| Summary: | Thesis (PhD)--Stellenbosch University, 2015. === ENGLISH ABSTRACT: Plants exhibit a flexible array of morphological, physiological and biochemical adaptations during
phosphorous limitation. Legumes are vulnerable to P deficiency, because it affects their ability to
fix atmospheric nitrogen (N2). In particular, legumes from nutrient-poor ecosystems, such as the
Fynbos in the Cape Floristic Region (CFR) evolved on P deficient soils and may therefore display
unique adaptations to soil P stress. In general, very few studies on legumes have focussed on the
belowground structures of nodules as a plant organ. Moreover, even less is known about the P
stressed responses in nodules from legumes in nutrient-poor ecosystems. The aim of this research
was to investigate the metabolic flexibility of organic acid and amino acid metabolism in the
nodulated root system of the Fynbos legume Virgilia divaricata, during low P stress.
Virgilia divaricata, which grows in the Cape Floristic Region, was used in this study to enhance
our knowledge regarding the vital role that the cytosolic enzyme, phosphoenol pyruvate
carboxylase (PEPC) plays in phosphoenol pyruvate (PEP) metabolism, in roots and nodules of this
legume during phosphate stress. V. divaricata was grown under glasshouse conditions (20 - 25°C)
in sterilized quartz sand for 2-3 months whilst being inoculated with the nitrogen fixation bacteria,
Burkholderia phytofirmans, which was isolated from V. divaricata nodules grown in fynbos soil.
Two phosphate treatments, 5 μM and 500 μM, were applied simulating low-phosphate and high
phosphate conditions respectively using a modified Long Ashton Nutrient Solution to simulate a
low nutrient ecosystem such as the Cape Floristic Region. Roots and nodules were then analysed
for growth kinetics, nutrient acquisition and distribution, enzyme activity and genetic responses. It
was shown that during phosphate deficiency, V. divaricata nodules experienced less Pi stress than
roots, due to increased metabolic phosphate conservation reactions during organic acid synthesis
via an increased PEPC activity. The increased PEPC activity resulted in an increase in downstream
metabolic products such as organic acids, (malic acid and citric acid), and amino acids (glutamate,
aspartate and asparagine). Although the biological nitrogen fixation (BNF) declined, the high
efficiency of BNF may be underpinned by these altered phosphate conservation pathways and
enhanced resource allocation during growth particularly under low phosphate (LP) conditions. Therefore, it can be concluded that the efficiency of the nodules via an increased allocation of
resources and P acquiring mechanisms in V. divaricata may be the key to the plant’s ability to
adapt to poor P environments and thus sustaining its reliance on BNF. From the data obtained as
well as previous findings, it has been established that the phosphate conservation mechanisms in roots and nodules, involve the non-adenylate requiring PEPC-bypass route. 13C Nuclear magnetic
resonance (NMR) gave us a better understanding regarding the incorporation rates of the PEPCderived
C into malate, α-ketoglutarate and asparagine. It therefore is suggested that V. divaricata
nodules may use their large PEPC-derived malate pool to prevent large declines in BNF under low
phosphate conditions. The nodules of V. divaricata were able to offset an excessive drop in BNF,
despite a decline in inorganic phophosphate (Pi) levels. It therefore appears that nodules have
evolved to acquire different mechanisms than roots to adapt to phosphate deficiency in order to
maintain their function. This was achieved via increased regulation of nodule PEPC and its
downstream products. This implies that compared to roots under low P, nodules alter the
metabolism of PEPC derived C, in order to maintain nodule respiration and amino acid synthesis.
This trait could be observed in the synthesis of larger 13C malate pools of nodules compared to
roots, from PEPC, which was underpinned by their different regulation mechanisms of enzyme
activity, of the same protein isoform. Since malate is a potent inhibitor of PEPC activity, roots
appear to have invested in more PEPC protein compared to nodules. In contrast, nodules with
lower PEPC protein, achieved greater enzyme activity than roots, possibly due to higher
phosphorylation in order to reduce the malate effect. The subsequent metabolism of this PEPCderived
malate, caused roots and nodules to synthesise asparagine via different pathways. These
findings imply that roots and nodules under P stress, synthesise their major export amino acid,
asparagine, via different routes. This research has generated new knowledge regarding the
physiological impact of the organic and amino acid metabolism, derived from PEPC-C in the roots
and nodules of legumes growing in nutrient poor ecosystems. It has demonstrated for the first time
that the nodules of legume from a nutrient-poor ecosystem rely on improved resource allocation,
Pi distribution, and PEPC-derived organic acids to maintain the efficient functioning of N
assimilation under P stress. This may be a consequence of having evolved in a nutrient-poor
ecosystem, so that nodule-bacteroid respiration and N metabolism can be maintained in P-poor
soils such as the Fynbos. === AFRIKAANSE OPSOMMING: Tydens fosfaat stremming maak plante gebruik van buigsame kombinasies van morfologiese,
fisiologiese en biochemiese aanpassings. Peulplante is sensitief vir fosfaat tekorte omdat dit die
vermoë om atmosferiese stikstof te kan fikseer, grootliks beïnvloed. Peulplante vanuit ekosisteme
met mineraal-arme gronde, soos Fynbos binne die Kaapse Blommeryk, het ontwikkel in grond met
lae fosfaatvlakke en mag dus unieke aanpassings tot fosfaat tekorte toon. Oor die algemeen is daar
baie min peulplant studies wat fokus op die ondergrondse strukture van wortelknoppies as ‘n plant
orgaan. Nog minder inligting is beskikbaar oor wortelknoppies, van peulplante, vanuit mineraalarme
ekosisteme, se reaksie teenoor ‘n fosfaat tekort. Die doel van hierdie navorsing was om die
metaboliese buigsaamheid van organiese- en aminosuur metabolisme in die (nodulated)
wortelknoppie-wortelstelsel van die Fynbos peulplant Virgilia divaricata, tydens fosfaat tekort te
ondersoek.
Virgilia divaricata wat voorkom in die Kaapse Blommeryk, was gebruik in hierdie studie om die
huidige kennis te verbeter van die essensiële rol wat die sitisoliese ensiem, fosfo-enol piruvaat
karboksilase (PEPC) in fosfo-enol piruvaat metabolisme tydens ‘n fosfaat tekort speel binne die
wortels en wortelknoppies van hierdie peulplant. V. divaricata was gegroei onder glashuis
toestande (20 - 25°C) in gesteriliseerde kwartssand vir 2-3 maande. Die plante was geïnokuleer
met die stikstoffikserende bakterie, Burkholderia phytofirmans, wat geïsoleer is vanaf V.
divaricata wortelknoppies wat in Fynbos grond gegroei is. Twee fosfaatbehandelings, 5μM and
500μM, was toegedien om lae en hoë fosfaat toestande, onderskeidelik, na te boots deur gebruik te
maak van ‘n aangepasde Long Ashton voedingstofmengsel om ‘n ekosisteem, soos die Kaapse
Blommeryk, met lae voedingstofvlakke na te boots. Die wortels en knoppies was geanaliseer ten
opsigte van die groeikinetika, opname en verspreiding van voedingstowwe, ensiemaktiwiteit en
genetiese aanpassings.
Dis is bewys dat tydens fosfaat tekort V. divaricata wortelknoppies minder fosfaat stres ervaar as
die wortels, as gevolg van die verhoogde metaboliese fosfaat bewaringsreaksies tydens organise
suur sintese via die styging in PEPC aktiwiteit. Die styging in PEPC aktiwiteit lei tot ‘n verhoging
in stroomaf metaboliese produkte soos organiese- (appel- en sitroënsuur) en aminosure (glutamaat,
aspartaat en asparagien). Alhoewel biologiese stikstoffiksering verlaag het, kan die hoë
doeltreffendheid daarvan ondersteun word deur díe aangepasde fosfaat bewarings weë asook
verhoogde hulpbron toekenning tydens groei onder lae fosfaat omstandighede. Dit kan dus afgelei word dat die doeltreffendheid van die wortelknoppies via die verhoging in belegging van
hulpbronne en fosfaat opname meganismes in V. divaricata moontlik die sleutel is tot die plant se
vermoë om aan te pas tot omgewings met lae fosfaatvlakke en sodoende die afhanklikheid van
biologiese stikstofbinding te kan onderhou.
Data in hierdie as ook vorige studies, wys dat die fosfaat bewaringsmeganismes in wortels en
wortelknoppies die PEPC-ompad roete, wat nie adenilaat benodig nie, gebruik. 13C NMR het meer
lig gewerp aangaande die vaslegging van koolstof vanaf PEPC na malaat, α-ketoglutaraat en
asparagien. Dit word voorgestel dat V. divaricata knoppies ‘n groot hoeveelheid malaat, afkomstig
van PEPC-werking, gebruik om groot dalings in biologiese stikstofbinding tydens fosfaat tekort, te
verhoed. Die wortelknoppies van V. divaricata kon ‘n oormatige verlaging in biologiese
stikstofbinding voorkom ten spyte van die verlaging in fosfaatvlakke. Dit wil voorkom dat
wortelknoppies ander meganismes as die wortels ontwikkel het om aan te pas tot fosfaat tekort en
sodoende dus hul funksie behou. Dit word bereik deur ‘n verhoging in die regulering van PEPC en
die stroomaf produkte in die wortelknoppies. Dit blyk dat wortelknoppies tydens fosfaat te kort, in
vergelyking met wortels, die metabolisme van die koolstof vanaf PEPC verander om sodoende
respirasie en aminosuursintese te onderhou. Dit wil voorkom dat hierdie meganismes verskil van
die van wortel meganismes.
Hierdie eienskap kan toegeskryf word aan die produksie van ‘n groter hoeveelheid van 13C malaat
vanaf PEPC in die wortelknoppies teenoor die wortels, wat ondersteun word die verskillende
reguleringsmeganismes van ensiemaktiwiteit van dieselfde proteïen isoform. Malaat is ‘n kragtige
inhibeerder van PEPC-aktiwiteit, dus blyk dit dat die wortels belê in meer PEPC proteïene as die
wortelknoppies. In teenstelling, toon die wortelknoppies met laer PEPC proteïene, ‘n hoër ensiem
aktiwiteit as die wortels. Dit kan wees as gevolg van hoër fosforilasie om die effek van malaat te
verlaag. Die metabolisme van die malaat vanaf PEPC het die sintese van asparagien in die wortels
en wortelknoppies via verskillende roetes tot gevolg gehad. Dit impliseer dat tydens ‘n tekort aan
fosfaat, wortels en wortelknoppies hul hoof uitvoer aminosuur, asparagien, deur verskillende
roetes sintetiseer.
Hierdie studie het nuwe kennis aangaande die fisiologiese impak van organiese- en aminosuur metabolisme met koolstof vanaf PEPC in die wortels en wortelknoppies van peulplante wat
voorkom in ekosisteme met lae voedingstofvlakke, voortgebring. Vir die eerste keer is dit bewys
dat die wortelknoppies vanaf peulplante wat voorkom in mineraal-arme ekosisteme, staatmaak op
verbeterde hulpbron beleggings, fosfaat verspreiding en organiese sure vanaf PEPC om die doeltreffendheid van funksionele stikstofassimilasie tydens fosfaat tekort, te onderhou. Dit mag
die gevolg wees van, om in ‘n voedingstof arme ekosisteem te ontwikkel sodat die wortelknoppiebakteroïed
respirasie en stikstofmetabolisme onderhou kan word in fosfaat arme grond soos die Fynbos.
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