>Magister Scientiae - MSc / Food security has always been one of the priority concerns in Africa, and it is mostly threatened by drought stress due to climate change. Drought-induced stress is one of the serious limiting factors of plant production, and it is known to impose oxidative stress as a consequence of excessive reactive oxygen species (ROS) accumulation that lead to lipid peroxidation, which is manifested as increased cell death. Hence, this study investigated the influence of drought stress on two contrasting canola genotypes (Agamax and Garnet), by monitoring their physiological and molecular changes. The results showed that the plant growth and biomass of both genotypes were significantly affected by drought stress as a consequence of excessive ROS accumulation (manifested as H₂O₂ and OH· content). However, under drought stress conditions, the reduction in biomass and shoot length was more pronounced in the Garnet genotype when compared to that of the Agamax genotype. This was further supported by the increase in lipid peroxidation and cell death, which were shown to be significantly higher in the Garnet genotype when compared to the Agamax genotype under drought stress. ROS scavenging ability which prevents oxidative stress and ultimately ROS-induced cellular damage. Hence, given the higher levels of antioxidant activity coupled with the reduction in ROS accumulation that was observed in the Agamax genotype, we suggest that the Agamax genotype might be slightly less susceptible to drought stress, when compared to the Garnet genotype. Furthermore, understanding the proteomic responses of these two contrasting genotypes that showed a marked difference in response to drought stress might help in unlocking complex biological networks of proteins underlying drought stress tolerance. Hence we use two-dimensional (2D) gel electrophoresis coupled with Matrix assisted laser desorption/ionisation-time of flight/time of flight tandem mass spectrometry (MALDI TOFTOF MS) analysis for this part of the study, in order to detect and analyze those differentially expressed proteins or proteins whose abundance levels were influenced as a consequence of drought stress. To gain additional insight into the leaf proteomes of the two canola genotypes, a protamine sulphate precipitation (PSP) method was used to remove RuBisCo and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. A total of 55 well resolved protein spots were selected for mass spectrometry analysis of which 31 (56%) were positively identified using the selective criteria analysis (SCA). All positively identified proteins were then classified into functional categories including protein folding (3%), photosynthetic (29%), detoxification and protection (20%), and energy related proteins whereas 16% could not be classified into any functional category. Apart from spot 32 (Fe superoxide dismutase) and spot 34 (chloroplast beta-carbonic anhydrase), no further significant difference in protein expression/abundance was observed for all the identified proteins for both genotypes in response to drought stress. Both proteins (spots 32 and 34) have been shown to contain antioxidant activity properties which suggest that they might play a crucial role in improving drought stress tolerance in canola plants.

Magister Scientiae - MSc (Biotechnology) / The mining industry in South Africa is of immense importance as this sector contributes largely to the countries income. In the Limpopo province, a large production of antimony (Sb) is generated per year. Antimony (Sb) is a trace element, which accumulates in the environment through anthropogenic activities, such as mining and smelting industries. Antimony is toxic to all living organisms and plants, and it is found to increase the peroxidation of membrane lipids and encourage an antioxidant response. Sb contamination in plants also accounts for DNA damage. The reduction in yield is due to the disruption of plant metabolism by reactive oxygen species (ROS). To combat abiotic stresses, plants have generated a signalling network that utilises multiple growth regulators that would offer protection against the stress. An increase in ROS is one of the responses to abiotic stresses. ROS is generated in response to the pants interaction with heavy metals, through the Harber-Weiss reaction. ROS compounds include: superoxide, hydrogen peroxide and hydroxyl radicals. Under normal conditions ROS molecules are produced as by-products, however, under stressful conditions the production of ROS molecules are increased to levels where they are detrimental to the plants. Therefore, the accumulation of ROS results in damage to proteins, lipids, carbohydrates and DNA which would lead to cellular death. ROS accumulation is thought to be a result of the disruption in the balance of ROS production and the anti-oxidation systems. The antioxidative system is thus introduced to restore the balance of ROS molecule production and to combat oxidative damage caused by the ROS molecules. The anti-oxidative system consists of various enzymes: superoxide dismutase, catalase, and ascorbate peroxidase and glutathione reductase. Each antioxidant scavenges one or two ROS molecules. / 2020-08-31

>Magister Scientiae - MSc / Sugargraze is a moderately drought tolerant sweet sorghum hybrid which is ideal for grazing, winter stand over and pit silage. A major advantage that Sugargraze has over other forages is its very high sugar content which improves feed quality thus increasing palatability and results in significantly reduced feed wastage. This study explored the influence of hyperosmotic stress on plant development, ROS accumulation, antioxidant capacity and the extent of cell death. Heat shock protein (Hsp70) expression immunoblotting assays were used to demonstrate whether the various treatment conditions induced stress within natural physiological parameters for the experimental material. This was coupled with the separation, visualization and identification of abundant proteins in Sugargraze leaves in response to hyperosmotic stress using two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry (MS). The results showed that hyperosmotic stress significantly influences plant development by reducing plant biomass and increasing the levels of ROS accumulation, proline content and subsequently reducing total chlorophyll content. An over accumulation of ROS in the form of hydrogen peroxide and lipid peroxidation was observed in the stressed plants which was supported by the extent of cell death. Although an increase in antioxidant enzyme activity (in the form of total enzymatic activity or individual isoform activity) in response to hyperosmotic stress was observed, this increase was not sufficient to counter the deleterious effects caused by the stress conditions hence the decrease in plant biomass and increase in cell death. Western blotting analysis of Sugargraze leaf tissues using Hsp70 antibodies showed that hyperosmotic stress induced Hsp70 expression to levels significantly higher than observed for the control plants. A total of thirteen CBB stained spots were selected for mass spectrometric identification, owing to their good resolution and abundance levels, and of these, nine were positively identified. Identified proteins were divided into functional categories including both known and novel/putative stress responsive proteins. Molecular and physiological functions of some of the proteins of interest identified will be subjected to further investigation via bioinformatic and molecular biology approaches.

>Magister Scientiae - MSc / Salinity is one of the major limiting factors to crop production, which consequently contributes to the risk of reduced food security. Among other factors, food security depends on availability of sufficient and nutritious food for humans. Livestock such as cattle and sheep are fed with various plant-based feeds; with Medicago sativa (commonly known as alfalfa or lucerne) being a very important forage/feed crop, so much that it is regarded as the queen of forage crops. However, alfalfa is severely affected by high soil salinity and thus its growth and yield are drastically reduced in soils with high NaCl content. Among the various alfalfa genotypes/varieties examined in this study, Agsalfa was identified as salt tolerant because it performed better under salt treatment compared to Magna601.

Thesis (PhD)--Stellenbosch University, 2012. / Salinity stress is one of the major environmental factors that lead to poor crop yield. This is due to overproduction of reactive oxygen species (ROS) which consequently lead to oxidative stress. Although these ROS may be required for normal physiological functions, their accumulation acts as a double edge sword, as they also cause oxidative damage to nucleic acids, lipids and proteins of plant cell membranes. Plants have evolved with an efficient antioxidant defensive system in order to protect and detoxify harmful effects of ROS. Ascorbate peroxidase (APX) is regarded as one of the major scavengers of H2O2. Although some studies have described the role of nitric oxide (NO) in diverse physiological processes in plants, there is still much to know as regards to modulation of APX activity by nitric oxide in salinity-induced stressed plants. For the purposes of this study, the effect of salt and exogenously applied NO on APX, dehydroascorbate reductase and antioxidant metabolite content was determined. This study investigated the use of NO donor 2,2'-(hydroxynitrosohydrazono) bis-ethanimine (DETA/NO) and diethylenetriamine (DETA) on soybean. The data obtained from this study shows that application of DETA/NO resulted in an increase of NO nodular content and also regulated APX activity. The NO-induced changes in APX enzymatic activity were coupled to altered nodule H2O2 content. Further analysis of APX enzymatic activity identified three APX isoforms for which augmented enzymatic activity occurred in response to NO. By supplementing salinity-induced stress soybeans with NO, this study shows that tolerance to salt stress is improved. The underlying mechanism of the NO-mediated tolerance to salt is shown to be its role in modulating the plant antioxidant defense system thus maintaining redox status under salinity-induced stress. Here, although there was increased APX activity in salt stressed plant, supplementing the salinity-induce stressed plants with NO resulted to even higher APX activity which was sufficient to detoxify ROS. Furthermore, this study shows that the NO-mediated effect is not limited in antioxidant enzymes but also involves regulating antioxidant metabolite ratio through modulating the antioxidant enzymes that are involved in the ascorbate -glutathione cycle.

Magister Scientiae - MSc (Biotechnology) / Brassica napus is a plant that is used for human and animal consumption. This plant is also used for phytoremediation due to its relatively higher level of heavy metal tolerance. In South Africa, mining is one of the main drivers of the economy. One of the major negative environmental impacts of mining is heavy metal contamination. Soil metal content can rise to levels that are quite high and can even have a negative impact on the yields of B. napus crop. The glucosinolate-myrosinase system of B. napus is a system that is used as defence against biotic stressors. Indole glucosinolate breakdown products have been proven to enhance the antioxidant capacity of plants. Some have also shown growth promoting properties in plants. We studied the effect of exogenous DIM on B. napus and it role in Zr induced heavy metal stress. Germination percentages revealed that DIM increased germination, Zr application decreased germination and the DIMZr treatment reversed the negative impact of Zr application on B. napus. The effect of treatments on the biomass of B. napus was assessed by determining the dry weights. Results show that exogenous DIM improves biomass. Zr application decreased biomass and DIM-Zr treatment ameliorated the effect of Zr application. / 2020-08-31

Pseudoperonospora cubensis (Bert. et Curt) Rost. is the causal agent of cucurbit downy mildew (CDM). It is the most damaging cucumber pathogen on the Eastern Shore of Virginia and eastern parts of the United States. Pseudoperonospora cubensis is an obligate oomycete pathogen, infecting crops within the Cucurbitaceae family. The disease is characterized by angular chlorotic lesions and a downy or felt-like appearance on the abaxial side of the leaf. Control of this pathogen includes use of resistant cucumber cultivars and costly fungicide programs. Continuous use has led to resistance to commonly used fungicides. This has become a major concern and in response, seed companies have developed cucumber cultivars which claim downy mildew resistance.  This study evaluates different cucumber cultivars and assesses their level of resistance to CDM. The results indicate that an integrated management approach of reduced fungicide application and the use of resistant cultivars can suppress levels of CDM and yield a cucumber crop. Additionally, a molecular study was conducted, comparing the relative expression of genes encoding a basic PR-1 protein, a cytosolic ascorbate peroxidase protein and three resistance (R) gene proteins, in nineteen cultivars. All of the selected genes were analyzed using real-time PCR. The relative expression levels of the R-genes varied between cultivars. The basic PR-1 protein decreased expression in the majority of the cultivars, suggesting no involvement in the first twenty-four hours. Cytosolic ascorbate peroxidase relative expression levels suggest an increase in susceptible cultivars and a decrease in tolerant cultivars. / Master of Science

Water stress is the most critical environmental factor limiting crop production in the US Piedmont. The presence of humic substances in composted organic amendments may increase crop tolerance to water stress through their hormone-like effects on plant metabolism. The objectives of this study were to calculate N mineralization rates of composted and non-composted organic materials used in this long-term field study, and to determine differences in soil physiochemical properties, corn and soybean leaf physical and biochemical properties yield and seed quality between organically amended and inorganically fertilized treatments. Nitrogen mineralization rates were greatest in the poultry litter (21%) and Panorama yard waste compost (4.5%) amended plots. Nitrogen uptake (120 mg/pot, 133 mg/pot, respectively) in these treatments were greater than that in the control (0N) (91.3 mg/pot) treatment. Wolf Creek biosolids compost and Huck's Hen Blend yard waste compost induced N immobilization (-5.0% and 0.18%, respectively), and had N uptake values similar to the control (92.6 mg/pot and 95.7 mg/pot). Rivanna biosolids compost immobilized N (-14.8%) but N uptake (136 mg/pot) was greater than that in the control due to the relatively high inorganic N content in the amendment. The total N concentration and C:N values were less reliable variables in predicting N mineralization when a significant portion of the total N was in the inorganic form. The annual application of poultry litter, Rivanna biosolids compost, and Panorama yard waste compost at 100% agronomic nitrogen and 30 % agronomic nitrogen rates in the field study improved soil fertility and increased total organic and humified carbon contents relative to the inorganically fertilized and control treatments. The amended treatments had slightly greater plant available water contents (average 10.0 cm/15 cm) than the control (8.38 cm/15 cm). Leaf water potential measurements revealed that neither crop experienced water stress during the sampling season. Treatment differences in leaf antioxidant activity were only observed in corn. All corn plants that were fertilized with amendments supplying the crop's nitrogen needs, regardless of the source, had greater leaf nitrogen (+29%), chlorophyll (+33%), and protein contents (+37%), lower superoxide dismutase (-29%) and ascorbate peroxidase (-17%) activities, and lower malondialdehyde (-33%) contents relative to the control and low nitrogen treatments. There were no observed differences in catalase activity, which was likely due to the evolutionary advantage of C4 metabolism. Yield was strongly related to midseason leaf nitrogen contents (R2=0.87, p<0.0001) and not soil humified carbon (R2=0.02, p=0.0543). There were no observed treatment differences in soybean leaf physiology and metabolism. Differences, however, were observed over time. As the leaves senesced, leaf chlorophyll, protein, superoxide dismutase and catalase activities decreased, and the malondialdehyde content increased. Ascorbate peroxidase activity slightly increased with time. Catalase activity in soybean was primarily driven by the oxidation of glycolate, a product of photorespiration, and not the formation of reactive oxygen species in the chloroplasts. The organically amended treatments had higher yields (9-21% increase), greater protein contents (4-9% increase), and seed weights (5-14% increase) relative to the fertilizer and control treatments. It was concluded that differences in soybean yield and seed quality were due to non-nutritive benefits of the organic amendments and not available water or plant nutrition. / Master of Science

>Magister Scientiae - MSc / There is an influx in heavy metals into soils and ground water due to activities such as increased mineral mining, improper watering and the use of heavy metal contaminated fertilizers. These heavy metals are able to increase the ROS species within plants which may result in plant metabolism deterioration and tissue damage. Heavy metals may also directly damage plants by rendering important enzymes non-functional through binding in metal binding sites of enzymes. The heavy metal focused on in this study was vanadium due to South Africa being one of the primary produces of this metal. Two related Brassica napus L cultivars namely Agamax and Garnet which are economically and environmentally important to South Africa were exposed to vanadium. Physiological experiments such as cell death, chlorophyll and biomass determination were conducted to understand how these cultivars were affected by vanadium toxicity. A low cost, sensitive and robust vanadium assay was developed to estimate the amount of vanadium in samples such as water, soils and plant material. The oxidative state as well as the antioxidant profile of the two cultivars were also observed under vanadium stress. A chlorophyll assay which was conducted on the two cultivars exposed to vanadium showed a marked decrease in chlorophyll A in the suspected sensitive cultivar which was Garnet. However, the suspected tolerant cultivar Agamax fared better and the decrease in chlorophyll A was much less. A similar trend was observed for the two cultivars when the cell death assay was conducted. The vanadium assay showed that Garnet had higher concentrations of vanadium within its leaves and lower concentrations in its roots when compared to Agamax. This observation displayed that Agamax had inherent mechanisms which it used to localize vanadium in its roots and which assisted in its tolerance to the vanadium stress. The oxidative state was determined by doing assays for the specific reactive oxygen species namely hydrogen peroxide and superoxide. It was observed that vanadium treated Garnet leaves had higher reactive oxygen species (ROS) production when compared to the Agamax treated leaves. In-gel native PAGE activity gels were conducted to determine the antioxidant profile for the two cultivars which were exposed to vanadium. The antioxidant enzymes which were under investigation were ascorbate peroxide (APX), superoxide dismutase (SOD) and glutathione-dependent peroxidases (GPX-like) as these enzymes are known to be responsible for controlling the ROS produced in the plants. The GPX-like profile consisted of three isoforms. No isoforms were inhibited by vanadium treatments but one isoform had increased activity in both the Garnet and Agamax treated samples. The SOD profile for Garnet consisted of six isoforms and Agamax had seven isoforms. One isoform which was visualized in both Agamax as well as Garnet was inhibited by vanadium treatments. Agamax also had two isoforms which were up-regulated however the corresponding isoforms in Garnet showed no change. The Ascorbate peroxidase profile consisted of seven isoforms for both Garnet and Agamax. No isoforms were inhibited by vanadium treatment. Three isoforms were up-regulated in Garnet and Agamax under vanadium treatments. Here, it is illustrated that Garnet lacked certain mechanisms found in Agamax (and thus experienced more cell death, yield and chlorophyll loss) and performed worst under high vanadium concentrations. Although Garnet increased the activity of some of its antioxidant isoforms in response to increasing ROS levels it was not adequate to maintain a normal oxidative homeostasis. This disruption in oxidative homeostasis lead to plant damage. Agamax was observed to produce less ROS than Garnet and was able to control the ROS produced more effectively than Garnet and thus less damage was observed in Agamax.

Using gentian and petunia as the experimental systems, potential alternative post-harvest treatments for cut flowers were explored in this project. Pulsing with GA₃ (1 to 100 µM) or sucrose (3%, w/v) solutions delayed the rate of senescence of flowers on cut gentian stems. The retardation of flower senescence by GA₃ in both single flower and half petal systems was accompanied by a delay in petal discoloration. The delay in ion leakage increase or fresh weight loss was observed following treatment with 5 or 10 µM GA₃ of the flowers at the unopen bud stage. Ultrastructural analysis showed that in the cells of the lower part of a petal around the vein region, appearance of senescence-associated features such as degradation of cell membranes, cytoplasm and organelles was faster in water control than in GA₃ treatment. In particular, degeneration of chloroplasts including thylakoids and chloroplast envelope was retarded in response to GA₃ treatment. In the cells of the top part of a petal, more carotenoids-containing chromoplasts were found after GA₃ application than in water control. In petunia, treatment with 6% of ethanol or 0.3 mM of STS during the flower opening stage was effective to delay senescence of detached flowers. The longevity of isolated petunia petals treated with 6% ethanol was nearly twice as long as when they were held in water. Senescence-associated petal membrane damage, weight decline, ovary growth and decrease in protein and total RNA levels were counteracted in ethanol-treated petals. The accumulation of ROS, particularly superoxide and hydrogen peroxide, was also inhibited or delayed by ethanol application. Anti-senescence mechanisms, particularly the changes of oxidative / antioxidant metabolism involved in petal senescence, were investigated. In gentian, activities of AP and SOD but not POD in the GA₃-treated petals were significantly higher than those of the control. In isolated petunia petals, the decreased trends of antioxidative SOD and AP activities during senescence were apparently prevented in response to ethanol treatment although the levels of ascorbate and photo-protective carotenoids were not affected. Furthermore, by optimizing a range of critical PCR parameters such as primer combinations, cDNA concentrations and annealing temperatures, a reliable protocol has been established for quantifying the expression level of Cu-Zn SOD gene in petunia petals using SYBR Green I based real-time RT-PCR. A 228 bp gene fragment of Cu-Zn SOD was isolated from petunia (var. 'hurrah') using RT-PCR. It was found that the mRNA level (relative to 18S rRNA level) of Cu-Zn SOD decreased significantly after 6 days in water. However, there was about a 55-fold increase in Cu-Zn mRNA level after 6 days of ethanol treatment when compared to water-treated petals. Similarly, down-regulation of the mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also observed during senescence of petunia petals. Increased vase life of petunia petals by ethanol treatment was correlated with promotion of GAPDH expression by a factor of about 16 on day 6. Taking together, the anti-senescence effects of GA₃ and ethanol are at least partially associated with an increased efficiency of petal system utilizing ROS since the selected antioxidants were significantly maintained when compared to the corresponding values for the control.