Biological Sciences

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Biochemical and biophysical indicators of chilling tolerance in some species of Eucalyptus.
(1993) Stewart, Gwethlyn Meriel.; Amory, Alan M.
Although Eucalyptus species are indigenous to Australia, they have been successfully cultivated in South Africa, where they are mainly used in the mining and paper industries. With the explosion in these industries it has been necessary to increase Eucalyptus plantations, often into areas which experience frost and chilling temperatures. To combat this, high yielding Eucalyptus species able to cope with these conditions would be desirable. The ability to rank species according to their chilling tolerance will enhance decisions as to the suitability of species for use in the field. To this aim, two biochemical and two biophysical parameters were chosen to investigate and characterise Eucalyptus nitens, E. smithii, E. macarthurii, E. grandis and E. grandis x nitens (GNI026). Ranking of these species in terms of chilling tolerance did not appear possible using the data from the biochemical parameters (proline concentration and glutathione reductase activity), but the biophysical parameters (fluorescence characteristics and onset of temperature of melt) gave results comparable to those obtained in field trials using these species. Fluorescence characteristics were particularly useful in assessing the chilling tolerance of the species in question. Fluorescence is the recommended technique for further studies as it is relatively inexpensive, rapid, does not require destructive sampling and can be used in both the laboratory and field.
The effect of elevated glutathione reductase and superoxide dismutase activities in stressed transgenic tobacco.
(1996) Penter, Mark Gavin.; Amory, Alan M.
Life as we know it would be impossible in the absence of oxygen. However, too much oxygen can be toxic to the aerobic organisms which depend on it for their very existence. This apparent paradox arises as a result of oxygen's ability to accept electrons, forming highly reactive (reduced) oxygen species such as superoxide, hydrogen peroxide and the hydroxyl radical. The toxicity of oxygen is greatly enhanced in illuminated plants, due to the photosynthetic reactions which produce both oxygen and highly energetic electrons in close proximity to one another. These problems are further exacerbated when plants are exposed to a variety of stress conditions, since these conditions reduce the ability of plants to utilise excess electrons. As a result of the danger posed by· these reactive oxygen species, plants have· evolved a complex antioxidant system for their scavenging. Research has shown that plants with naturally elevated levels of the components of the antioxidant system are better equipped to deal with stress conditions which enhance the production of reactive oxygen species. A considerable amount of research has thus been dedicated to the elucidation of the antioxidant system. Almost as much research has been dedicated to enhancing the antioxidant system, with the aim of improving plant productivity under stress conditions. This study sought to evaluate plants carrying elevated levels of two of the enzymes of the antioxidant system. For these purposes, tobacco was transformed with the gene for E. coli glutathione reductase (GR), an enzyme believed to catalyse the rate limiting reaction in the scavenging of hydrogen peroxide. This gene was fused to the gene for the RUBISCO small subunit transit peptide - a peptide capable of targeting proteins to the chloroplast. Due to the presence of this peptide the transformed plants exhibited high chloroplastic levels of GR activity. These plants were crossed with a second tobacco transformant carrying high levels of chloroplastic tomato superoxide dismutase (SOD) - an enzyme responsible for the scavenging of superoxide. These hybrid plants were shown to exhibit high GR and SOD activities in the chloroplast .- the subcellular compartment most susceptible to damage caused by reactive oxygen species. The transgenic hybrids were evaluated for their ability to tolerate oxidative stress by treating them with paraquat - a herbicide whose mode of action involves the production of large quantities of activated oxygen. Under stress conditions, plants carrying just E. coli GR showed a slight improvement in their ability to deal with oxidative stress. In contrast to this, the SOD transformants showed more cellular damage than untransformed control plants. This was attributed to the inability of other enzymes in the antioxidant pathway to deal with the increased flow of metabolites through the pathway. The hybrid transformants showed enhanced stress tolerance in the initial stages of oxidative stress, but this declined with ongoing exposure to stress conditions. As with the SOD transformants, this decline in protection was . ascribed to the relatively low activities of the other enzymes in the antioxidant pathway. It was concluded that elevated levels of the two enzymes conferred greater stress tolerance than just one of the enzymes, but for true stress tolerance it will be necessary to evaluate the antioxidant system and enhance the activity of further enzymes in the pathway. It may also be necessary to improve the regulation of transgene expression, ensuring that none of the enzymes are overwhelmed by the increased flow of metabolites through the system.
The effect of H2O2 on the photosynthetic biochemistry of Pisum sativum.
(1990) Ford, Lesley.; Pammenter, Norman William.; Amory, Alan M.
To study the short-term effect of hydrogen peroxide on plant metabolism aminotriazole (2 mM), a catalase inhibitor was applied through the transpiration stream of pea seedlings and the gas exchange characteristics, ascorbate peroxidase, glutathione reductase and catalase activities, and the levels of hydrogen peroxide, glutathione and formate were determined. CO2 assimilation rates were inhibited after the addition of aminotriazole: photorespiratory conditions (high 02 and low CO2 concentrations, high light intensities) exascerbated the inhibition. CO2 response curves showed that aminotriazole reduced both the RuBP regeneration rate and the efficiency of the carboxylation reaction of Rubisco. The inhibition of the CO2 assimilation rate during the first 100 minutes after the addition of aminotriazole was alleviated by feeding PGA through the transpiration stream. Catalase activity was completely inhibited within 200 minutes of aminotriazole application, but there was no concomitant increase in the hydrogen peroxide concentration, suggesting that hydrogen peroxide is metabolised by an alternative mechanism. The treatment had no effect on ascorbate peroxidase and glutathione reductase activities or total glutathione pool size, but greatly increased formate levels. These results suggest that hydrogen peroxide is metabolised by reacting with glyoxylate to form formate and CO2, The increased production of formate may reduce the flow of carbon through the normal photorespiratory pathway and may be used anapleurotically as a precursor of products of l-C metabolism other than serine. This would prevent the return of photorespiratory carbon to the BensonCalvin cycle in the form of 3-PGA. It is proposed that when RuBP regeneration levels are low high formate levels may be an effector of Rubisco, and competitively inhibit the binding of CO2 and 02. These results suggest that under photorespiratory conditions the uninterrupted flow of carbon through the photorespiratory pathway is fundamental to the functioning of photosynthetic metabolism.
Investigation of the processes involved during the photoinhibition of Zea mays L. seedlings.
(1990) Ripley, Bradford Sherman.; Amory, Alan M.; Pammenter, Norman William.
It has been proposed that the protective systems (photorespiration, the anti-oxidant system and non-radiative energy dissipation) alleviate or reduce photoinhibitory damage under high light conditions. To investigate the role of these mechanisms in C4 photosynthetic species, nine day old Zea mays seedlings were photoinhibited (30 minutes of 2500 J,Lmol m-2 s-1 PPFD) in the presence of various concentrations of 02 or CO2; or by photoinhibiting leaves in N2 after they had been fed glycolate or phosphoglycerate via the transpiration stream. The extent of the photoinhibition and the subsequent recovery from the photoinhibitory treatments was monitored with both CO2 gas exchange and chlorophyll fluorometry. Photoinhibitory treatments resulted in both a decrease in the rate of CO2 fixation and an interruption of PSII electron transport. CO2 response curves were used to monitor the efficiency of the carboxylation processes and the level of carbon metabolism substrate cycling during recovery following photoinhibitory treatments. Both were decreased by the treatment and recovered once leaves were returned to normal conditions. Low concentrations of 02 (2%) markedly reduced the extent of the photoinhibition. This protection could not be accounted for by photorespiration, which would be inoperative at such a low 02 concentration. Leaves fed glycolate exhibited enhanced photoinhibtion. It is also unlikely that the anti-oxidant system (Mehler reaction and associated glutathione and ascorbate reactions) could utilize sufficient reductant at such low 02 concentrations to produce the observed protection. Leaves inhibited in the presence of 02 had decreased maximum fluorescence yields (Fm) and little altered initial fluorescence yields (F0)' resulting in decreased PSlI efficiency (Fv/Fm)' Photoinhibition resulted in a small increase in the slow relaxing component (60 minute) of non-radiative energy dissipation. This component became more predominant as the 02 concentration was increased. The rate constant for photochemistry was also decreased by the inhibitory treatment. Leaves supplied with CO2 at a concentration above 50 J,Lmol mol-1 exhibited little photoinihibition suggesting that the protection was not due to a quantitative utilization of energy. PGA, fed via the transpiration strea~ enhanced the photoinhibition, suggesting that more than just the Benson-Calvin cycle is required to protect C4 plants from photoinhibition. At CO2 concentrations below this, the Fv/FID ratio was decreased due to large increases in the F0 values. Fm was little altered. These changes are characteristic of a decrease in the rate constant for photochemistry. The rate constant for non-radiative energy dissipation was little altered by the photoinhibition. The protection observed in the presence of either CO2 or 02 was not due to a quantitative utilization of energy and the different responses of F0' Fm and the rate constants KD and Kp, suggest that different mechanisms were operative in the presence or absence of oxygen.
Studies of transgenic tobacco plants containing Escherichia Coli glutathione reductase.
(1996) Dlamini, Zodwa Lawrentia.; Amory, Alan M.
Glutathione reductase (GR) and superoxide dismutase (SOD) enzymes are thought to play an important role in the plant chloroplast antioxidant system. Tobacco plants transformed with E. coli glutathione reductase and superoxide dismutase genes were used to investigate the role of these gene products (enzymes) in the chloroplast antioxidant system. These plants were T1318 (transformants with increased levels of cytoplasmic glutathione reductase activity) and GOR1OT (transformants with increased levels of cytoplasmic' glutathione reductase activity and chloroplastic superoxide dismutase). In addition, 10~M methyl violegen (paraquat), was used to perturb the system experimentally under high light, low light and in darkness. During these experiments GRA (glutathione reductase activity) was assayed and the results expressed as mg-1protein, mg-1 chlorophyll and g-1 tissue, using different types of transgenic plants. T131 B-cytosolic GOR transformants had a higher GRA under high light intensity. Under low light intensity T131B had a small increase in GRA compared to controls (T131 Bs in 1mM CaS04). Also leaf discs in the dark showed similar GRA as did controls. The three treatments had no effect on the GRA of untransformed plants. GOR1OT (cytoplamic GOR and chloroplastic SOD transformants) had a slight increase in GRA under high light intensity and in darkness. At low light intensity GOR10T showed similar results to controls. The results indicate the overall absolute increase in GRA in transgenic plants after methyl violegen treatment. The higher activity than that of nontransgenic controls indicate that bacterial GRA must have also increased following exposure to methyl violegen.
The use of a virtual world to address misconceptions held by students regarding photosynthesis and respiration.
(1998) Adams, Jillian Claire.; Amory, Alan M.; Criticos, C.
In an effort to contribute to the improvement of Matric Biology education, a survey was conducted in 1996 and 1997 of Matric pupils and first year students at several tertiary institutions, in order to identify those topics which learners found most difficult. Photosynthesis and respiration were among the topics with which Matric Biology learners experienced many conceptual difficulties. The aim of this project was twofold: firstly to identify specific misconceptions students had regarding these topics, and secondly, to develop and evaluate a learning tool that would address these misconceptions. In order to identify the most common specific misconceptions, a quantitative research approach was taken. A three-tiered multiple choice questionnaire was developed, and administered to first year students in the 1998 intakes at MLSultan Technikon and the Biology Department of the University of Natal, Durban. It was also administered to students at the end of their first, second and third years of Cell Biology (University of Natal), Analysis of the questionnaires revealed that students did not understand the complementary relationship between photosynthesis and respiration. Computer-based virtual worlds provide constructivist learning environments, in which visualisation and problem solving in a complex system is possible. It was proposed that use of a virtual world would be an effective means of addressing the misconceptions we identified. A game was developed that presented students with authentic tasks of filling an oxygen cylinder (as an air supply) and a carbon dioxide cylinder (which would later be used to extinguish a fire). In order to do this students were required to solve a series of three puzzles, all of which related to the processes of photosynthesis and respiration. To account for different learning styles, the puzzles were based on three of Gardner's multiple forms of intelligence. Evaluation of the virtual world made use of a combination of quantitative and qualitative research methods. Students' understanding of the processes was measured with the use of the questionnaire. A deeper evaluation of their understanding and affective response to the game was obtained through interviews. It was found that students who had played the game had a clearer understanding of the complementary relationship between photosynthesis and respiration, and understood that respiration in plant cells is a continuous process. Students also showed greater confidence in their grasp of the processes, and reported that playing the game had been an enjoyable way of complementing their traditional lecture material in order to master these concepts. The virtual world was an effective learning tool for addressing the misconceptions students held regarding photosynthesis and respiration.

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Browsing Biological Sciences by Author "Amory, Alan M."