Thesis (PhD (Plant biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: In soils, the concentration of available phosphate (P) for plants is normally very low
(ca. 1 µM in the soil solution), because most of the P combines with iron, aluminium
and calcium to form relatively insoluble compounds. Inorganic P (Pi)-deficiency is
thought to be one of the limiting factors of nitrogen fixation due to the high energy
requirement for nitrogenase function of plants taking part in nitrogen fixation. Pideficiency
has important implications for the metabolic Pi and adenylate pools of
plants, which influence respiration and nitrogen fixation. An alternative route of
pyruvate supply during Pi stress has been proposed involving the combined activities
of phosphoenolpyruvate carboxylase (PEPc), malate dehydrogenase (MDH) and
NAD-malic enzyme (ME) supplying pyruvate to the mitochondrion during Pi stress.
Previously, three isoforms of PEPc were isolated from lupin nodules and roots, with
two forms being nodule specific. The aim of this project was to determine the effect of
Pi stress on these PEPc isoforms in Lupinus luteus at transcript and protein
expression level with a view to produce genetically modified crops for nutrient-poor
soils.
Cytosolic P levels were measured over a time course to give an indication of
temporal development of P stress in nodules. The changes in enzyme activities of
PEPc, MDH and PK (pyruvate kinase) under P stress were measured and the
downstream effect on amino and organic acid pools were analysed. Two novel PEPc
isoforms, LUP1 (AM235211) and LUP2 (AM237200) were isolated from nodules,
followed by transcriptional and protein expression analyses.
Nodules under P stress had lower amounts of metabolically available Pi and as P
stressed developed, the amount of Pi decreased. This decline in Pi levels was
associated with lower growth, but higher biological nitrogen fixation (BNF). A greater
proportion of root-nodule respiration was devoted to nutrient acquisition than to new
growth. A typical P-stress response is higher anaplerotic carbon fixation via PEPc.
However, in this study, no significant differences were found for PEPc, MDH or PK in
P-stressed plants compared to P-sufficient plants which would lead to an increase in
organic acids. An increase in key amino acids was reported along with unchanged
levels of organic acids. These levels of organic and amino acid are in congruence
with the increases in BNF under P-starvation. No significant differences were found in expression of PEPC1 or PEPC2 at 12 and
20 days for both P-sufficient and P-stressed plants which further supported the lack
of engagement of the PEPc-MDH-ME bypass. PEPc activity appeared not to be
regulated by gene expression or phosphorylation indicating that other posttranslational
modifications such as a decrease in protein degradation may be of
importance. / AFRIKAANSE OPSOMMING: Die konsentrasie van fosfaat (P) beskikbaar vir opname deur plante vanuit die grond
is gewoonlik baie laag (in die omgewing van 1 µM) aangesien die P onoplosbare
komplekse vorm met katione soos yster, aluminium en kalsium. ‘n Tekort aan
anorganiese P (Pi) word gereken as een van die beperkende faktore van
stikstofbinding as gevolg van die hoë energie behoefte wat nitrogenase plaas op
plante wat van gefikseerde stikstof gebruik maak. Hierdie P-tekort het ook belangrike
betrekking op die metaboliese fosfaat- en adenilaatpoele wat weer op hul beurt
respirasie en stikstofbinding beÏnvloed. ‘n Alternatiewe roete van pirovaatvoorsiening
aan mitochondria tydens fosfaatstres is voorgestel wat bestaan uit die aktiwiteite van
fosfoenolpirovaat karboksilase (PEPc), malaat dehidrogenase en NAD-malaat
ensiem. Vantevore is drie isovorme van PEPc uit Lupinus luteus wortelknoppies en
wortels geïsoleer, met twee van die isovorme wat wortelknoppie-spesifiek was. The
doel van hierdie projek was om die invloed van P-tekort op die transkripsie en
proteien uitdrukkingsvlak van hierdie PEPc isovorme te bepaal met die doel van
gemodifiseerde gewasse vir arm gronde ingedagte.
Sitoplasmiese P konsentrasies is gemeet oor tyd om ‘n aanduiding te gee van die
ontwikkeling van P-tekort oor tyd. Veranderinge in ensiemaktiwiteite van PEPc, MDH
en pirovaatkinase (PK) is gemeet gedurende P-tekort as ook die moontlike effek van
hierdie ensiemaktiwiteite op aminosuur en organiese suur poele. Twee nuwe PEPc
isovorme, LUP1 (AM235211) en LUP2 (AM237200) is uit wortelknoppies geïsoleer
en gekarakteriseer. Transkripsie en proteïenuitdrukking is geanaliseer.
Wortelknoppies wat P-tekort behandeling ontvang het, het laer vlakke van metabolise
beskikbare Pi gehad en soos die P-tekort ontwikkel het oor tyd, het die Pi vlakke
gedaal. Hierdie afname in vlakke van Pi was geassosieer met laer groei, maar met ‘n
toename in biologiese stikstofbinding. ‘n Groter proporsie van respirasie is
toegestaan aan minerale opname as aan nuwe groei. ‘n Tipiese reaksie op P-tekort
is hoër anaplerotiese koolstofbinding via PEPc. Alhoewel, in hierdie studie is geen
gevind betekenisvolle verandering gevind in die aktiwiteite van PEPc, MDH en PK
nie in plante wat P-tekort ervaar het nie. Verhoogde aktiwiteit van hierdie ensieme
sou verhoogde organise suur konsentrasies tot gevolg hê. ‘n Toename in aminosuur
konsentrasies is gevind tesame met onveranderde vlakke van organiese sure.
Hierdie toename in aminosure word onderskryf deur die verhoogde biologiese
stikstofbinding tydens P-tekort. Geen betekenisvolle verskille is gevind in die geenuitdrukking van pepc1 en pepc2
by beide 12 en 20 dae van P-tekort nie, wat verder die afwesigheid van die PEPc-
MDH-ME alternatiewe roete beaam het. Dit blyk dat PEPc aktiwiteit nie deur
geenuitdrukking of proteïenfosforilering beheer word nie, maar eerder dat ander posttranslasie
modifikasies soos ‘n verlaagde afbraak van proteïen ‘n rol speel.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/5184 |
| Date | 12 1900 |
| Creators | Kleinert, Aleysia |
| Contributors | Valentine, Alexander Joseph, Kossmann, Jens, University of Stellenbosch. Faculty of Science. Dept. of Botany and Zoology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 111 p. : ill. |
| Rights | University of Stellenbosch |
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