Glycerophosphodiesters are products of phospholipase B-mediated hydrolysis of phospholipids. Their transport and metabolism is elaborated in the fungal pathogen, Candida albicans, as compared to the non-pathogenic Saccharomyces cerevisiae. C. albicans contains four ORFs (CaGIT1-4) predicted to encode transporters for glycerophosphodiesters, compared to one (ScGIT1) in S. cerevisiae. Here I have identified the gene products responsible for glycerophosphoinositol (GroPIns) and glycerophosphocholine (GroPCho) transport. C. albicans strain lacking ORF 19.34, which codes for CaGit1, is unable to transport intact GroPIns. Transport activity can be rescued by reintegration of one copy of CaGIT1 back into the genome. Similarly, a strain lacking CaGIT3 (ORF 19.1979) and CaGIT4 (ORF 19.1980) is unable to transport intact GroPCho into the cell. Reintegrating one copy of either CaGIT3 or CaGIT4 can rescue GroPCho transport activity. Initial transport assays and kinetic analyses indicate that CaGit3 is responsible for the majority of GroPCho transport activity. In addition, I present evidence that CaGDE1 (ORF 19.3936) codes for an enzyme with glycerophosphodiesterase activity against GroPCho. Homozygous deletion of CaGDE1 results in a buildup of internal GroPCho, which is restored to wild type accumulation by reintegration of one copy of CaGDE1 into the genome. The transcriptional regulator, CaPho4, was shown to positively regulate the expression of CaGIT1, CaGIT3, CaGIT4, and CaGDE1. Finally, glycerophosphodiester transport and metabolism was active under physiological relevant conditions that C. albicans may experience in the human host. / Bayer School of Natural and Environmental Sciences; / Biological Sciences / PhD; / Dissertation;

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.

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells, while the steroid 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] inhibits the rapid stimulation by 1,25(OH)2D3. Previous work in this laboratory has indicated that 24,25(OH)2D3 inhibits phosphate uptake in isolated intestinal cells and perfused duodenal loops. It is critical to show this effect in the whole animal to determine the presence of any confounding factors. Studies were therefore undertaken to determine if 24,25(OH)2D3 had a similar effect in vivo. 24,25(OH)2D3 has two isomers which are 24R, 25dihydroxyvitamin D3 [24R,25(OH)2D3] and 24S, 25dihydroxyvitamin D3 [24S,25(OH)2D3]. We studied these two isomers separately and tested them over a time course of 1, 5, 10, 15, and 18 h after steroid using chicks on regular diet, but fasted, and chicks on a lower vitamin D diet. All chicks were anesthetized prior to surgical exposure of the duodenal loop and injection of a solution containing H3 32PO4 into the lumen. An initial time course study of phosphate transport determined that 3 to 9 min of absorption in vivo was in a linear range, as judged by serum levels of radioactivity. Chicks were then injected with either 200 μg of 24R,25(OH)2D3, 20 μg of 24S,25(OH)2D3, or vehicle for control groups within the same time course studies. We found that the isomers had different effects on phosphate absorption. 24R,25(OH)2D3 had a hypophosphatemic effect in vivo. The serum levels of radionuclide revealed hypophosphatemic effects at 1, 5, 15, and 18 h time points with a decrease of 20%, 42%, 39%, and 43%, respectively, (P< 0.05) compared with controls; chicks raised on a low vitamin D diet also showed a decrease in phosphate absorption at 10 h time point by 33%. In contrast, 24S,25(OH)2D3 stimulated intestinal phosphate absorption at the 5-h time point by 64%, but had no other effects at the other time points tested. Because 24S,25(OH)2D3 was largely ineffective, dose-response studies were undertaken with only the 24R,25(OH)2D3 isomer. In comparing phosphate absorption in chicks fasted 18 h, and dosed with vehicle, 100 μg, 200 μg, or 300 μg of steroid 1 h prior to experimentation it was found that the lowest dose increased absorption to 99% of controls, while the 200 μg and 300 μg doses decreased phosphate absorption. (48 pages)

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Inorganic Phosphate (Pi) is one of the key nutrients required by all living organisms on earth. This nutrient is of vital importance to higher plants but it is not readily available for uptake from the soil, implying constant stress on plants. During photosynthetic dark and light reactions, phosphate is a prerequisite for all reactions to occur and to ensure plant survival. This statement implies that a careful homeostatic control of this nutrient is necessary in order to maintain a balanced carbon flow in all sub-cellular plant compartments. Phosphate limitation is a threat to plant survival and one way of addressing this nutritional hurdle is by feeding plants with fertilizer. This method of crop development and general plant maintenance by humans has a devastating effect on the environment, as phosphate causes eutrophication and various other consequences which are detrimental to animal life. Plants, however, are naturally equipped with Pi transporters which are activated conditionally depending on the external Pi availability. These transporters are present in most sub-cellular compartments and some of them have been identified and characterised, while others remain to be a prediction. If these transporters are characterised accordingly it might eventually mean that the use of fertilizers may no longer be necessary. In order to contribute to successful Pi-efficient crop development, a clearer understanding of P-dynamics in the soil and its recycling ability inside the plant itself is necessary. During this study it was attempted to characterise a putative high affinity Pi transporter, PHT1;5, from Arabidopsis thaliana via a Escherichia coli and yeast heterologous expression system and its Km value predicted in order to verify/hypothesise whether it is a high or low affinity transporter. This transporter is expressed in leaves and could be a promising tool for future carbon partitioning studies during phosphate limitation. / AFRIKAANSE OPSOMMING: Anorganiese fosfaat (Pi) word beskou as een van die belangrikste nutriente benodig vir alle lewe op aarde. Dit vervul ‘n hoof rol in talle noodsaaklike prosesse in hoër plante en is veral ‘n voorvereiste vir fotosintetiese reaksies om plaas te vind. In ‘n plant se natuurlike omgewing is anorganiese fosfaat nie geredelik bekskikbaar in grond nie en dus word daar vermoed dat plante onder konstante fosfaat stres gevind word. Omdat fosfaat so ‘n belangrike rol speel tydens fotosintese is dit noodsaaklik dat daar ‘n balans op sellulêre vlak gehandhaaf word, veral wat die verspreiding van koolhidrate tussen die verskillende kompartemente van die sel betref. Plante se oorlewing word bedreig deur ‘n tekort aan fosfaat in die omgewing en die enigste onmiddelike oplossing daarvoor is deur die toediening van bemestingstowwe. Hierdie metode van landery ontwikkeling en algemene instandhouding van plante deur die mensdom het ’n baie negatiewe effek op die omgewing. ‘n Oormaat fosfaat lei tot eutrifikasie en het verkeie ander negatiewe nagevolge wat dodelik is vir die dierelewe. Plante beskik ook oor natuurlike interne fosfaat transporters om hierdie tekort te oorkom. Hierdie transporters word op grond van eksterne fosfaat beskikbaarheid ge-aktiveer of ge-deaktifeer. Die transporters is teenwoordig in meeste sub-sellulêre kompartemente en sommige is al ge-identifiseer en gekarakteriseer, terwyl ander slegs ‘n voorspelling bly. Gedurende hierdie studie was ‘n poging aangewend om ‘n anorganiese fosfaat transporter van Arabidopsis thaliana, PHT1;5, te karakteriseer met behulp van mikro-organismes soos Escherichia coli en gis. Die poging het ingesluit om ‘n Km waarde vir hierdie transporter te voorspel en sodoende ‘n hipotese daar te stel van of dit hoë of lae affiniteit het vir fosfaat. Die transporter word groot en deels aangetref in blare en kan dus dien as ‘n belowende apparaat vir toekomstige koolhidraat uitruiling studies gedurende fosfaat tekort.