The graduation theses theme was to determinate changes of initial activity, total activity and Rubisco enzyme content in spinach leaves. Ascertained activities and Rubisco enzyme content were determined from extracts of spinach leaves, which were collected during two distinctively climatic different days. Both activities were determined by spectrometric method and content of enzyme Rubisco by SDS-PAGE method. The findings were compared with findings determined in earlier done works.

No description available.

In this diploma thesis, the diurnal changes of initial and total Rubisco activity and Rubisco enzyme content in beech (Fagus sylvatica) were studied under conditions of ambient (A) CO2 concentration (350 µmol.mol-1) and elevated (E) CO2 concentration (700 µmol.mol-1) during the day. Samples were taken on July 8th (from 10:00 to 21:30), on July 9th (from 04:00 to 12:00) and then on July 22th (from 04:00 to 21:30). The initial and total activity were measured spectrophotometrically and the activation level was calculated from the ratio of initial and total activities. Rubisco enzyme content was determined by SDS-PAGE method and the initial and total specific activity were calculated from the ratio of initial or total activity and Rubisco enzyme content. In our experiment no statistically significant difference was found between Rubisco activities in beeches cultivated under conditions of ambient CO2 concentration and elevated CO2 concentration, so any down-regulation of Rubisco activity did not appear under the influence of ambient CO2 concentration. Diurnal changes of Rubisco activities showed only statistically nonsignificant fluctuation. After daylight no significant increase of total Rubisco activity was observed, which demonstrates the absence or the immaterial effect of CA1P night inhibitor in beech. Intensive fluctuation of the activation level (40–90 %) proves the regulation of photosynthesis during the day via Rubisco enzyme carbamylation. Rubisco enzyme content in beech cultivated under conditions of ambient CO2 concentration was lower than that one in beech cultivated under conditions of elevated CO2 concentration. Then down-regulation of Rubisco enzyme content is presented under conditions of ambient CO2 concentration. Before daylight Rubisco enzyme content showed an indistinctive minimum on the level of statistical significance, during the day only nonrelevant fluctuation was noticed.

Little is known of the physiological and biochemical mechanisms underlying competitive interactions between alien invasive grasses and native taxa, and how these are affected by resource supply. Consequently, this study compared photosystem II (PS II) function, photosynthetic gas and water exchange, enzyme and pigment concentrations, flowering and biomass accumulation in an indigenous geophyte, Tritonia crocata (L.) Ker. Gawl., grown in monoculture and admixed with the alien grass, Lolium multiflorum Lam., at different levels of water and nutrient supply. Diminished stomatal conductances were the primary cause of reduced net C02 assimilation rates, and consequent biomass accumulation in T. crocata admixed with L. multiflorum at all levels of water and nutrient supply with one exception. These corresponded with decreased soil water contents induced presumably by more efficient competition for water by L. multiflorum, whose biomass was inversely correlated with soil water content. Biochemical impairments to photosynthesis were also apparent in T. crocata admixed with L. multiflorum at low levels of water and nutrient supply. These included a decline in the density of working photosystems (reaction center per chlorophyll RC/ABS), which corresponded with a decreased leaf chlorophyll a content and a decreased efficiency of conversion of excitation energy to electron transport (¥0 / l-^o), pointing to a reduction in electron transport capacity beyond QA~, a decline in apparent carboxylation efficiency and Rubisco content. At low nutrient levels but high water supply, non-stomatal induced biochemical impairments to photosynthesis (decreased RC/ABS, chlorophyll a and Rubisco content) were apparent in T. crocata admixed with L. multiflorum. These attributed to a reallocation of fixed carbohydrate reserves to floral production which increased significantly in T. crocata under these conditions only and associated with a corresponding reduction in the mass of its underground storage organ (bulb). The results of this study did not support the hypothesis that under conditions of low water and low nutrient supply invasive annual grasses would have a lesser impact on the growth and physiology of native geophytes than under resource enriched conditions that favor growth of these grasses. Unresolved is whether resource limitation and allelopathic mechanisms functioned simultaneously in the inhibition of the native geophyte by the alien grass. / Thesis (M. Environmental Science (Ecological Remediation and Sustainable Utilisation))--North-West University, Potchefstroom Campus, 2008.

No description available.

Little is known of the physiological and biochemical mechanisms underlying competitive interactions between alien invasive grasses and native taxa, and how these are affected by resource supply. Consequently, this study compared photosystem II (PS II) function, photosynthetic gas and water exchange, enzyme and pigment concentrations, flowering and biomass accumulation in an indigenous geophyte, Tritonia crocata (L.) Ker. Gawl., grown in monoculture and admixed with the alien grass, Lolium multiflorum Lam., at different levels of water and nutrient supply. Diminished stomatal conductances were the primary cause of reduced net C02 assimilation rates, and consequent biomass accumulation in T. crocata admixed with L. multiflorum at all levels of water and nutrient supply with one exception. These corresponded with decreased soil water contents induced presumably by more efficient competition for water by L. multiflorum, whose biomass was inversely correlated with soil water content. Biochemical impairments to photosynthesis were also apparent in T. crocata admixed with L. multiflorum at low levels of water and nutrient supply. These included a decline in the density of working photosystems (reaction center per chlorophyll RC/ABS), which corresponded with a decreased leaf chlorophyll a content and a decreased efficiency of conversion of excitation energy to electron transport (¥0 / l-^o), pointing to a reduction in electron transport capacity beyond QA~, a decline in apparent carboxylation efficiency and Rubisco content. At low nutrient levels but high water supply, non-stomatal induced biochemical impairments to photosynthesis (decreased RC/ABS, chlorophyll a and Rubisco content) were apparent in T. crocata admixed with L. multiflorum. These attributed to a reallocation of fixed carbohydrate reserves to floral production which increased significantly in T. crocata under these conditions only and associated with a corresponding reduction in the mass of its underground storage organ (bulb). The results of this study did not support the hypothesis that under conditions of low water and low nutrient supply invasive annual grasses would have a lesser impact on the growth and physiology of native geophytes than under resource enriched conditions that favor growth of these grasses. Unresolved is whether resource limitation and allelopathic mechanisms functioned simultaneously in the inhibition of the native geophyte by the alien grass. / Thesis (M. Environmental Science (Ecological Remediation and Sustainable Utilisation))--North-West University, Potchefstroom Campus, 2008.

This project was expected to contribute to the understanding of abscisic acid (ABA) perception in plants through identification of new ABA-binding proteins. The novel, biotinylated ABA derivative PBI686 (of biological activity comparable to natural ABA) has served as an affinity probe for isolation of ABA-binding proteins. Photoaffinity labeling in conjunction with affinity chromatography (streptavidin-biotin based) was used for specific identification of target proteins from complex mixtures of cytosolic and membrane-bound proteins. Proteins of interest were identified by Mass Spectrometry through peptide mass fingerprinting and MS/MS ion search.<p> Ribulose bisphosphate carboxylase/oxygenase (Rubisco) was identified as an ABA binding partner, and its interaction with ABA was initially confirmed by its ability to block the photoaffinity labeling reaction with PBI686. In addition, Surface Plasmon Resonance (SPR) experiments with ABA and Rubisco were performed, which provided further evidence for selective interaction between the two binding partners, with a very small preference towards (+)-ABA over (-)-ABA. SPR has also yielded the value of equilibrium dissociation constant (KD) being 5 nM for (+)-ABA and 7 nM for (-)-ABA. This was further confirmed by [3H] (±)-ABA binding assays, which have also shown that non-radiolabeled (+)-ABA and (-)-ABA (at concentration 1000 fold higher) were able to displace [3H] (±)-ABA from binding to Rubisco. Compounds other than ABA such as PA (phaseic acid) or trans-(+)-ABA were not able to displace [3H] (±)-ABA, which has suggested the selectivity of binding. Further, Rubisco enzymatic activity in the absence of ABA was compared to that in the presence of ABA at various concentrations. The results have clearly indicated the effect of ABA on Rubiscos enzymatic activity. This was reflected on the enzymes Km values being increased by seven fold in the presence of 10 mM ABA and 1 mM substrate (RuBP). The interpretation of changes in enzyme kinetics upon inhibition by ABA most resembles allosteric inhibition. The biological function of this newly discovered interaction is interpreted as ABAs ability to regulate plant growth during abiotic stress by its direct action on the photosynthetic machinery - hypothesis often suggested in the literature.

La enzima Rubisco es uno de los sitemas biológicos más importantes. Por un lado controla la fotosíntesis y por otro cataliza la reacción que inicia el proceso de la fotorrespiración. Este comportamiento dual y aparentemente contradictorio de la enzima la ha hecho objeto de numerosos estudios. En la Tesis que se presenta hemos aplicado las técnicas propias de la Química Teórica para estudiar su comportamiento. En primer lugar, nuestros resultados indican que la razón para el comportamiento bifuncional de la enzima radica en la geometría tensionada que tiene el sustrato en el interior del centro activo, que hace que tanto el estado electrónico triplete como el singlete se encuentren accesibles y participen en los procesos químicos. En segundo lugar, hemos proporcionado una explicación al fenómeno de autoinactivación de la enzima observado experimentalmente. En tercer lugar, hemos obtenido las estructuras de transición correspondientes a las reacciones de enolización, carboxilación y oxigenación catalizadas por la enzima, caracterizando su acción catalítica y obtieniendo claves para interpretar su comportamiento: el sustrato se encuentra preparado para experimentar adición de CO2 o de O2, y el hecho de que ocurra una u otra reacción sólo depende de la molécula de gas que alcance el centro activo. De esto se desprende que el proceso de oxigenación es inevitable, una vez enolizado el sustrato, y está indisolublemente ligado al de carboxilación.

This project was expected to contribute to the understanding of abscisic acid (ABA) perception in plants through identification of new ABA-binding proteins. The novel, biotinylated ABA derivative PBI686 (of biological activity comparable to natural ABA) has served as an affinity probe for isolation of ABA-binding proteins. Photoaffinity labeling in conjunction with affinity chromatography (streptavidin-biotin based) was used for specific identification of target proteins from complex mixtures of cytosolic and membrane-bound proteins. Proteins of interest were identified by Mass Spectrometry through peptide mass fingerprinting and MS/MS ion search.<p> Ribulose bisphosphate carboxylase/oxygenase (Rubisco) was identified as an ABA binding partner, and its interaction with ABA was initially confirmed by its ability to block the photoaffinity labeling reaction with PBI686. In addition, Surface Plasmon Resonance (SPR) experiments with ABA and Rubisco were performed, which provided further evidence for selective interaction between the two binding partners, with a very small preference towards (+)-ABA over (-)-ABA. SPR has also yielded the value of equilibrium dissociation constant (KD) being 5 nM for (+)-ABA and 7 nM for (-)-ABA. This was further confirmed by [3H] (±)-ABA binding assays, which have also shown that non-radiolabeled (+)-ABA and (-)-ABA (at concentration 1000 fold higher) were able to displace [3H] (±)-ABA from binding to Rubisco. Compounds other than ABA such as PA (phaseic acid) or trans-(+)-ABA were not able to displace [3H] (±)-ABA, which has suggested the selectivity of binding. Further, Rubisco enzymatic activity in the absence of ABA was compared to that in the presence of ABA at various concentrations. The results have clearly indicated the effect of ABA on Rubiscos enzymatic activity. This was reflected on the enzymes Km values being increased by seven fold in the presence of 10 mM ABA and 1 mM substrate (RuBP). The interpretation of changes in enzyme kinetics upon inhibition by ABA most resembles allosteric inhibition. The biological function of this newly discovered interaction is interpreted as ABAs ability to regulate plant growth during abiotic stress by its direct action on the photosynthetic machinery - hypothesis often suggested in the literature.

Little is known of the physiological and biochemical mechanisms underlying competitive interactions between alien invasive grasses and native taxa, and how these are affected by resource supply. Consequently, this study compared photosystem II (PS II) function, photosynthetic gas and water exchange, enzyme and pigment concentrations, flowering and biomass accumulation in an indigenous geophyte, Tritonia crocata (L.) Ker. Gawl., grown in monoculture and admixed with the alien grass, Lolium multiflorum Lam., at different levels of water and nutrient supply. Diminished stomatal conductances were the primary cause of reduced net C02 assimilation rates, and consequent biomass accumulation in T. crocata admixed with L. multiflorum at all levels of water and nutrient supply with one exception. These corresponded with decreased soil water contents induced presumably by more efficient competition for water by L. multiflorum, whose biomass was inversely correlated with soil water content. Biochemical impairments to photosynthesis were also apparent in T. crocata admixed with L. multiflorum at low levels of water and nutrient supply. These included a decline in the density of working photosystems (reaction center per chlorophyll RC/ABS), which corresponded with a decreased leaf chlorophyll a content and a decreased efficiency of conversion of excitation energy to electron transport (¥0 / l-^o), pointing to a reduction in electron transport capacity beyond QA~, a decline in apparent carboxylation efficiency and Rubisco content. At low nutrient levels but high water supply, non-stomatal induced biochemical impairments to photosynthesis (decreased RC/ABS, chlorophyll a and Rubisco content) were apparent in T. crocata admixed with L. multiflorum. These attributed to a reallocation of fixed carbohydrate reserves to floral production which increased significantly in T. crocata under these conditions only and associated with a corresponding reduction in the mass of its underground storage organ (bulb). The results of this study did not support the hypothesis that under conditions of low water and low nutrient supply invasive annual grasses would have a lesser impact on the growth and physiology of native geophytes than under resource enriched conditions that favor growth of these grasses. Unresolved is whether resource limitation and allelopathic mechanisms functioned simultaneously in the inhibition of the native geophyte by the alien grass. / Thesis (M. Environmental Science (Ecological Remediation and Sustainable Utilisation))--North-West University, Potchefstroom Campus, 2008.