Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: This study aimed to assess the contribution of anaplerotic C prOVISIOn VIa phosphoenolpyruvate carboxylase (pEPc, EC 4.1.1.31), during Pi stress in the root and nodule components of Lupinus angustifolius. The role of PEPc in DIC metabolism in roots and nodules of phosphate-starved plants was studied. The symbioses involving leguminous plants and species of Rhizobium and Bradyrhizobium bacteria form an integral part of effective management ofN in the environment. In agricultural settings, roughly 80% of this biologically fixed N2 comes from this type of symbiotic relationship. Nitrogen-fixing bacteria in concert with legumes fix atmospheric nitrogen, which is then available to the infected plant. Worldwide, legumes are grown on approximately 250 Mha and they fix about 90 Tg (90 billion tons) of N, per year. The overall stoichiometry for nitrogen assimilation in the nodule requires one molecule of oxaloacetate to be converted to one molecule of asparagine per dinitrogen molecule fixed. One possible source for the required oxaloacetate is the reaction catalysed by PEPc. The reaction catalysed by PEPc is a major source of anaplerotic carbon for the plant and it is expected that this reaction will be even more important to plants under Pi stress, as the reaction is not ATP-dependent. Seeds of Lupinus angustifolius (cv. Wong a) were inoculated with Rhizobium sp. (Lupinus) bacteria and grown in hydroponic culture. Tanks were supplied with either 2 11MP04 (LP) or 2 mM P04 (HP) and air containing 360 ppm CO2. Roots experienced pronounced P stress with a greater decline in Pi, compared to nodules. 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. LP roots also synthesised more pyruvate from malate than LP nodules. The role of pyruvate accumulation under Pi stress, was further highlighted by the metabolism of PEP via both the pyruvate kinase (PK, Ee 2.7.1.40) and PEPc routes. The enhanced PK activities supported these high pyruvate levels. The results show unequivocally that nodules do not experience P stress to the same extent as roots. Implications of the findings are that nodules require low P to function normally. Maintenance of phosphate levels in nodules may be at the expense of host. It can be suggested that when nodules are P-starved they can become aggressive scavengers for available P and even out-compete roots. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om die bydrae van anaplerotiese koolstof-voorsiening via fosfo-enolpirovaatkarboksilase (pEPc, EC 4.1.1.31), tydens fosfaatstremming in die wortels en wortelknoppies van Lupinus angustifolius te bepaal. Die rol van PEPc in die metabolisme van opgeloste anorganiese koolstofdioksied in fosfaat-beperkte wortels en wortelknoppies is ondersoek. Die simbiose tussen peulplante en spesies van Rhizobium en Bradyrhizobium bakterieë vorm 'n integrale deel van die doeltreffende bestuur van stikstof in die omgewing. In die landbou word ongever 80 %van biologies-gefikseerde stikstof deur hierde simbiotiese verhouding geproduseer. Stikstotbindende bakterieë, in simbiose met peulplante, fikseer atmosferiese stikstof, wat dan beskikbaar is vir die geïnfekteerde plant. Wêreldwyd fikseer peulplante ongeveer 90 biljoen ton stikstof per jaar. Die algehele stoïgiometrie vir stikstof-fiksering in wortelknoppies vereis dat een molekule oksaalsuur na een molekule asparagien omgesit word per stikstofmolekule wat gefikseer word. Een moontlike bron vir die benodigde oksaalsuur is die reaksie wat deur PEPc gekataliseer word. Die reaksie wat deur PEPc gekataliseer word is 'n belangrike bron van anaplerotiese koolstof vir die plant en dit word vermoed dat hierdie reaksie van nog groter belang sal wees vir plante onder fosfaatstremming, omdat die reaksie nie ATP-afhanklik is nie. Sade van Lupinus angustifolius (cv. Wonga) is geïnokuleer met Rhizobium sp. (Lupinus) bakterieë en gekweek in waterkultuur. Tenke is voorsien met óf 2 !lM P04 (LP), óf 2 mM P04 (HP) en lug wat 360 ppm CO2 bevat het. Wortels het skerp fosfaatstremming ervaar, met 'n groter afname in Pi, vergelykbaar met wortelknoppies. Tydens fosfaatstremming het die aktiwiteit van PEPc toegeneem in wortels, maar nie in wortelknoppies nie en hierdie veranderinge was nie verwant aan die uitdrukking van die ensiem nie. PEPc van wortels en wortelknoppies is nie gereguleer deur uitdrukking nie, maar moontlik deur post-translasie kontrole. Wortels onder 'n lae-fosfaat voorsiening het ook meer pirodruiwesuur vanaf malaat gesintetiseer as wortelknoppies. Die rol van pirodruiwesuur-akkumulering tydens fosfaatstremming is verder beklemtoon deur die metabolisme van PEP via beide die pirovaatkinase- (PK, EC 2.7.1.40) en PEPc- roetes. Die verhoogde PK-aktiwiteite verklaar hierdie hoër vlakke van pirodruiwesuur. Die resultate toon ondubbelsinnig dat wortelknoppies me tot dieselfde mate fosfaatstremming ervaar as wortels nie. Dit impliseer dat wortelknoppies min fosfaat benodig om normal te funksioneer. Handhawing van fosfaatvlakke in wortelknoppies mag ten koste van die wortel wees. Dit is moontlik dat, wanneer wortelknoppies fosfaatbeperk is, hulle aggressiewe opruimers word vir beskikbare fosfaat en selfs beter funksioneer as die wortels.

In recent years, the rhizobial taxonomy changed significantly with the discovery of novel symbiotic associations between legumes and nodulating bacteria. This was aided by the focus shift from studying only agricultural crops to legumes indigenous to certain regions, ultimately to discover new inoculant strains and to uncover the secrets of the rhizobium¬legume symbiosis. In previous studies on the diversity of South African rhizobia, it has become clear that our country has a wealth of rhizobia. Cyclopia is a legume genus, which belongs to the fynbos biome of South Africa. Honeybush tea is a herbal infusion manufactured from the leaves and stems of certain Cyclopia spp. Commercial cultivation of this potentially new agricultural crop is now developed to protect the natural Cyclopia spp. populations from harvesting and ultimately extinction. Superior inoculant strains are necessary for these commercial seedlings. The diversity of root-nodulating strains isolated from 14 Cyclopia spp. was determined using 16S-23S IGS-RFLP and partial 16S rDNA base sequencing. Based on 16S-23S IGS-RFLP and partial 16S rDNA base sequencing most of the isolates, with the exception of seven strains, were found to belong to the genus Burkholderia. More extensive phylogenetic, symbiotic and phenotypic studies of selected strains were performed using near full-length 168 rDNA base sequencing, nodA base sequencing and substrate utilisation analysis. In the genus Burkholderia, the isolates belonged to the novel root-nodulating species Burkholderia tuberum and several novel, undescribed Burkholderia genotypes. However, no new Burkholderia species could formally be proposed, since DNA-DNA hybridisation analysis, which is a prerequisite for the description of new species could not be performed in our laboratory. The seven strains not affiliated with the Burkholderia genus belonged to two Bradyrhizobium genospecies, R tropici and a possibly new genus in the a-Proteobacteria. The nodA sequences of all the Cyclopia isolates corresponded to a large extent, indicating that different chromosomal genotypes harbour the same symbiotic genotype. All the isolates of the Cyclopia genus appear to be acid-tolerant, which is in agreement with the acidic nature of the soil from which the strains were isolated. / Thesis (PhD(Microbiology))--University of Pretoria, 2004. / Microbiology and Plant Pathology / unrestricted