Graduate

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No description available.

Phenylketonuria and hyperphenylalanemia are autosomal recessive inborn errors of phenylalanine metabolism that are caused by mutations in the phenylalanine hydroxylase gene. Due to the stringency of the present dietary therapy, alternative treatments are being studied. Phenylalanine ammonia-lyase (PAL) is one of the potential dietary supplements for these patients. PAL is a well-studied plant enzyme which breaks down phenylalanine into trans-cinnamic acid and ammonia (Camm and Towers, 1973). It is found in the cytoplasm of the plant cells and is naturally encapsulated by plant cell walls which may protect it against the acidic pH environment in the gastrointestinal tract. It presumably degrades ingested Phe that circulates in the intestinal lumen. In this study, red spring wheat seedlings (Triticum aestivum L.) found to contain high PAL activity naturally were investigated as a potential alternative oral therapy. Specifically, the objectives were (1) to evaluate different drying methods on generating concentrated and dried preparation of wheat seedlings containing high levels of PAL activity; (2) to examine the retention of PAL activity over three months of storage under various storage conditions; (3) to determine the stability of PAL activity in simulated human digestion condition to establish if further study of using plant source enzyme in vivo is warranted. Freeze-drying (FD) was found to have retained the most activity (>90 % recovery dry wt basis) compared to air-drying (AD) and vacuum-microwave drying (VMD) for both leaf and residual seed/root samples. Pre-freezing of leaf tissues at -18 °C before FD significantly retained the highest PAL activity compared to pre-freezing at -25 °C, -35 °C, and -80 °C (P<0.05). Over three months of storage, 60-80 % of PAL activity was recovered in leaf and —100 % was recovered in residual seed/root tissues after storage at -20 °C. After in vitro protein digestion, 36% and 42 % of PAL activity was recovered in fresh leaf and root tissues respectively; however, FD tissues were found to be susceptible to proteases and acidic environment and no activity was recovered after three hours of in vitro protein digestion. High performance liquid chromatography (HPLC) analysis of the residual Phe after in vitro protein digestion confirmed that fresh tissues had significantly higher conversion of Phe than that of FD tissues. Together, these results suggest that red spring wheat seedlings may have potential as a dietary supplement for phenylketonuric patients while further study to enhance PAL activity in plant preparations is required.

Pinus patula Schiede ex Schlect. & Cham. is the most widely planted softwood species for both pulpwood and saw timber in the South African forestry industry. High mortality of this species, often in excess of 20%, following planting is currently of major concern and has the potential to limit future deployment for commercial timber. Water stress is often reported to be a cause of mortality during regeneration in commercial forestry plantations yet, prior to 2007, there was no published research on the water relations of P. patula during regeneration in South Africa. This, together with questions raised by the industry as to the role of using water in the planting operation, initiated the series of studies conducted for this thesis. Water planting (application of water into the planting hole at the time of planting) of P. patula seedlings has been used commercially to reduce post-planting water stress and buffer against potentially extreme weather conditions immediately after planting. However, the primary role of the water, as well as its success in increasing survival following planting, has never been critically assessed. Since the use of water in the planting operation is expensive, it was essential that the benefits to using water were quantified, in terms of survival and growth, and justified, in terms of any monetary investment. In addition, there was a lack of local studies investigating the physiological characteristics of P. patula seedlings, particularly their tolerance to low soil water availability. To understand the role of water during the regeneration of P. patula in terms of plantation management and seedling physiology, a variety of research methodologies were used that included: applied field trials, multivariate methods (a retrospective investigation), pot trials and the development of a simple financial model. Four field trials were implemented to test the response in P. patula survival to water applied at planting. Two trials each were situated in the KwaZulu-Natal (KZN) Midlands and Mpumalanga Escarpment. The first trial at each site was planted in spring (October) and the second in summer (February). Watering treatments consisted of different quantities of water used in the planting operation and included 0.5 litres, 2 litres, 4 litres and no water (dry plant). Only at the spring planted trial in the KZN Midlands was survival of the dry planted seedlings significantly lower than that of the seedlings planted with water, at 90 days after planting. This may have been due to low rainfall during the week before and two weeks after planting, or the small size of the seedlings used in the trial. Application of 0.5 litres of water to the planting pit was sufficient to increase survival to a level equivalent to that where 2 or 4 litres of water was used, yet only increased soil moisture in the area immediately surrounding the seedling. This suggested that the role of the water applied during planting was increased root to soil contact. Overall, these four trials indicated that planting with water had the potential to increase survival only when soil water availability was low and rainfall sporadic. There was no effect of water applied at planting on early tree growth. While the results of the four field trials provided an indication of the effect of planting with water on subsequent survival of P. patula seedlings, there was concern that the results of the four trials may not be a true reflection of a dynamic situation. Survival in response to water applied at planting may vary from year to year and across forestry regions due to the unpredictable nature of rainfall and high air temperatures during the planting season, as well as the wide range of forestry sites across which P. patula seedlings are planted. To improve our understanding, a database of 58 trials was compiled where water and dry planting had been carried out. In this way it was possible to investigate whether the results from the four field trials were reflected in a range of previously conducted field trials implemented across time and space. The trials incorporated into the dataset were all planted to P. patula between 1990 and 2005 in the summer rainfall region of southern Africa. Data related to the climate, local weather, physiography and site management at each trial were also included. Summary statistics, linear correlation and multiple regression were used to determine if site-associated variables were related to an increase in survival in the water relative to the dry planted treatments. The analyses indicated that for all 58 trials, survival was lowest during the summer months, regardless of planting treatment. Planting with water was most likely to increase survival when used during spring, autumn and winter planting, although (as with the four applied field trials) there was no overall significant relationship between water planting and survival. Based on these results it was anticipated that an understanding of the water stress physiology of P. patula seedlings was required to explain the observed trends from a more fundamental perspective; if planting with water did not always increase survival, why not? Three pot trials were conducted to increase the understanding of the water relations of P. patula seedlings. These trials were also used to provide benchmark physiological data related to stressed (water) and unstressed seedlings. The first pot trial highlighted the importance of root plug moisture at the time of planting for increasing subsequent survival. The subsequent two pot trials were aimed at investigating the interaction between planting stock quality (as determined by measures of size) and soil water availability and the effect on survival, growth and physiology of P. patula seedlings. These results indicated that P. patula seedlings were not as sensitive to high air and soil temperatures (above 30°C) and low soil water availability (below -1.5 MPa) as previously thought. The seedlings were able to tolerate low soil water availability for several weeks and, following rewatering, were able to recover from moderate and severe water stress (a shoot water potential of below -1.5 MPa). This data supported the results from the four applied field trials and retrospective study of 58 trials, where the application of water to the seedlings at planting did not substantially increase survival. In the pot trials, stomatal conductance started to decrease when shoot water potential approached -0.8 to -0.9 MPa. Stomatal closure occurred at a shoot water potential between -1.2 MPa to -1.5 MPa. Mortality due to water stress occurred only in response to extended periods of low soil water and was associated with a shoot water potential of below -3.0 MPa. There was variability between seedlings in their potential for survival and growth. Inherently bigger seedlings had a greater capacity for new root growth following planting. New root growth, as well as a greater mass of new roots, was associated with higher shoot water potentials and higher rates of transpiration under conditions of low soil water availability. This indicated that seedling quality, as determined by size, may play a role in sensitivity to water stress. The field trials, retrospective study and pot trials indicated that the practice of planting with water was not always critical to the survival of P. patula seedlings. A simple financial model was developed to estimate whether planting with water represented a cost that could be used as a decision criterion, given certain growth parameters and management scenarios. The data projected by the model were also compared to actual research data for water versus dry planting (and the inclusion of an insecticide in the water). While these comparisons were specific to the parameters included in the model for this study, as well as the results of the research trials used in the benchmarking exercises, the model indicated that; 1) costs for planting with water were likely to be recovered only when no blanking (replacing of dead trees) was carried out, with capital invested at a low return rate (3%), 2) including an insecticide in the water increased the likelihood of cost recovery, and 3) site quality had an impact on the increase in survival required to recover planting method costs, with a greater percentage increase in survival required on lower quality sites. Lower quality sites often have a lower mean annual precipitation (associated with higher rainfall variability), or shallow soils (associated with lower soil water availability) and therefore are also likely to be sites where foresters may want to use water to reduce (drought related) mortality. The impact of site quality is thus also an important factor to include in any decisions regarding planting methods (i.e. using water) and their costs. Further investigations should be aimed at examining; 1) the interaction of root plug size (as determined by container type) and soil water availability on growth and physiology of P. patula seedlings, 2) the methods of grading seedlings within a population to select those that have a high potential for survival and growth, and 3) the effects of soil water availability on the physiology, survival and growth of P. patula cuttings, as well as other pine species and hybrids grown in South Africa, such as P. elliottii, P. elliottii x P. caribaea and P. patula x P. tecunumanii. It is likely that the proportion of forestry regions planted to these hybrids will increase in the future. / Thesis (Ph.D.)-University of KwaZulu-Natal, 2008.

This thesis quantifies aspects of shade tolerance in tree seedlings of species native to the Caledonian pinewood ecosystems of Glen Affric (Highland Region, Inverness-shire). Growth, allocation and morphological responses of 15 species to irradiance under simulated forest canopy light were investigated in a nursery-based shade house experiment. The same responses of four of the 15 species (Ilex aquifolium, Alnus glutinosa, Sorbus aucuparia and Betula pubescens) to different developmental stages of Pinus sylvestris woodland were investigated in the field. The spatial and temporal growth responses of naturally regenerating S. aucuparia seedlings to shade and gap microhabitats were also studied. Data from the shade house experiment enabled further detailed exploration of the relationship between relative growth rates (RGR) and irradiance and potential cross-overs of ranks of growth in high and low light conditions.

Phenylketonuria and hyperphenylalanemia are autosomal recessive inborn errors of phenylalanine metabolism that are caused by mutations in the phenylalanine hydroxylase gene. Due to the stringency of the present dietary therapy, alternative treatments are being studied. Phenylalanine ammonia-lyase (PAL) is one of the potential dietary supplements for these patients. PAL is a well-studied plant enzyme which breaks down phenylalanine into trans-cinnamic acid and ammonia (Camm and Towers, 1973). It is found in the cytoplasm of the plant cells and is naturally encapsulated by plant cell walls which may protect it against the acidic pH environment in the gastrointestinal tract. It presumably degrades ingested Phe that circulates in the intestinal lumen. In this study, red spring wheat seedlings (Triticum aestivum L.) found to contain high PAL activity naturally were investigated as a potential alternative oral therapy. Specifically, the objectives were (1) to evaluate different drying methods on generating concentrated and dried preparation of wheat seedlings containing high levels of PAL activity; (2) to examine the retention of PAL activity over three months of storage under various storage conditions; (3) to determine the stability of PAL activity in simulated human digestion condition to establish if further study of using plant source enzyme in vivo is warranted. Freeze-drying (FD) was found to have retained the most activity (>90 % recovery dry wt basis) compared to air-drying (AD) and vacuum-microwave drying (VMD) for both leaf and residual seed/root samples. Pre-freezing of leaf tissues at -18 °C before FD significantly retained the highest PAL activity compared to pre-freezing at -25 °C, -35 °C, and -80 °C (P<0.05). Over three months of storage, 60-80 % of PAL activity was recovered in leaf and —100 % was recovered in residual seed/root tissues after storage at -20 °C. After in vitro protein digestion, 36% and 42 % of PAL activity was recovered in fresh leaf and root tissues respectively; however, FD tissues were found to be susceptible to proteases and acidic environment and no activity was recovered after three hours of in vitro protein digestion. High performance liquid chromatography (HPLC) analysis of the residual Phe after in vitro protein digestion confirmed that fresh tissues had significantly higher conversion of Phe than that of FD tissues. Together, these results suggest that red spring wheat seedlings may have potential as a dietary supplement for phenylketonuric patients while further study to enhance PAL activity in plant preparations is required.

Thesis (M.Sc.(Agric.))(Horticulture)--University of Pretoria, 2006. / Includes summary. Includes bibliographical references. Available on the Internet via the World Wide Web.

Made available in DSpace on 2014-10-09T12:28:42Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:56:04Z (GMT). No. of bitstreams: 0 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP / FAPESP:08/55680-8