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Aluminium toxicity is the main limiting factor in crop production on acid soils. The main symptom of aluminium toxicity is a rapid inhibition of root growth, but the mechanism of root growth cessation remains unclear. In this diploma thesis we deal with the question of whether phospholipases PLDα1 and PLDδ may play a role in the mechanism of aluminium toxicity. We compared the responses of plants lacking PLDα and PLDδ with WT plants. Growth analysis of roots was performed in hydroponic conditions. The most sensitive part of roots was transient zone in which cells were dying earlier. It was further found that pldα1 plants were less sensitive on aluminium toxicity because their roots showed less growth inhibition than WT. Pldδ plants did not differ from WT plants in their response to aluminum. During further analysis of the pldα1 reactions, it was found that the root cells were capable of cell expansion during aluminum toxicity, and the cellular malformations were formed on the roots. This phenomenon was associated with faster reorientation and even depolymerization of cortical microtubules in response to toxic aluminium in pldα plants compared to WT plants. The results indicated that PLDα1 molecule affects the stability of cortical microtubules. Microtubules were less stable and they depolymerized...

Aluminium (Al) and silver (Ag), through human activities, are present in the environment at concentrations sufficient to cause toxicity. The aim of this study was to administer Al and Ag to the short lived model organism Drosophila melanogaster, so that developmental toxicity and potential ameliorative interventions could be examined over a compressed timescale relative to mammalian models. Aluminium was administered to Drosophila in food as either the chloride salt or citrate complex at concentrations of 1, 10 and 100 mM and various developmental parameters were assessed. The lowest concentration to delay pupation relative to the control was 10 mM but this depended upon the food in which it was administered. Higher whole body tissue levels of Al were seen following Al citrate administration compared to AlCl3, but Al citrate was less toxic as this did not did not impair larval viability at 100 mM; 100 mM AlCl3 resulted in 100% mortality. Eclosion success was significantly impaired with either form of Al at 10 mM, but no difference was seen between the forms of Al. When Drosophila were fed AlCl3 over their entire lifespan, a small but significant reduction in the lifespan of male flies was seen. No behavioural toxicity could be demonstrated. Existing studies have demonstrated significant tissue Al concentrations and toxicity whereas these have been minimal in this study. It is suggested that these differences may have a genetic component, with food composition exerting an influence also. Silver, either as AgNO3 or Ag nanoparticles (AgNPs) was administered in concentrations up to 500 micromolar and 10 mM, respectively. Either form of Ag, at 50 micromolar was sufficient to significantly retard pupation rate, although pupation or eclosion success was not impaired until 100 micromolar. The concentration-response relationship for AgNO3 was steep with pupation success dropping to nearly zero by 300 micromolar; Drosophila in this study were far more sensitive to AgNO3 than those in other reports. Animals exposed to AgNPs were still able to pupate at 500 micromolar, but these pupae were almost all non-viable when exposed to 400 micromolar AgNPs. At 1 mM and above, AgNPs, however, showed reduced toxicity compared to lower concentrations. The reasons for this are unclear. Both forms of Ag caused de-pigmentation in adults after larval exposure that may be explainable by inhibition of polyphenol oxidase enzymes by Ag (I) ions. The de-pigmentation was preventable by pre-loading larvae with Cu. Ascorbate prevented the de-pigmentation caused by AgNPs but not AgNO3 suggesting that AgNP toxicity is due to Ag (I) ion release. Oxidation of AgNPs was found to be greatly accelerated by Fe (III) and Cu (II) ions in the presence of Cl- ions. Although some of the results here conflict with the literature, developmental toxicity has been observed here, for both Al and Ag, and the variability across studies may provide an opportunity for dissecting the mechanisms behind Al and Ag toxicity through identification of the traits that confer sensitivity or resistance.

Okra (Abelmuschus esculentus (L) Moench.) is one of the most popular crops within the Malvaceae family of plants. It is a common vegetable eminently cultivated in regions experiencing constraints to manage climate change. In South Africa climate change coupled with aluminium-enriched soils are responsible to drawbacks crop performance. Therefore, it is worthwhile to whether okra will thrive as an alternative crop in the country. Many studies have identified potential of okra to improve yields of resource poor farmers in Africa. The physiological responses of okra seed to variations in aluminium ions and temperature were not determined. Therefore, a study with okra, cv. Clemson Spineless, seed coated and uncoated with B. subtilis, was initiated to assess germination on moist filter paper in 90mm diameter Petri plates. Germination medium consisted of various concentrations of aluminium chloride (AlCl3), 0M, 0.001M, 0.01M, 0.05M and 0.1M. Each aluminium treatment was allocated into incubators adjusted to 22°C, 25°C and 37°C temperatures. This resulted into a 5 x 3 x 2 factorial experiment with five replicates and was conducted in three cycles. Daily scores of germinated seeds were assessed from the second to the fifth day after initiation of germination. During termination, five days after the initiation of the experiment 10 seeds with the longest coleoptiles had their coleoptiles measured using a digital caliper. At the fifth day after initiation of the experiment, coleoptile lengths from 10 seeds per treatment were measured using digital caliper. A total of 50 plates (10 from 37°C in Cycle 1; 30 from 22°C, 25°C and 37°C from Cycle 2; 10 from 37°C in Cycle 3), were selected and germinated were ground and stored at - 20°C before 1H NMR analysis. Metabolites were extracted from 50mg ground seed material with 750 μL methanol-D4 and 750 μL buffer (deuterium oxide + potassium dihydrogen phosphate). The mixture was vortexed for three minutes, sonicated for 20 minutes, centrifuged at 18000 rpms for 20 minutes and the supernatant filtered through cotton wool. Then the supernatant was dispensed into NMR tubes for further 1H NMR spectroscopic processing using a 600 MHz NMR xiii Varian spectrometer to generate magnetic spectra of the fifty samples. Results of this study demonstrated that in all the experimental cycles, regardless of aluminium concentration and bacterial seed coating, 37°C inhibited germination percentages and coleoptile lengths in okra seed germination. Germination percentages and coleoptile lengths of bacteria-coated seeds growing in 25°C were most stimulated at all aluminium concentrations, but not at 0.1M. In this temperature germination percentages and coleoptile lengths were highly influenced by the interaction of aluminium concentrations and bacterial coating, respectively. 1H NMR metabolomic association showed no distinct grouping, but clusters across treatments showed to be linked through a subset of metabolites amongst aluminium concentrations, bacterial seed coating and temperatures, respectively. This infers that treatment variations in both seed and bacterial physiological responses were associated through shared metabolic pathways. In conclusion, the study proved that 25°C provide temperature environment within which B. subtilis can be able to stimulate growth and remediate physiological constraints from aluminium ions during okra seed germination. / Agriculture, Animal Health and Human Ecology / M. Sc. (Agriculture)

Okra (Abelmuschus esculentus (L) Moench.) is one of the most popular crops within the Malvaceae family of plants. It is a common vegetable eminently cultivated in regions experiencing constraints to manage climate change. In South Africa climate change coupled with aluminium-enriched soils are responsible to drawbacks crop performance. Therefore, it is worthwhile to whether okra will thrive as an alternative crop in the country. Many studies have identified potential of okra to improve yields of resource poor farmers in Africa. The physiological responses of okra seed to variations in aluminium ions and temperature were not determined. Therefore, a study with okra, cv. Clemson Spineless, seed coated and uncoated with B. subtilis, was initiated to assess germination on moist filter paper in 90mm diameter Petri plates. Germination medium consisted of various concentrations of aluminium chloride (AlCl3), 0M, 0.001M, 0.01M, 0.05M and 0.1M. Each aluminium treatment was allocated into incubators adjusted to 22°C, 25°C and 37°C temperatures. This resulted into a 5 x 3 x 2 factorial experiment with five replicates and was conducted in three cycles. Daily scores of germinated seeds were assessed from the second to the fifth day after initiation of germination. During termination, five days after the initiation of the experiment 10 seeds with the longest coleoptiles had their coleoptiles measured using a digital caliper. At the fifth day after initiation of the experiment, coleoptile lengths from 10 seeds per treatment were measured using digital caliper. A total of 50 plates (10 from 37°C in Cycle 1; 30 from 22°C, 25°C and 37°C from Cycle 2; 10 from 37°C in Cycle 3), were selected and germinated were ground and stored at - 20°C before 1H NMR analysis. Metabolites were extracted from 50mg ground seed material with 750 μL methanol-D4 and 750 μL buffer (deuterium oxide + potassium dihydrogen phosphate). The mixture was vortexed for three minutes, sonicated for 20 minutes, centrifuged at 18000 rpms for 20 minutes and the supernatant filtered through cotton wool. Then the supernatant was dispensed into NMR tubes for further 1H NMR spectroscopic processing using a 600 MHz NMR xiii Varian spectrometer to generate magnetic spectra of the fifty samples. Results of this study demonstrated that in all the experimental cycles, regardless of aluminium concentration and bacterial seed coating, 37°C inhibited germination percentages and coleoptile lengths in okra seed germination. Germination percentages and coleoptile lengths of bacteria-coated seeds growing in 25°C were most stimulated at all aluminium concentrations, but not at 0.1M. In this temperature germination percentages and coleoptile lengths were highly influenced by the interaction of aluminium concentrations and bacterial coating, respectively. 1H NMR metabolomic association showed no distinct grouping, but clusters across treatments showed to be linked through a subset of metabolites amongst aluminium concentrations, bacterial seed coating and temperatures, respectively. This infers that treatment variations in both seed and bacterial physiological responses were associated through shared metabolic pathways. In conclusion, the study proved that 25°C provide temperature environment within which B. subtilis can be able to stimulate growth and remediate physiological constraints from aluminium ions during okra seed germination. / Agriculture, Animal Health and Human Ecology / M. Sc. (Agriculture)