Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: This study investigated the activities of several of the enzymes involved in the alternative
route of PEP metabolism via PEPc (EC 4.1.1.31). This reaction circumvents the
adenyl ate-controlled PK (EC 2.7.1.40) reaction of the conventional glycolytic network
under conditions of P stress. It was hypothesized that the synthesis of pyruvate under Pi
stress would induce the PEPc alternative route and that C for pyruvate synthesis would
primarily be imported via this route. This was assessed by looking at how total enzyme
activities are perturbed under P stress and also by following the route of radioactive
labelled 14C02 under sufficient (2 mM) and deficient P (2 JlM) conditions in either roots
or nodules. The significance of the pathway under P stress, was further assessed by
determining pool sizes of pyruvate that was synthesized from PEPc-derived C. The
experiments were conducted under glasshouse conditions, as two separate studies: one to
investigate the phenomenon of Pi stress and its consequences for PEPc-derived C
metabolism, and the other one to study the enzymes involved. Seeds of Lupinus
angustifolius (cv. Wonga) were inoculated with Rhizobium sp. (Lupinus) bacteria and
grown in hydroponic culture. Tanks were supplied with either 2 JlM P04 (LP) or 2 mM
P04 (control) and air containing 360 ppm CO2.
Roots experienced pronounced P stress with a greater decline in Pi, compared to nodules.
LP roots synthesized more pyruvate from malate than LP nodules, indicating the
engagement of the PEPc route under Pi stress. In this regard, pyruvate is considered as a
key metabolite under Pi stress. The role of pyruvate accumulation under Pi stress, was further highlighted by the metabolism of PEP via both the PK and PEPc routes. The
enhanced PK activities supported these high pyruvate levels. Under P stress, PEPc
activities increased in roots but not in nodules and these changes were not related to the
expression of the enzyme. Root and nodular PEPc were not regulated by expression, but
possibly by posttranslational control.
The novelty of our results for symbiotic roots demonstrates that using metabolically
available Pi is indeed a more sensitive indicator ofP stress. These results show that under
Pi stress, nodules are able to maintain their Pi and adenylate levels, possibly at the
expense of the root. It is suggested that nodules do not experience P stress to the same
extent as roots or alternatively function optimally under conditions of low P availability.
The increase in concentration of pyruvate synthesized from malate, indeed suggest that
under LP conditions there is an increase requirement for pyruvate. It is clear from this
data that the operation of bypass route in nodules should be investigated further.
Nevertheless, this study provided incentives for understanding the role of C pathways in
Ni-fixation, in particular under conditions ofP limitation. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om die aktiwiteite van verskeie ensieme van die
alternatiewe metaboliese roete via phosphoenolpirovaat karboksilase (pEPc, EC 4.1.1.31)
te ondersoek. Dié reaksie omseil die adenilaat-beheerde pirovaatkinase (PK, EC 2.7.1.40)
reaksie van die konvensionele glikolitiese weg onder toestande van fosfaat (P)
stremming. Dit is gepostuleer dat die sintese van pirovaat onder toestande van Pstremming
die alternatiewe roete via PEPc sou induseer en dat die koolstof (C) vir
pirovaatsintese gevolglik hoofsaaklik vanaf hierdie roete sou kom. Dit is bepaal deur die
veranderinge in die totale ensiemaktiwiteite wat sou plaasvind onder P-stremming te
ondersoek. Daar is ook gekyk na die roete' wat radioaktiewe C C4C02) sou volg in
wortles en wortelknoppies wat behandel is deur blootsteling aan eerder lae fosfaat (2 1lM)
of genoegsame fosfaat (2 mM; kontrole), Die betekenis van die alternatiewe roete is ook
ondersoek deur die poel-groottes van pirovaat, soos gesintetiseer via die PEPc reaksie, te
bepaal.
Twee eksperimente is in 'n glashuis uitgevoer. Eerstens is die verskynsel van Pstremming,
asook die invloed daarvan op PEPc-afgeleide C-metabolisme, bepaal.
Tweedens is die betrokke ensieme bestudeer.
Sade van Lupinus angustifolius (cv. Wonga) is geïnokuleer met Rhizobium sp. (Lupinus)
bakterieë en in 'n waterkultuur gekweek. Die houers is voorsien met óf2 IlM P04 (LP) óf
2 mM P04 (HP) en lug wat 360 ppm C02 bevat het. Wortels, anders as wortelknoppies, het 'n betekenisvolle afname in anorganiese P (Pi)
ervaar. Onder P-stremming, het lae fosfaat wortels meer pirovaat vanaf malaat
gesintetiseer as wortelknoppies, wat 'n definitiewe bydrae vanaf die PEPc roete
impliseer. Hiervolgens is pirovaat 'n sleutel metaboliet onder P-stremming. Die
belangrikheid van die akkumulering van pirovaat onder P-stremmende toestande is verder
beklemtoon deur die toename in metabolisme van PEP via beide die PK- en die PEPcreaksies.
Die toename in PK-aktiwiteite is goed gekorreleer met die verhoogde produksie
van pirovaat. Onder toestande van P-stremming het die aktiwiteit van PEPc in wortels
verhoog, maar nie in wortelknoppies nie. Dit was nie die gevolg van 'n verhoogde
uitdrukking van die ensiem nie. Wortel- en wortelknoppie- uitdrukking van PEPc is
derhalwe nie gereguleer deur die uitdrukking daarvan nie, maar eerder deur post-tranlasie
kontrole.
Hierdie resultate vir wortels met wortelknoppies demonstreer dat metaboliese Pi 'n beter
maatstaf is om P-stres aan te dui. Hierdie resultate toon dat wortelknoppies beter daartoe
instaat is om hul Pi-vlakke en adenilaatvlakke te reguleer, en dit mag ten koste van die
gasheerwortel wees. Ons stel voor dat wortelknoppies nie P-stremming tot dieselfde mate
ervaar as die gasheerwortel nie en dat dié knoppies optimaal funksioneer by lae Pi
vlakke. Die verhoogde konsentrasie van pirovaat, wat vanaf malaat gesintetiseer is,
impliseer dat daar 'n groter vereiste is vir dié metaboliet onder toestande van Pstremming.
Hierdie studie het die rol van koolstofmetabolismein stikstofbindende
organismes, spesifiek onder toestande van fosfaat-tekort, beklemtoon.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/50060 |
| Date | 03 1900 |
| Creators | Le Roux, Marcellous Remarque |
| Contributors | Valentine, A. J., Botha, F. C., Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB). |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 124 pages : illustrations |
| Rights | Stellenbosch University |
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