differences seen in the phosphate alteration in both N. benthamiana leaves and potato tubers indicates that in Solanaceae species these proteins may have a slightly altered specificity when compared with Arabidopsis, although they are undoubtedly involved in starch degradation. The effect of silencing SEX4 or LSF2 on cold-induced sweetening was also investigated, with no effect being found. This may be because of functional redundancy between the proteins and a better approach in terms of blocking cold sweetening would be to simultaneously repress SEX4 and LSF2. Overall, these enzymes seem to play similar roles in leaves of Solanum species as has been described in Arabidopsis. The starch from the engineered plants did have an altered phosphate ratio and further analysis is needed to determine if this leads to improved or additional functionality.
Analysis 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).
Analysis of sugar accumulation under cold stress in Physcomitrium patens
(Stellenbosch : Stellenbosch University, 2021-12) Jacobs, Desren Brando; Lloyd, James Richard; Peters, Shaun W.; 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.