Molecular studies on phosphate homeostasis in higher plants

Zwiegelaar, Jacobus Petrus (2010-03)

Thesis (PhD (Genetics))--University of Stellenbosch, 2010.

Dissertation presented for the degree of Doctor of Philosophy Stellenbosch University.

Thesis

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.

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