Doctoral Degrees (Genetics)

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    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.
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    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.
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    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.
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    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.
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    Analysis of the molecular and physiological effects following treatment with BC204 in Arabidopsis thaliana and Solanum lycopersicum
    (Stellenbosch : Stellenbosch University, 2020-12) Loubser, Johannes; Hills, Paul N.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology
    ENGLISH ABSTRACT: Plant biostimulants have been earmarked as one of the pivotal role players in the next much-needed agricultural revolution. Plant biostimulants are mostly from natural sources and they do not directly provide the plant with any nutrients. To date, many different biostimulants have been produced and tested on several different plant species. Although several reports indicate that they elicit an increase in overall plant growth, induce resistance to both abiotic and biotic stresses, increase crop yield and improve fruit/vegetable quality, the molecular data to back up these claims has generally been missing. One such plant biostimulant, BC204, is a citrus-based plant extract used on a variety of crop species in South Africa, China and Australia. There are internal reports from tests conducted by the producers of BC204 which show that it elicits physiological responses such as an increase in crop yield and fruit quality. One postgraduate research study reported that Croplife, a product closely related to BC204, has the potential to improve water utilisation efficiency in table grape cultivars (Van Zyl, 2007). However, no molecular data is available to explain the specific mechanisms associated with the increase in plant growth and tolerance to environmental stresses. Environmental stress is predicted to worsen due to climate change, but also due to irrigation practices on arable land areas, which can result in soil salinity. Although some progress has been made towards understanding plant mechanisms towards salt tolerance in efforts to combat the negative effects of salinity, these mechanisms are still a long way from being fully understood. BC204, like other plant biostimulants, could be a short-term alternative whilst salt tolerance and other abiotic stress mechanisms in plants are further unravelled. Such biostimulants can also be used to study salt tolerance, as the first part of this study provides preliminary evidence that BC204 significantly alleviates salt stress in Arabidopsis thaliana. BC204 treatment increased chlorophyll content, fresh and dry weights, whilst reducing proline, anthocyanin and malondialdehyde content in the presence of 10ds·m-1 EC salt stress. Stomatal conductivity was also reduced by BC204 in source leaves. In addition, BC204 had a significant effect on the expression of salinity-related genes, stimulating the expression of salinity-related genes RD29A and SOS1 independently of NaCl-stress, whilst suppressing the expression of SOT1 and P5CS1. In the second part of study, an RNA-seq approach was adopted to elucidate the effects of BC204 at the molecular level in the model plant species, Arabidopsis thaliana. BC204, applied via a soil drench at a low concentration of 0.01% (v/v), stimulated above-ground biomass production whilst eliciting a large change in gene expression levels across several biochemical pathways in Arabidopsis thaliana. Of the entire transcriptomic profile examined, a total of 8.212% of genes were significantly differentially expressed between the treated and control groups, of which 5.136% were upregulated and 3.076% downregulated. Most notably, genes involved photosynthesis, several aspects of cell wall metabolism, carbohydrate metabolism, signalling, stress and secondary metabolism were upregulated, which could explain the increase in plant growth. Genes related to transcription and RNA regulation were both strongly up- and downregulated, which suggests that BC204 plays a role in inducing and suppressing several pathways. In the third part of this study, the same RNA-seq approach was adopted to elucidate the effect of BC204 in Solanum lycopersicum, an important model crop species, at the molecular level under unstressed conditions. BC204, applied via foliar spray at a concentration of 0.05% (v/v), stimulated tomato root and shoot biomass production, root and shoot length and stem width compared to the untreated control plants. Of the 33308 transcripts analysed, a total of 18.059% genes were significantly differentially expressed between the control and treated groups, of which 8.776% were upregulated and 9.283% downregulated. Most notably, genes involved in signalling, stress and protein metabolism were upregulated, which could explain the increased growth that was observed. In both plant species, BC204 seemed to induce pathways involved in several environmental stresses. Together, the results of this study provide evidence that BC204 elicits a major change in a variety of metabolic processes which forms part of a complex network activating a broad priming response. These priming responses seem to start with enhanced photosynthesis, allowing additional energy to be channelled towards complex metabolic changes through RNA regulation and signalling. Very few metabolic plant processes seem to be unaffected by BC204 treatment.
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    Characterisation of citrus tristeza virus-induced stem pitting in citrus
    (Stellenbosch : Stellenbosch University, 2023-12) Aldrich, Dirk Jacobus; Maree, Hans Jacob; Bester, Rachelle; Burger, Johan Theodorus; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Citrus tristeza virus (CTV) is the most important viral pathogen of citrus and causes several disease syndromes in different citrus hosts. CTV-induced stem pitting leads to substantial economic losses in sensitive citrus varieties, including grapefruit. The exact mechanisms of stem pitting development in CTV-infected citrus remain unclear. This study aimed to utilise CTV infectious clone mutants in a reverse genetics approach to study stem pitting induction. A panel of recombinant CTV clones was generated focussing on the open reading frames (ORFs) implicated in stem pitting induction and pathogenicity, namely p33, p18, p13 and p23. ORF replacements from severe- and mild-pitting South African CTV isolates were introduced into the mild-pitting infectious clone (CTV-fl6 - genotype T36) to determine if severe stem pitting could be induced. Stem pitting assessments were complemented with the determination of virus concentration ratios in ‘Mexican’ lime and ‘Duncan’ grapefruit to relate stem pitting outcomes to virus concentration. The various infectious clone and wild type CTV infections yielded a broad range of stem pitting outcomes and specific ORF replacements associated with enhanced stem pitting were identified. Plant responses to different stem pitting pressures were further assessed by untargeted metabolite profiling and the quantitation of the stress-responsive phytohormones, abscisic acid, jasmonic acid and salicylic acid. In both citrus hosts, the metabolite profiling yielded fourteen statistically significant compounds that differed between stem pitting groups. These compounds were mainly phenolic acids and phenolic glycosides and are known to function as antioxidant and stress signalling molecules. Significant differences in phytohormone content were also found between test groups, particularly in plants that were severely stunted. Stem pits were also characterised at the molecular anatomical level using a combination of known and novel techniques to better understand the nature of the xylem and phloem tissues impacted by severe pitting. Established methods such as biological staining and fluorescence microscopy were used to visualise CTV-induced stem pitting and virus localisation. The utility of two novel technologies that have not previously been used to study CTV-induced stem pitting, namely high-resolution CT scanning and serial block face scanning electron microscopy was also evaluated. This study contributed to our understanding of CTV-induced stem pitting in citrus. Importantly, the use of CTV infectious clones for South African CTV research was established, which can allow for targeted follow-up experiments to further characterise the local citrus-CTV pathosystem. This can also provide novel opportunities for potentially employing CTV infectious clones as a crop protection tool against other important citrus diseases on the African continent.
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    Characterisation of EMS mutagenic bread wheat (Triticum aestivum l.) lines to investigate their water deficit stress tolerance and adaptability
    (Stellenbosch : Stellenbosch University, 2023-03) Kayaga, Helen Ninsiima; Botha-Oberholster, Anna-Maria; Van der Vyver, Christell; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Wheat is a staple food for 2.5 billion people worldwide and is the second most important cereal grain grown in South Africa. Water deficit stress has adverse effects on wheat productivity in the world. This is exacerbated under current unpredictable climatic patterns due to global warming. In 2017, most of the wheat production areas in South Africa were lost to alternate crops due to prevailing drought conditions. This created a need to develop water deficit stress tolerant wheat lines using ethyl methanesulfonate (EMS) because currently, no officially released varieties in the country carry such a trait. This study aimed to characterise newly developed mutagenic water stress-resilient bread wheat lines. The agro-morphological traits of three wild types and ten M3 lines were determined based on wheat descriptors in a randomised experiment with three replicates in a greenhouse. The chemical properties of flour were measured using the Inframatic 9500 NIR Grain Analyser machine, and the sedimentation tests of flour were quantified using the AACC International Method 56-60.02. The M3 lines were screened for tolerance to water deficit stress in a split-plot (3x5 factorial) experimental design that induced stress at the following growth stages: stress at seedling growth (emergence), tillering (forty days after planting), anthesis (flowering), milk development stage (seed set), and control (well-watered), and 13 subplots (bread wheat lines). Results from the analysis indicated a variation in the agro- morphological traits (18 traits) of mutants from the wild types (WTs) aside from ten traits that were similar across all bread wheat lines. Baking quality analysis showed that mutant Big 8.3 had the most desirable moisture content percentage of flour, a high protein, and wet gluten. Hence its dough has good elasticity and extensibility. The mutants performed better than WTs under water deficit stress. Big 395.1 was the most tolerant at emergence and forty days after planting, while Big 8.1 and Big 8.3 were most tolerant to water deficit stress at flowering and seed set, respectively. Previously identified drought-related genes, Sal1 (an inositol polyphosphate 1- phosphatase encoding gene) and Era1 (enhanced response to Abscisic acid 1), were analysed for single nucleotide polymorphism (SNPs) variants induced by chemical mutagenesis through sequencing complementary DNA (cDNA) of the genes and aligning them to the reference wheat genome of “Chinese Spring” available on the National Centre for Biotechnology Information (NCBI) web-based platform. I could not attain results from the Era1 sequences, and alignment results showed no differences between the Sal1 sequences of the mutant lines to that of reference genome “Chinese Spring”. In conclusion, chemical mutagenesis increased the genetic diversity of the parental bread wheat lines through induction of point mutations resulting in desirable new mutant lines like Big 8.3 with a desirable agrotype, baking quality characteristics, and high tolerance to water deficit stress at seed set.
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    Characterisation of sucrose synthase activity in the sugarcane culm
    (Stellenbosch : University of Stellenbosch, 2004-04) Schafer, Wolfgang Erich; Botha, F. C.; Rohwer, J. M.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB).
    ENGLISH ABSTRACT: This study had three main goals: 1. to investigate the occurrence on the protein level of sucrose synthase (SuSy) isoforms in sugarcane sink tissue, 2. to determine the kinetic properties of these isoforms, 3. to establish the tissue localisation of SuSy in the sugarcane culm The results are summarised below: Three SuSy isoforms were obtained from leaf roll tissue. The SuSyA and SuSyB isoforms differed in terms of charge characteristics, with SuSyA not binding to an anion exchange column that bound SuSyB and SuSyC under the same conditions. Both SuSyB and SuSyC isoforms were eluted at 180 mM KCl. The SuSyA and SuSyB isoforms were present during autumn, but during winter only the SuSyC isoform could be isolated. Even though they eluted at the same salt concentration, SuSyB and SuSyC were different isoforms, because they had different kinetic parameters, as well as different immunological properties. SuSyB and SuSyC could not have been mixtures of the same isoforms, since a polyclonal antiserum against SuSyB, which inactivates native SuSyB, did not inactivate SuSyC. All three isoforms had significantly different kinetic parameters, with the SuSyA isoform also having a much lower sucrose breakdown/synthesis ratio than the other two isoforms. Therefore, at least three SuSy isoforms occur in sugarcane leaf roll tissue on the protein level. The SuSyC isoform was subsequently kinetically characterised in detail. Data showed that the enzyme employs an ordered ternary complex mechanism, with UDP binding first and UDP-glucose dissociating last. These experimentally obtained kinetic parameters were then used to extend a kinetic model of sucrose accumulation. Data show that when the experimentally determined SuSy kineticparameters were entered into the model, a 40 % increase in sucrose concentration and 7 times reduction in fructose concentration resulted. These data illustrate the pronounced physiological effects that may result from the presence of different SuSy isoforms. SuSy protein localisation data, obtained by an immunohistochemical approach, indicated that SuSy protein was present in both storage parenchyma and vascular tissue of young, intermediate, and mature internodes. SuSy enzyme activity in different parts of the internodes was similar, except for internode 3, which had much higher activity in the bottom part of the internode, possibly because growth is faster here, hence a higher demand for sucrose cleavage exists here.
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    Characterization of citrus tristeza virus variants and their influence on symptom expression in grapefruit
    (Stellenbosch : Stellenbosch University, 2019-04) Cook, Glynnis; Maree, H. J.; Burger, Johan T.; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Citrus tristeza virus (CTV), a member of the family Closteroviridae, was responsible for large scale destruction of citrus, especially in the Americas, due to tristeza disease and necessitated a production switch to less sensitive rootstocks. CTV however continues to affect citrus through the stem-pitting disease phenotype which is especially problematic in sweet orange, grapefruit and lime cultivars. In South Africa, the productive lifespan of grapefruit trees was severely affected by stem-pitting, requiring early tree replacement with an associated lag in production. This affect was later mitigated by applying cross-protection, a management strategy using non-stem-pitting sources of CTV, but without prior knowledge of which CTV strains were responsible for stempitting or which strains were present in the cross-protection sources. To understand the disease and unravel mechanisms underlying cross-protection, it is necessary to characterise CTV strains to investigate both virus-host- and strain-interactions. The aim of this study was firstly to identify single-strain isolates belonging to different strains, to characterise them biologically and to determine full-genome sequences. These characterised CTV isolates were further used in a complementation study to investigate possible synergistic interactions affecting stem-pitting. Complete viral genomes of eight single-strain isolates were determined during the study. Two commercial grapefruit cultivars, ‘Star Ruby’ and ‘Marsh’, were used in a glasshouse trial to evaluate the ability of specific strains to induce stem-pitting in single or mixed infections. Evaluation over four years showed that symptom expression of mild strains did not result in altered symptom expression when in combination with each other. Importantly demonstrating that there was no additive effect on stem-pitting expression with multiple isolates. Relative quantitation of the strains in ‘Marsh’ and ‘Star Ruby’ plants indicated that the individual strain concentrations were not significantly altered when in combination with the other strains. A valuable discovery made within this project was the characterisation of two variants of the T68 strain, derived from the same GFMS12 source, but displaying differences in stem-pitting severity in grapefruit. This finding demonstrates the co-existence of severe and mild variants of the same strain in one source and provides an explanation for the presumed strain segregation event observed for the GFMS12 cross-protection source that resulted in the discontinuation of the source for use in cross-protection of grapefruit. The characterisation of these variants will further assist in the identification of the sequence determinants for stem-pitting in grapefruit.
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    Characterization of SUMO proteases and other proteins involved in water stress responses in Triticum aestivum L.
    (Stellenbosch : Stellenbosch University, 2021-03) Le Roux, Marlon-Schylor; Botha-Oberholster, Anna-Maria; Kunert, Karl; Cullis, Christopher; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Currently, approximately 4.5 billion people in developing countries consider wheat (Triticum aestivum L.) as a staple crop as it is a key source of daily calories. Therefore, it is ranked the second most important grain crop in the developing world. Meanwhile, climate change associated with severe drought conditions and the rising global mean temperatures has resulted in sporadic soil water shortages. Water deficit due to drought creates concerns because, historically, drought is the leading cause of yield loss in wheat. Thus, a more comprehensive understanding of the mechanisms underlying wheat drought responses is imperative. One of the latest avenues in plant-drought response is SUMOylation, which is a post-translational modification. SUMOylation is responsible for affecting essential proteins during water deficit stress changing their functionality, thus contributing to the plant succumbing to drought. SUMO proteases can counter the process by acting directly within the SUMOylation pathway. Therefore, SUMO proteases are an ideal target for manipulating stress-responses. In this Ph.D. study, novel findings have confirmed the overarching hypothesis “that soil water deficit stress influences an array of physiological, metabolic, and proteomic mechanisms mitigated by reducing levels of SUMOylation, consequently delaying protein turn over, thereby increasing drought tolerance in the cereal crop wheat.” Proof for this hypothesis has been that overexpression of an A. thaliana cysteine protease (Overly Tolerant To Salt-1, OTS1) (At1g60220), in wheat leads to improved plant growth under drought conditions. These overexpressing plants had an improved stomatal conductance and photosynthesis rate (Fv/Fm), accompanied by a higher total chlorophyll content than the controls. More importantly, these overexpressing plants had a reduced level of SUMOylated proteins with delayed senescence under drought conditions, allowing these plants to survive up to 14 days without water (with a final soil water content ± 15%). This finding further suggests that SUMO proteases may influence an array of mechanisms in wheat to the advantage of the crop to be more tolerant to water deficit stress caused by drought. This is the first report to elucidate SUMOylation effects in the hexaploid crop wheat. Furthermore, this Ph.D. work is also in agreement with various other studies that showed water deficit stress constrains almost every part of developing plants, inducing morphological and physiological changes, and cellular biochemical alterations as an adaptative response. To substantiate the aforementioned, random mutagenesis was also applied to produce two new wheat mutants, RYNO3926, and BIG8-1, with both expressing water deficit stress tolerance. While the BIG8-1 mutant survived three weeks without water, the RYNO3936 mutant could only endure two weeks without any water but rapidly recovered fully, despite leaves being completely dry/dehydrated after exposure to water deficit stress. Qualitative proteomic analysis further revealed that both the mutant and control have an improve regulation of SUMOylation when compared to non-mutant plants and in addition to having more ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Quantitative proteomic analysis also revealed that RYNO3936 mutant plants expressed a large number of proteins to endure drought conditions such as the abscisic stress-ripening protein, cold-induced protein, cold-responsive protein, dehydrin, Group 3 late embryogenesis, and a lipoprotein (LAlv9) belonging to the family of lipocalins. Moreover, BIG8-8 mutant uniquely expressed ABC transporter permeases and Xylanase inhibitor protein during severe water deficit stress. Collectively the research supports the idea that there is a multifaceted nature of how plants react to drought, which is a non-linear response, as it involves multiple pathways related to genomics, transcriptomics, proteomics, and phenomics responses. Though this dissertation does not argue a comparative analysis of which of the lines is more superior, it does advocate that each developed line will thrive better with specific dryland conditions, adding to the knowledge for future breeding programs to improve drought tolerance.
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    Characterization of the Sucrose Synthase and class II Trehalose 6-Phosphate Synthase gene families in the moss Physcomitrella patens.
    (Stellenbosch : Stellenbosch University, 2020-12) Wiese, Anna Johanna; Lloyd, James Richard; Stellenbosch University. Faculty of AgriSciences. Department of Genetics. Institute of Plant Biotechnology.
    ENGLISH ABSTRACT: Plant sugars have dual functionality, in that they play a role in primary metabolism and partake in signal transduction pathways. As it relates to their signaling function, they relay information to the nucleus regarding energy status, allowing the plant to adapt accordingly. Most of what is known about these functions of sugars have come from research in vascular plants, leaving aspects thereof unaddressed in non-vascular plants. Certain bryophytes (non-vascular plants) have been developed over into model plants, with Physcomitrella patens representing one such model. This plant has become popular in studies of evolutionary development and non-vascular plant biology. In this study, I characterized two gene families in P. patens, namely sucrose synthases (SUS) and trehalose 6-phosphate synthases (TPS), whose homologs in higher plants are implicated in sugar metabolism and signaling. Sucrose, the end-product of photosynthesis, is central to primary carbon metabolism. Its synthesis and degradation are tightly controlled to balance out supply and demand throughout the plant. Sucrose synthases are implicated in phloem loading and sink strength in vascular plants, where its cleavage products can enter primary metabolism or be used in the synthesis of complex carbohydrates. Little is known about SUS function in non-vascular plants, and in this study, I report the characterization of four putative SUS homologs in P. patens. Phylogenetic classification of land plant SUS sequences revealed the existence of 5 clades, one of which contained only bryophyte-sequences including all those from P. patens. Analysis of the amino acid sequences revealed that residues involved in SUS regulation in higher plants were conserved in PpSUS proteins. I was able to demonstrate SUS activity in crude protein extracts, however, detailed kinetic characterization was hindered by protein expression in E. coli. Localization studies revealed that all PpSUS proteins were cytosolic, while expression analyses indicated that PpSUS genes have overlapping and unique expression patterns. Another sugar which is implicated in signaling is trehalose 6-phosphate (Tre6P), an intermediate in the trehalose biosynthesis pathway. Levels of this sugar phosphate change in parallel to that of sucrose, with researchers proposing that it plays a role in communicating sucrose availability. The second part of this study focussed on the proteins involved in Tre6P synthesis, namely trehalose 6-phosphate synthases (TPSs), which are divided into two classes, with class I proteins containing catalytically active polypeptides (Leyman et al., 2001; Lunn, 2007). Very little is known about the class II proteins, and in this study, I characterized members of this class in P. patens. Physcomitrella contains six TPS genes in its genome, four of which encode class II proteins. Phylogenetic classification differentiated TPS sequences from land plants into 2 clades (I and II) consisting of 7 sub-clades (IA-B and IIA-E), suggesting the existence of 1 ancestral TPS gene. Functional complementation revealed weak TPS catalytic activity for one of the class II TPS proteins, a first for any plant class II protein studied to date. Subcellular localization experiments conducted on three of the class II proteins revealed that they were cytosolic, while yeast two hybrid analyses indicated that these proteins do not form complexes with each other or the class I proteins. Finally, expression analyses indicated that class II genes have overlapping expression patterns. This study provides novel insights into the evolution of SUS and TPS genes in P. patens and, will serve as a platform for the design of future experiments related to these gene families in non-vascular plants.
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    Defining the QTL for chill requirement during dormancy and dormancy release in apple (Malus x domestica Borkh.).
    (Stellenbosch : Stellenbosch University, 2021-12) Cornelissen, Stephanie; Burger, Johan T.; Rees, Jasper; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Dormancy is a physiological stage that all deciduous fruit trees experience during winter. It consists of three sub stages, para-, endo- and ecodormancy. During the endodormic stage, apple trees need to be exposed to cold temperatures for their chill requirement to be fulfilled. Most apple varieties have a unique chill requirement, for instance ‘Anna’ requires less than 300 hours. After this requirement is fulfilled, the plant will break dormancy if the environmental conditions are optimal, i.e. the less the chill requirement the earlier the plant will bud. This study looks at the genetic component of chill requirement by firstly determining the region of the apple genome that is primarily associated with budbreak and thereafter the genes within this region and their expression patterns. A mapping population was created by crossing ‘Lady Williams’ with ‘Anna’. ‘Lady Williams’ is a medium to high chill variety and ‘Anna’ is a low chill variety that requires less than 300 chill units. This population was used to generate a genetic map and subsequent QTL analysis, as well as association mapping, were used to determine the region of the apple genome that is associated with budbreak. A major QTL in the first 10Mb distal region of Linkage Group/chromosome 9 was discovered. This region consists of 741 genes, of which 27 were differentially expressed over time when exposed to cold. An RNA time series analysis was performed by periodically extracting RNA from the meristems of cuttings from ‘Anna’, incubated at 4°C for up to 800 hours. The expression patterns of the 27 genes grouped into seven distinct clusters. The major observation that could be made is that there is a difference between the expression patterns before and after the theoretical time of budbreak (300 hours) in ‘Anna’. The 27 genes were differentially expressed before budbreak but there was little to no differential expression after budbreak. Another observation that was made, is that the biggest differences in gene expression were between 0-hours and a 100-hours. This indicated that the plant could have experienced cold shock by transferring it to low temperature incubation and subsequently the onset of endodormancy. Known dormancy-associated DAM genes, were not differentially expressed over time. However, an ortholog of the predicted transcription factor ICE1-like, bHLH, and the gene RING domain ligase 2, that have been shown to be associated with the regulation of ICE1-like, was differentially expressed over time when first introduced to cold. Furthermore, genes regulating the expression of the plant hormones ethylene and auxin were differentially expressed. Other notable genes include ones that encode Tubulin alpha-2 chain and xyloglucan endotransglucosylase/hydrolase 9 proteins, both known to regulate transport through cell wall/membrane, and 5'-adenylyl phosphosulfate reductase 2 that is associated with oxidation-reduction homeostasis. This study serves as a starting point for further investigations of the complex nature of the onset of dormancy, chill accumulation during dormancy and the eventual budbreak, by identifying genes that are associated with dormancy.
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    The development and characterisation of grapevine virus-based expression vectors
    (Stellenbosch : University of Stellenbosch, 2010-03) Du Preez, Jacques; Burger, J. T.; Goszczynski, D. E.; Stephan, D.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Grapevine (Vitis vinifera L.) is a very important agricultural commodity that needs to be protected. To achieve this several in vivo tools are needed for the study of this crop and the pathogens that infect it. Recently the grapevine genome has been sequenced and the next important step will be gene annotation and function using these in vivo tools. In this study the use of Grapevine virus A (GVA), genus Vitivirus, family Flexiviridae, as transient expression and VIGS vector for heterologous protein expression and functional genomics in Nicotiana benthamiana and V. vinifera were evaluated. Full-length genomic sequences of three South African variants of the virus (GTR1-1, GTG11-1 and GTR1-2) were generated and used in a molecular sequence comparison study. Results confirmed the separation of GVA variants into three groups, with group III (mild variants) being the most distantly related. It showed the high molecular heterogeneity of the virus and that ORF 2 was the most diverse. The GVA variants GTG11-1, GTR1-2 and GTR1-1 were placed in molecular groups I, II and III respectively. A collaboration study investigating the molecular divergence of GVA variants linked to Shiraz disease (SD), described two interesting GVA variants of group II, namely GTR1-2 and P163-M5 (Goszczynski et al., 2008). The group II variants were found to be closely linked to the expression of SD. GTR1-2 was isolated from a susceptible grapevine plant that never showed SD symptoms (Goszczynski 2007). The P163-M5 variant that resulted in exceedingly severe symptoms in N. benthamiana and is that used as SD positive control by the grapevine industry, was found to contain a 119 nt insert within the native ORF2. Comparative analysis performed on the complete nt and aa sequences of group II GVA variants suggested that the components in the GVA genome that cause pathogenicity in V. vinifera are more complex (or different) to those that cause pathogenicity in N. benthamiana. The three South African variants (GTR1-1, GTG11-1 and GTR1-2) were assembled into fulllength cDNA clones under control of CaMV 35S promoters. After several strategies were attempted, including a population cloning strategy for GTR1-2, none of the clones generated were able to replicate in N. benthamiana plants. A single amino acid substitution at position 13 (Tyr/Y Cys/C) in ORF 5 of the GTR1-2 cDNA clone was shown to abolish or reduce replication of the virus to below a detectable level. Two infectious clones of Israeli variants of GVA (T7-GVA-GR5 and T7-GVA118, obtained from M. Mawassi) were brought under control of a CaMV 35S promoter (35S-GVA-GR5 and 35S-GVA118). Both clones were infectious, able to replicate, move systemically and induce typical GVA symptoms after agroinfiltration in N. benthamiana. These Israeli clones served as backbone for further experiments in characterisation of transient expression and VIGS vectors. The use of GVA as gene insertion vector (35S-GVA118) and gene exchange vector (35S-GVA-GR5- ORF2+sgMP) in N. benthamiana and V. vinifera was compared. The gene insertion vector, 35S-GVA118 was based on the full-length GVA genome. The gene exchange vector, 35SGVA- GR5- ORF2+sgMP, was constructed in this study by elimination of ORF 2 and insertion of a sgMP and unique restriction sites to facilitate transgene insertion. In N. benthamiana both vectors showed similar GUS expression levels and photobleaching symptoms upon virus-induced NbPDS silencing. In V. vinifera limited GUS expression levels and VIGS photobleaching symptoms were observed for the gene insertion vector, 35SGVA118. No GUS expression was observed for the gene exchange vector 35S-GVA-GR5- ORF2+sgMP in this host. As for silencing, one plant, agroinfiltrated with 35S-GVA-GR5- ORF2-VvPDS+sgMP, developed photobleaching symptoms in 3 systemic infected leaves after 4 months. This study showed that GVA can be used as gene insertion and gene exchange vector for expression and VIGS in N. benthamiana, but in grapevine its use is limited to expression and silencing of genes in the phloem tissue. It is also the first report that ORF 2 of GVA is not needed for long distance movement in grapevine. To investigate the possible role of the P163-M5 119 nt insertion and the GVA ORF 2 (of unknown function), in expression of symptoms in plants, ORF 2 of a 35S-GVA-GR5 cDNA clone was removed and subsequently substituted by the corresponding ORFs of four South African GVA variants. Upon agro-infiltration into N. benthamiana leaves, all chimaeric GVA constructs were able to move systemically through the plant. At this stage no correlation could be found between severity of symptoms, the presence of the P163-M5 insert and the specific GVA ORF 2 present in the chimaeras, indicating that other factors in the viral genome or the host plant probably play a crucial role. This study contributed to the pool of available in vivo tools for study and improvement of the valuable grapevine crop. It also opened several exciting research avenues to pursue in the near future.
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    Development of a transformation system for sugarcane (Saccharum spp. hybrids) in South Africa using herbicide resistance as a model system
    (Stellenbosch : Stellenbosch University, 2002-12) Snyman, Sandra Jane; Botha, F. C.; Huckett, B.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics ​and Institute of Plant Biotechnology.
    ENGLISH ABSTRACT: Please refer to fulltext for abstract
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    Diuraphis noxia biotypes ascribed through genotypic characterization and utilization of their endosymbiont, buchnera aphidicola
    (Stellenbosch : Stellenbosch University, 2022-11) Burger, Nicolaas Francois Visser; Oberholster, Anna-Maria; Stellenbosch University. Faculty of AgriSciences. Dept. of Forest and Wood Science. Genetics.
    ENGLISH ABSTRACT: The aphid Diuraphis noxia (more commonly known as the Russian wheat aphid), is a pest of wheat and other small grains which has successfully spread to all global wheat producing regions. Feeding damage of the aphid results in characteristic longitudinal streaking, leaf rolling, and head trapping which all contribute to a lowered crop yield. Although wheat cultivars resistant to the aphid have been successful in curbing yield losses, the aphid has overcome most of these resistant lines through the process of biotypification. As reproduction of the aphid is limited to cyclical parthenogenesis, and thus unable to generate genetic diversity through chromosomal crossover, the exact means of how biotypification occurs remains elusive. With the availability of the only known genealogically linked D. noxia biotypes SA1 and SAM, this study attempted to ascertain their genomic backgrounds to better understand how they could occupy the extremes of the virulence scale. Sequencing, assembly, and comparisons of the genomes of these biotypes revealed an unexpected number of genetic polymorphisms and an extremely AT rich genome. The roughly 400 Mb genome, containing just over 32,000 protein coding genes, appears to have many polymorphisms in genes responsible for protein expression and turnover, which supports previous results indicating that biotypes differ in their ability to regulate their transcription. The unexpected number of polymorphisms would indicate that there is a yet unknown mechanism that enables these aphids to induce genomic variation in the absence of sexual reproduction. Further analysis of the salivary protein genes shared between the biotypes indicates that copy number and polymorphisms in genes associated with defence against plant volatiles also play a role in how these biotypes interact with their wheat host. Through comparative transcriptomics with other aphid transcriptomes, this study was also able to identify genes preferentially expressed in aphid head and salivary tissue through a proof-of-concept comparison, thereby enabling the future discovery of D. noxia salivary effectors. Lastly, this study was able to assemble and compare the genome of the sole endosymbiont of D. noxia, Buchnera aphidicola, with that of nine other genomes. From this the overall importance of several genic features such as gene length and percentage GC content was found to be critical for the maintenance of Buchnera genes when compared to their closest free-living relative, Escherichia coli. It was also revealed that D. noxia contains multiple Buchnera isotypes and that their genes are polyadenylated which may indicate a mechanism for their regulation. With the availability of these datasets, the as of yet unanswered question of the process of biotypification within D. noxia is becoming clearer.
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    Drought tolerance studies in spring wheat cultivars produced in South Africa
    (Stellenbosch : Stellenbosch University, 1999-12) Strauss, Johannes Abraham; Agenbag, G. A.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics & Institute of Plant Biotechnology.
    ENGLISH SUMMARY: Drought is considered, worldwide, to be the most important factor limiting crop yields. Spring wheat produced in the Western Cape region of South Africa is also affected by water stress. The onset of global warming may cause periods of water stress in the winter rainfall regions of South Africa more frequently, which makes it important to identify spring wheat cultivars that can withstand these conditions. The aim of this study was to determine whether cultivar differences in drought tolerance could be detected through the use of a series of physiological tests on spring wheat plants subjected to water stress at different growth stages. Furthermore to determine whether differences in physiological responses would be of any commercial value. To evaluate this, plants were analyzed to compare biomass production, yield and quality of stressed and control (unstressed) plants. A preliminary trial showed that the withholding of water might be more appropriate in the induction of water stress than the use of polyethylene glycol. The trial also showed that the reduction of 2,3,5-triphenyltetrazoliumchloride (modified method) was not sensitive enough to detect water stress in spring wheat. The evaluation of the physiological parameters showed that differences in drought tolerance do exist in spring wheat cultivars produced in the Western Cape region. The accumulation of proline and the water content of leaves proved to be the most sensitive parameters tested. A combination of these parameters may provide valuable information in newly bred spring wheat cultivars. In a study on biomass production it was shown that the above ground biomass was reduced by applied water stress. No clear distinctions in drought tolerance could, however, be made between cultivars. The use of leaf area (rate of leaf abscission) shows promise as a method to distinguish between drought tolerant cultivars. Yield and the yield components of all cultivars tested were severely reduced by water stress at both the flag leaf-, milky kernel growth stage. Although reductions in yield and yield components was shown, no single cultivar proved more tolerant than the other. The application of water stress resulted in a general increase in kernel protein content. Flour yield was lower, due to a relatively smaller production of non-protein components in the kernel. Although protein content increased with increased water stress, no significant differences were noted in micro-loaf volumes. The results of the mixograph parameters tested were also similar.
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    Elucidating functional interactions between the Russian wheat aphid (D. noxia Kurjumov) and bread wheat (Triticum aestivum L.)
    (Stellenbosch : Stellenbosch University, 2014-12) Schultz, Thia; Botha-Oberholster, Anna-Maria; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
    ENGLISH ABSTRACT: The Russian wheat aphid (Diuraphis noxia, Kurdj., Hemipetra, Aphididae, RWA) is an important pest of wheat, causing large-scale damage and yield losses. Various studies have been done at a transcriptomics level, including complementary DNA-amplified fragment length polymorphisms (cDNA-AFLPs), suppressive subtractive hybridization (SSH) and micro-array, which have identified genes putatively involved in RWA resistance. Even though these candidate genes have been identified, their role in host defence still needs to be verified using a functional genetics approach. In this study virus induced gene silencing (VIGS) using a barley stripe mosaic virus (BSMV) vector, has been utilized to knock-down candidate genes of interest in a wheat cultivar with the Dn1-resistance gene (TugelaDN). In this study it was hypothesized that genes involved in the hypersensitive response (HR) may contribute towards resistance and were thus targeted for silencing. These include glutathione-S-transferase (GST), superoxide dismutase Cu/Zn (SOD) and thylakoid-associated ascorbate peroxidase (tAPX). However, since aphid feeding also results in wounding, the genes were also analyzed under wounding only. Aphid fecundity is considered an indicator of involvement in RWA resistance, as susceptible plants result in higher aphid fertility. Findings in the study suggest that with wounding only, that Dn1 containing plants produce a greater hypersensitive response than susceptible controls. Ascorbate peroxidase was found to be important for wounding-induced resistance in Dn1 wheat plants. Under infestation conditions, silencing of superoxide dismutase Cu/Zn (SOD) and thylakoid-associated ascorbate peroxidase (tAPX) was found not to have an effect on aphid fertility and thus are not directly involved in resistance signaling. Knock-down of a phi-class glutathione-S-transferase F6 (TaGSTF6) transcripts however, had a large effect on aphid nymph numbers and thus may contribute to Dn1-resistance. Putative resistance genes silenced under aphid infestation conditions were a nucleotide binding protein (NBP) and resistance gene analogue 2 (RGA2). Analysis of NBP revealed its identity as a part of the iron homeostasis machinery in the cytosol, responsible for Fe-cluster assembly. Silencing of both NBP and RGA2 resulted in the expression of a susceptible phenotype. T10rga2-1A is an NBS-LRR protein known to be required for rust resistance in concert with resistance gene Lr10. T10rga2-1D silenced treatments resulted in susceptibility and plant death after aphid infestation, suggesting that T10rga2-1D may be a good up-stream candidate in Dn1-resistance.
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    The evolutionary history of the genus Seriola and the phylogeography and genetic diversity of S. lalandi (yellowtail) across its distribution range
    (Stellenbosch : Stellenbosch University, 2014-04) Swart, Belinda Louisa; Roodt-Wilding, Rouvay; Bester-van der Merwe, Aletta Elizabeth; Von der Heyden, Sophie; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
    ENGLISH ABSTRACT: The genus Seriola includes several important commercial fish species, yet the phylogenetic relationships between species have not been fully investigated to date. This study reports the first molecular phylogeny for this genus based on two mitochondrial (Cytb and COI) and two nuclear gene (RAG1 and Rhod) fragments for all extant Seriola species (nine species, n = 27). The phylogenetic patterns resolved three main lineages: a ((S. fasciata and S. peruana), S. carpenteri) clade, a (S. dumerili and S. rivoliana) clade and a (S. lalandi and S. quinqueradiata) clade. The closure of the Tethys Sea (12 - 20 MYA) coincides with divergence of the ((S. fasciata and S. peruana), S. carpenteri) clade from the rest of the Seriola species; while the uplifting of the Isthmus of Panama (± 3 MYA) played an important role in speciation between S. fasciata and S. peruana. The climate and water temperature fluctuation in the Pliocene played important roles during the divergence of the remainder of the Seriola species. This study is also the first to describe the evolutionary history of the commercially important species Seriola lalandi across its distribution range. Global patterns of genetic variation within S. lalandi (n = 190) were examined using three genes fragments (mitochondrial DNA COI, Cytb and nuclear RAG1). Three distinct clades were identified, corresponding to three different geographic regions (North-western Pacific - Japan, North-eastern Pacific - USA, and the southern hemisphere clade). These groupings correspond with the previously identified subspecies of S. lalandi (North-western Pacific – S. lalandi aureovittata, North-eastern Pacific – S. lalandi dorsalis, and the southern hemisphere clade - S. lalandi lalandi). AMOVA results and pairwise FST values revealed significant population differentiation between these groups. The population subdivision between these clades in all probability is maintained by biogeographic or oceanographic barriers (such as the equator and East Pacific Barrier) that disrupt gene flow. The southern hemisphere clade comprised of samples from the southern Pacific (AUS, NZL and Chile) and the southern Atlantic (SA). No haplotypes were shared between these areas in the southern hemisphere. This southern hemisphere clade was further investigated with six microsatellite markers. The analyses revealed the South African populations as genetically distinct from populations of the South Pacific oceans (AMOVA, FCA and STRUCTURE results). In summary, the South African and southern Pacific grouping could be the result of recent vicariant events during the Pleistocene glacial / interglacial periods and / or contemporary oceanographic forces acting on these populations. Further population differentiation was found within the South African samples, but not in the South Pacific. In the southern Pacific clade this lack of population structure is the result of high gene flow (analysed with MIGRATE) between the sampling localities. This is the first report on the genetic structure of this commercial important species for South African populations. Five sampling localities from the west- to the east coast of South Africa were sampled (n = 201). The microsatellite analyses revealed two potentially genetically distinct groups. AMOVA, FST and FCA results suggest small but significant differentiation between populations from the west coast and from the south- and east coast, suggesting a potential genetic break in the Cape Point region (BARRIER). However, the program STRUCTURE showed a high level of admixture along the South African coast and the migration results (MIGRATE and BAYESASS) also suggest a high degree of gene flow between these regions.
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    Functional characterization of a putative signalling peptide TAXIMIN in the model plant Arabidopsis thaliana and a medicinal plant Sutherlandia frutescens L. R. Br.
    (Stellenbosch : Stellenbosch University, 2016-03) Colling, Janine; Makunga, N. P.; Goossens, A.; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB).
    ENGLISH ABSTRACT: Secondary metabolite production in plants assists with protection against predators and attraction of pollinators. Manipulation of secondary metabolite pathways towards increased production of compounds of interest has become a target. The techniques to assist with understanding regulation of these pathways are therefore important. Several factors influence metabolite synthesis in plants including age, developmental stage, tissue type and environmental factors. In this study we describe a technique, cDNA-AFLP, which can be applied to study changes in whole genome expression to identify genes which are differentially expressed during stress conditions. We also describe the study of the function of a novel signalling peptide TAXIMIN in the model plant Arabidopsis thaliana. This peptide was discovered by cDNA-AFLP analysis of Methyl Jasmonate (MeJA) elicited Taxus baccata cell suspension cultures. TAXIMIN represents a novel signalling peptide which belongs to the cysteine rich peptides and has an N-terminal signal peptide and a C-terminal peptide with six conserved cysteines and three conserved prolines. Two TbTAX homologs (TAX1 and TAX2) were discovered in A. thaliana. Fusion of the full length peptide to the Venus fluorescent protein targeted the peptide to the plasma membrane-cell wall interface and this movement was abolished when the N-terminal signal was removed. Single and double mutants lacked a visible phenotype which can be related to functional redundancy with other genes or lack of environmental factors to induce a phenotypic response. Fusion of the TAX promoters to the GUS reporter gene revealed that TAX2 was expressed in vasculature tissue, whilst TAX1 expression was found in anthers, nectaries, roots and the base of the organs of the paraclade junctions indicating the neo-functionalization of the two peptides. Constitutive expression of TAX1 resulted in a fusion phenotype in the paraclade junctions and a fruit phenotype. Fruits were shorter and wider at the tip which co-insided with a wider replum as well as seed-stacking in this region. The fusion phenotype was similar to the phenotype observed for a mutant of the MYB transcription factor LATERAL ORGAN FUSION (LOF1) which plays a role in boundary formation. However, TAX1 overexpression (TAX1 OE) did not result in reduced LOF expression in paraclade junctions and TAX1 expression was similar to wild type plants in the lof1lof2 mutant paraclade junctions. Dexamethasone induction of TAX1 overexpression also did not result in changes in LOF1 expression in seedling or in the shoot apical meristem. No changes in LOF2 driven GUS expression level or pattern was observed when crossing to the TAX1 overexpression background. TAX1 therefore appears to regulate boundary formation independently from LOF1. However, these pathways may converge later in development. Metabolite analysis of the primary metabolite profiles of the leaf and roots of TAX1 OE lines indicated increases in phosphate (leaves) and serine (root and leaves) levels which were observed in all lines. TAX1 OE lines also appeared to be sensitive to the length of the photoperiod and this may be related to a reduced abundance of sinapoyl malate in leaves. cDNA-AFLP was applied to study changes in genome expression of nitrogen, salinity or MeJA-stressed Sutherlandia frutescens shoots cultivated in vitro. Results indicated that pathways involved in polyamine biosynthesis or regulated by plant hormones such as ethylene or abscisic acid are differentially expressed. Salinity stress caused a reduction in nitrogen uptake, but did not affect photosynthesis or the carbon: nitrogen ratio. On the metabolite level an increase in arginine and proline content was observed. This might be related to the reduction of ammonium toxicity effects or the osmotic response to reduce the damage due to accumulating ions. Plants were generally tolerant to low levels of salinity and no significant changes in sutherlandioside B abundance were observed. Constitutive expression of TAXIMIN genes from T. baccata and Medicago truncatula did not alter the abundance of sutherlandins, sutherlandiosides or soyasaponins in S. frutescens hairy roots. This suggests that these peptides do not directly affect the biosynthesis pathways of these compounds in S. frutescens. Application of MeJA enhanced soyasaponin production confirming previous reports on induction of these pathways by MeJA elicitation. This study describes the establishment of a platform which can be used to study changes in the transcriptome in response to the application of stress in non-model plants and includes the use of tools to study the function of uncharacterized genes in the model plant A. thaliana. This study also describes the transformation of a non-model medicinal plant (S. frutescens) which could be used to study the effects of ‘novel’ heterologous genes on the metabolism of these plants.
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    Functional roles of raffinose family oligosaccharides: Arabidopsis case studies in seed physiology, biotic stress and novel carbohydrate engineering
    (Stellenbosch : Stellenbosch University, 2015-12) Loedolff, Bianke; Peters, Shaun W.; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB)
    ENGLISH ABSTRACT: The raffinose family of oligosaccharides (RFOs) are α1,6-galactosyl extensions of sucrose (Suc-Galn) unique to the plant kingdom. Their biosynthesis is mediated via α1,6-galactosyltransferases which catalyse the formation of raffinose (Raf, Suc-Gal1), stachyose (Sta, Suc-Gal2) and higher oligomers (Suc-Galn, n ≥ 13) in a stepwise manner. RFOs are well known for their historical roles as phloem translocates and general carbon storage reserves. In recent years their physiological roles have expanded to include potential functions in global plant stress-responses, where correlative mass increases are associated with abiotic stresses such as desiccation, salinity and low temperatures and, to a lesser extent biotic stress (pathogen infection). This study focused on (i) the functional characterisation of a putatively annotated stachyose synthase from Arabidopsis seeds (RS4, At4g01970), (ii) dissection of the proposed functional role of the RFO precursor galactinol in biotic stress tolerance using the Arabidopsis/Botrytis cinerea pathosystem and, (iii) an attempt to engineer long-chain RFOs into Arabidopsis by constitutive over-expression of the unique RFO chain elongation enzyme galactan:galactan galactosyltransferase (ArGGT) from Ajuga reptans. In Arabidopsis Raf is the only RFO known to accumulate in leaves, strictly during conditions of abiotic stress. However, seeds accumulate substantial amounts of both Raf and Sta. While RFO physiology in Arabidopsis leaves and roots is quite well characterised, little is known about the RFO physiology in the seeds. Apart from a single enzyme being described to partially contribute to seed Raf accumulation (RS5, At5g40390), no other RFO biosynthetic genes are known. In this work we functionally characterised an α1,6-galactosyltransferase putatively annotated as a stachyose synthase (RS4, At4g01970) in the Arabidopsis database. Using two insertion mutants (atrs4-1 and 4-2) we demonstrated Sta deficiency in mature seeds. A double mutant with the recently characterised RS5, shown to partially be responsible for Raf accumulation in mature seeds was completely deficient in seed RFOs. This provided the first hint that RS4 could potentially also be involved in Raf biosynthesis. Seed specific expression of RS4 was deregulated by constitutive over-expression in wild-type (Col-0) and the atrs5 mutant background (RS and Raf deficient). Both Raf and Sta unusually accumulated in Col-0 leaves over-expressing RS4, under normal growth conditions. Further, leaf crude extracts from atrs5 insertion mutants (RS and Raf deficient) over-expressing RS4 showed enzyme activities for both RS and SS, in vitro. Collectively our findings have physiologically characterised RS4 as a RFO synthase responsible for Sta and, partially Raf (along with RS5) accumulation during Arabidopsis seed development. The galactosyl donor in RFO biosynthesis, galactinol (Gol) has recently been implicated in biotic stress signalling (pathogen response) in cucumber, tobacco and Arabidopsis. Those studies focused exclusively on Gol in their experimental approaches using both over-expression (tobacco, Arabidopsis) and loss-of-function (Arabidopsis) strategies. However, they did not address the invariable accumulation of Raf that is routinely obtained from such over-expression strategies. We therefore investigated if Raf could play a functional role in induced systemic resistance (ISR), a well-studied mechanism employed by plants to combat necrotrophic pathogens such as Botrytis cinerea. To this end we looked to the RS5 mutant backgrounds (Raf deficient but Gol hyper-accumulating) reasoning that the Gol accumulating mutants should be resistant to B. cinerea (as previously described for transgenic over-expression of GolS1 isoforms in tobacco and Arabidopsis). Such findings would then preclude a role for Raf, since the system would be Raf deficient. Surprisingly, two independent T-DNA insertion mutants for RS5 (atrs5-1 and 5-2) were equally hypersensitive to B. cinerea infection as two independent T-DNA insertion mutants for GolS1 (atgols1-1 and 1-2). The hyper-sensitivity of the GolS1 mutant background has previously been demonstrated. The RS5 mutant backgrounds accumulate substantial amounts of Gol, comparable to those reported for transgenic plants (tobacco and Arabidopsis) where pathogen resistance was reported. Further, during the course of our investigations we discovered that both AtGolS1 mutants also accumulated substantial amounts of both Gol and Raf under normal growing conditions. This was not reported in previous studies. Collectively our findings argue against a role for either Gol or Raf being responsible for the induction/signalling of ISR. However, we do not preclude that the RFO pathway is somehow involved, given the previous reports citing pathogen resistance when GolS1 genes are over-expressed. We are further investigating a potential role for the GolS transcript and/or protein being the component of the suggested signalling function in ISR. The unique enzyme from A. reptans (galactan:galactan galactosyltransferase, ArGGT) is able to catalyse the formation of higher oligomers in the RFO pathway without the use of Gol as a galactosyl donor but rather, using RFOs themselves as galactose donors and acceptors (Gol-independent biosynthesis). We constitutively over-expressed ArGGT in Arabidopsis as a way to engineer long-chain RFO accumulation to further dissect a role for them in improving freezing tolerance. To this end we have been unsuccessful in obtaining RFOs higher than Sta (which occurred in extremely low abundance) in the leaves. Since ArGGT would appear to show substrate preference for Sta, and Arabidopsis seeds accumulate substantial quantities of Sta, we further analysed the seed water soluble carbohydrate (WSC) profiles of three independent transgenic lines but detected no additional RFO oligomers beyond the normally accumulating Raf and Sta. We suggest further strategies to improve this approach (Chapter 4). Collectively this work represents case studies of RFOs in seed physiology, their abilities/requirement in biotic stress and the use of unique enzymes to engineer long-chain RFO accumulation using the Arabidopsis model. At the time of submission of this dissertation the following contributions have been made to the general scientific community: (i) Presentation of chapter 2 at the 26th International Conference for Arabidopsis Research (26th ICAR, 2015, Paris, France) and, (ii) Submission of chapter 2 as a manuscript presently under peer review for possible publication in Plant and Cell Physiology.