Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / Dissertation presented for the degree of Doctor of Philosophy Stellenbosch University. / ENGLISH ABSTRACT: Phosphorus (P) is essential for the survival of all living organisms and forms part of several key biological molecules and processes. The basic biological function of all cells depends on the availability of P as structural element in phospholipids and nucleic acids. P plays a central role in the energy metabolism of the cell by activating metabolic intermediates of carbohydrate metabolism and by acting as an energy currency in the form of adenosine tri-phosphate (ATP). ATP is produced during photosynthesis from the energy derived from sunlight, probably the most important biological process on earth. The balance of P supply and demand is of critical importance here. Plants assimilate P in the form of orthophosphate (Pi) via its roots and utilises complex mechanisms to redistribute and balance the Pi concentrations throughout the plant. These processes are collectively known as phosphate homeostasis and in this study we utilised molecular techniques to study some key aspects of this complex network of mechanisms in the plant Arabidopsis thaliana. When the role of the PHT1;5 Pi transporter was investigated in photosynthesis under Pi limitation a new mechanism utilised by plants to supply Pi for the production of ATP in the chloroplast was discovered. During periods of adequate Pi supply plants make use of the triose phosphate / phosphate translocator (TPT) to exchange Pi for phosphorylated carbon intermediates. This transporter does, however, not function at the low Pi concentrations present during Pi limitation and the plant therefore express an alternative transporter i.e. PHT1;5. Together with this transporter several genes were identified that was expressed to allow the export of carbon intermediates from the chloroplast via starch turnover. Amongst these, several alternative isoforms of the enzymes responsible for starch turnover are expressed during Pi limiting conditions. It is therefore suggested that the products of starch degradation, e.g. glucose and maltose are the potential candidates for carbon export from chloroplasts under Pi limiting conditions. In an attempt to perturb the Pi concentrations in the Arabidopsis vacuole we expressed the three genes of a newly discovered polyphosphate (PolyP) polymerase from the yeast Sacharomyces cerevisiae in Arabidopsis. This enzyme complex accumulates PolyP in the yeast vacuole and since the plant vacuole is playing a key role in buffering Pi concentrations we anticipated some observable effects that could lead to the elucidation of the mechanisms involved. Production of PolyP was conclusively shown in plant callus, but it was only at very low concentrations with no detectable perturbing effect and undetectable in whole plants. With the aim to apply this technology to the PolyP and PHT1;5 lines developed in the other parts of this study, newly developed fluorescent indicator protein nanosensors (FLIPPi) were evaluated as a method for detecting and monitoring in vivo Pi concentrations in multicellular plant organs. This technique is capable of detecting changes in metabolite concentrations in real-time and it was applied to the roots of Arabidopsis seedlings subjected to Pi limitation. We specifically looked at changes in the cytosol, but our results revealed no detectable changes occurring in the Pi concentrations in this compartment. This was interpreted to indicate lower levels of Pi in this compartment as was previously expected. / AFRIKAANSE OPSOMMING: Fosfaat (P) is essensieël vir die oorlewing van alle organismes en maak deel uit van etlike kern biologiese prosesse en molekules. Die basiese biologiese funksionering van alle selle hang direk af van die beskikbaarheid van P as strukturele element van fosfolipiede en nuklëinsure. Fosfaat speel 'n sentrale rol in die energie metabolisme van 'n sel deur metaboliese intermediante te aktiveer en deur op te tree as die geld eenheid van sellulere energie in die vorm van adenosien tri-fosfaat (ATP). ATP word gegenereer gedurende fotosintese vanaf die energie wat van sonlig vasgevang word, dit is waarskeinlik die belangrikste biologiese proses op aarde. Dit is van kritiese belang dat die fosfaat vraag en aanbod hier fyn gebalanseer word. Plante assimileer P in die vorm van ortofosfaat (Pi) deur hulle wortels en maak gebruik van komplekse meganismes om Pi deur die plant te versprei en konsentrasies te balanseer. Hierdie prosesse staan gesamentlik bekend as fosfaat homeostase en in die huidige studie het ons gebruik gemaak van molekulêre tegnieke om 'n paar belangrike aspekte van hierdie komplekse netwerk van prosesse in die plant Arabidopsis thaliana te bestudeer. Toe die rol van die PHT1;5 Pi transporter in fotosintese onder toestande van Pi tekort bestudeer is, is 'n nuwe meganisme ontdek waarmee plante Pi verskaf aan chloroplaste vir die proses van fotosintese onder toestande van Pi tekort. Gedurende periodes wat die plant genoegsame Pi tot sy beskikking het, word van die triose fosfaat / fosfaat uitruiler (TPT) gebruik gemaak om Pi uit te ruil vir gefosforileerde koolstof metaboliete. Hierdie transporter kan egter nie onder die lae Pi konsentrasies wat voorkom in die sitoplasma onder Pi tekort toestande funksioneer nie, en gevolglik moet die plant van 'n alternatiewe transporter naamlik PHT1;5 uitdruk. Verskeie ander gene is ook geidentifiseer wat saam met hierdie transporter onder toestande van Pi tekort uitgedruk word en die plant toelaat om koolstof tussengangers uit die chloroplaste uit te vervoer via die proses van stysel produksie en afbraak. Onderandere is verskeie alternatiewe isoforme van die gene wat verandwoordelik is vir stysel produksie en afbraak identifiseer wat uitgedruk word onder toestande van Pi tekort. In 'n poging om die Pi konsentrasies in die Arabidopsis vakuool te versteur is drie gene van die nuut ontdekte polifosfaat (PolyP) polimerase kompleks van die gis Sacharomyces cerevisiae in Arabidopsis uitgedruk. Hierdie ensiem kompleks is verandwoordelik vir die akkumulasie van PolyP in die gis vakuool en siende die plant vakuool 'n kern rol speel in die buffering van Pi konsentrasies in die plant, het ons sekere waarneembare gevolge verwag wat kon lei tot die ontrafeling van die meganismes hierby betrokke. Die produksie van PolyP in plant kallus is duidelik gedemonstreer, maar dit was slegs teen baie lae konsentrasies met geen waarneembare versteuringseffek nie, en kon glad nie in heel plante waargeneem word nie. Met die oog daarop om hierdie tegnologie toe te pas op die bestudering van die PolyP en PHT1;5 lyne wat in die ander dele van hierdie studie ontwikkel is, is 'n nuut ontwikkelde fluoresente indikator protein nanosensor (FLIPPi) tegnologie evalueer as 'n metode om Pi konsentrasies in vivo in multisellulere plant organe waar te neem en te monitor. Hierdie tegniek is in staat daartoe om veranderinge in Pi konsentrasies in selle direk te monitor en is gevolglik op die wortels van Arabidopsis saailinge onder Pi tekort toestande toegepas. Daar is spesifiek na veranderinge in die sitosol gekyk, maar ons resultate kon geen waarneembare veranderinge in Pi konsentrasies in hierdie kompartement uitwys nie. Hierdie resultaat beteken waarkeinlik dat die Pi konsentrasies in hierdie kompartement waarskeinlik baie laer is as wat voorheen verwag is.

De part ses propriétés physico-chimiques, le fer est un cofacteur de choix pour de nombreuses enzymes, impliquées dans de multiples processus biologiques, comme la photosynthèse ou la respiration. Cependant, sa capacité à gagner/perdre des électrons le rend très réactif, et potentiellement toxique. Son homéostasie doit donc être finement régulée. Chez A. thaliana, le gène de ferritine AtFER1 est régulé transcriptionnellement par le fer, et son expression est régulée par le rythme circadien. Au début de ce travail, aucun facteur de transcription impliqué dans cette régulation n'était identifié. Des cribles simple hybride en levure ont été réalisés, permettant l'identification de deux facteurs de transcription régulant le gène AtFER1: AtPHR1 et AtPIF7.PHR1 (Phosphate starvation Response1) et son homologue PHL1 (PHr1 Like 1) sont des facteurs de transcription impliqués dans la réponse à la carence en phosphate. Ils régulent directement le gène AtFER1, en se fixant sur l'élément 2 du promoteur d'AtFER1. Cette régulation ne fait pas intervenir l'IDRS et est indépendante du statut en fer des plantes. Par ailleurs, l'homéostasie du fer est affectée dans le double mutant phr1 phl1. Ces résultats montrent l'existence d'un lien moléculaire direct entre les homéostasies du fer et du phosphate.PIF7 (Phytochrome Interacting Factor 7) est un facteur de transcription de type bHLH impliqué dans la régulation circadienne des gènes DREB. Il se fixe probablement sur la G-box présente dans l'élément 5 du promoteur d'AtFer1. Dans un mutant perte de fonction pour le gène PIF7, l'amplitude des oscillations de l'expression du gène AtFer1 en cycles jour/nuit est augmentée. Un résultat similaire est obtenu lorsque l'élément 5 est muté dans des lignées trasngéniques exprimant le gène rapporteur LUC sous le contrôle du promoteur d'AtFer1. Ces résultats montrent que PIF7 est un répresseur de l'expression d'AtFer1. / Due to its redox properties, iron is a major cofactor for numerous proteins involved in many biological processes such as photosynthesis or respiration. Nevertheless, its ability to easily gain or lose electrons makes it highly reactive with oxygen and potentially toxic. Iron homeostasis has to be tightly regulated. In A. thaliana, AtFER1 ferritin gene is regulated at the transcriptional level by iron, and its expression is regulated by the circadian clock. Before this work, no transcription factor involved AtFer1 regulation has been identified. A yeast one hybrid was performed and allowed us to identify PHR1 and PIF7 as transcription factors involved in AtFER1 regulation. PHR1 (Phosphate starvation Response1) and its homolog PHL1 (PHr1 Like 1) are transcription factors involved in phosphate starvation response. They directly regulate AtFER1 expression and bind to the element 2 present in AtFER1 promoter. This regulation does not involve the IDRS sequence and is independent of iron status of plants. Moreover, iron homeostasis is affected in phr1phl1 double mutant. These results highlight a direct molecular link between iron and phosphate homeostasis.PIF7 (Phytochrome Interacting Factor 7) is a bHLH transcription factor involved in the circadian regulation of DREB genes. PIF7 probably interacts with the G-box found in element 5 in AtFER1 promoter region. In a pif7 knock-out mutant, amplitude of AtFer1 oscillations during light dark cycles are increased. Such a result was also obtained with transgenic lines expressing LUC reporter gene under the control of AtFER1 promoter region harboring a mutation in element 5. These results show that PIF7 is a repressor of AtFer1 expression.

The capacity of a calcareous desert soil, Thiokol silt loam, to retain natural , as well as added , orthophosphate-P was measured by equilibrium adsorption employing a batch technique and evaluated using the two-slope Langmuir adsorption isotherm. From these data, corrected to account for indigenous soil P, a hypothesis was formulated as to the nature of retention of P by the soil, including the identification of two interfacial reactions involving P, and a value calculated for the adsorption maximum as defined by the Langmuir isotherm equation f o r P with soil at each of two soil depths and t h ree constant temperatures within the range of biological activity . The initial reaction was considered to be surface adsorption , where phosphate ions interact with the clayand lime mineral surfaces at definite sites. The activity of the second mechanism was identified as adsorption, and in addition, the heterogeneous nucleation of metal phosphates on the lime mineral surfaces. In addition to these quantitative studies, the flux of P in the soil was also investigated for the same soil and temperatures by means of kinetic experiments conducted to identify the nature (mechanisms) and measure the rates and release maxima of indigenous P release from the soil. These experiments were carried out using an anion-exchange resin as an infinite sink for P, again applying a batch technique. Ultimately, the release of indigenous P from the soil under saturated conditions was attributed to three simultaneous first-order reactions. 'll1e rate constants of the three reactions were found to be of orders of 10-4, 10-5, and 10-6 (1/sec), and did not vary significantly with soil depth or ternperature. The three reactions above were identified as dissolution of poorly crystalline or amorphous calcium phosphates, the desorption of surface site adsorbed or labile P, and the slow dissolution of calcium hydroxyapatite, respectively.

Phosphorylated molecules play vastly important roles in the environment and biological systems. The main focus of this work sought to expand the current collection of synthetic phosphate receptors to include the binding of oligosaccharide phosphates. To this end, the synthesis of a boronic acid - appended porphyrin whose selectivity could be tuned through nucleic acid selection was pursued through a number of synthetic routes. Though difficulties were encountered with synthetic reactivities and product solubility, these efforts culminated in the development of a bis-boronic acid-substituted porphyrin soluble in DMSO/water mixtures that displays fluorescence quenching upon the addition of specific saccharide derivatives. In efforts to ease the synthetic burden, the creation of a DNA-based self-assembled receptor system was also investigated. Further, this work included the synthesis of phosphorylated derivatives of gemcitabine, a nucleoside chemotherapeutic drug, with the goal of developing targeted delivery systems for the improved treatment of cancer. Progress in each of these areas is discussed. / text

10 pp. / Phosphorus is the primary fertilizer nutrient needed by alfalfa in Arizona. The objective of this study is to determine the effect of P fertilizer rate on alfalfa yield and soil test P. A phosphorus fertilizer rate study was conducted with alfalfa in Buckeye, AZ where 11-52-0 was applied at 0, 200, 400, and 800 lb fertilizer/acre in February, 2014 after the first cutting. Alfalfa hay yield was increased by phosphorus fertilizer application up to the cutting on July 30, but not thereafter. No differences in yield were found among the fertilizer rates of 200,400, and 800 lb 11-52-0/acre. Soil test phosphorus increased directly proportional to fertilizer rate, but eventually decreased close to deficient levels 3-5 months after fertilizer application. It is not known if additional fertilizer applications throughout the season would increase yield. Fertilizer rates higher than 200 lb 11-52-0/acre were not beneficial under the conditions of this study.

Orientador: Ademércio Antonio Paccola / Banca: Wolmar Apparecida Carvalho / Banca: Sandra Maria Barbalho / Banca: Silas Macedo Silva / Banca: Rosana Cavalcante dos Santos / Resumo: O processo de solubilização, dissolução de minerais do solo e suas interações com os processos de adsorção e dessorção de fosfato são de alta importância devido às possibilidades de otimização no processo de adubação do solo para o plantio. Alguns autores sugerem que a dissolução de ferro nos solos ocorre por redução microbiana e complexação, interferindo diretamente no processo de adsorção e dessorção do fosfato. O objetivo deste trabalho foi investigar a relação existente entre liberação de ferro, a neoformação de óxidos de ferro e adsorção e dessorção de fosfato em um Latossolo Vermelho distrófico de região Tropical Úmida. A relação foi estudada em solos sob diferentes tipos de ocupação: cana de açúcar, café, milho, reflorestamento de eucalipto e floresta natural. Amostras de solo foram coletadas nos horizontes A e B, tratadas com solução de sacarose a 3% e mantidas em repouso. O processo redox ocorreu em todas as amostras tratadas o que foi comprovado pela análise de pH onde se obteve uma acidificação do meio e posterior aumento do pH, indicando o estabelecimento de um equilíbrio químico do processo, mantendo assim o pH estável. Análises quantitativas de ferro, zinco, cobre, manganês e fósforo foram realizadas no solo natural, no solo sob tratamento e na fase líquida das amostras. A estes solos foram adicionadas duas concentrações de fosfato: 200 mg.dm3 e 400 mg.dm3 e calculadas as adsorções. A comparação dos resultados destas análises mostrou que a fixação de fósforo foi maior nos solos submetidos ao tratamento. Isto pode ser explicado pela decomposição microbiana dos compostos orgânicos, na interface com a atmosfera oxidante, que provocou a precipitação dos óxidos e hidróxidos dos metais onde os íons fosfato foram fixados pelos óxidos e hidróxidos reprecipitados. / Abstract: The solubilization process, minerals dissolution and its interactions with the phosphate adsorption/desorption processes are of high importance due to the possibilities of improvement in the process of the soil manuring in the plant crops.Some authors suggest that the iron dissolution in the soils occurs by microbial reduction and complexation, interfering directly in the phosphate adsorption /desorption process.The purpose of this work was to investigate the existent connection between the iron liberation, the neoformation of iron oxides and the phosphate adsorption/ desorption in a "Latossolo Vermelho Distrófico"of humid tropical region.This connection was studied in soils by different crops: sugar cane, coffee, corn, reforestation with eucalyptus and natural forest. Soil's samples were collected in A and B horizon, treated with sucrose 3% solution and maintained in repose.Redox process occurred in all the treated samples, what was proved by the pH analysis, in where were obtained some acidification of the environment and subsequent pH increase, indicating the establishment of a chemical balance of the process, maintaining like this the pH stable. Quantitative analyses of iron, zinc, copper, manganese and match were accomplished in the natural soil, in the soil under treatment and in the liquid phase of the samples.Two phosphate concentrations: 200 mg.dm3 and 400 mg.dm3 were added to these soils and the adsorptions were calculated. The comparison of these analyses results shows that the phosphorous fixation was higher in the soils submitted to the treatment. This can be explained by the microbial decomposition of the organic compounds, in the interface with the oxidant atmosphere, which nettled the precipitation of the oxides and hydroxides of the metals in where the phosphate ions were fixated by oxides and hydroxides reprecipitated. / Doutor

Detailed analysis of the site-specific phosphorylation of a glucan phosphate from Saccharomyces cerevesiae has been carried out by 2D NMR techniques. Phosphorylation has been shown to be limited to the C-6 and C-2 positions, with the C-6 resonance showing two slightly different environments. Phosphorylation at C-4 is hindered due to proximity effects with the neighboring glucose ring oxygen. Noncovalently bound, nondialyzable phosphate appears to be coordinated to the nonphosphorylated HO-2 group of the helical polymer.

No description available.

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published_or_final_version / abstract / Dentistry / Doctoral / Doctor of Philosophy