Phosphoenolpyruvate (PEP) metabolism in roots and nodules of Lupinus angustifolius under P stress

Le Roux, Marcellous Remarque (2004-03)

Thesis (MSc)--Stellenbosch University, 2004.

Thesis

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.

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