Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Sutherlandia frutescens (L.) R. Br., also regarded as Lessertia frutescens, is a leguminous, perennial shrub indigenous to South Africa. Extracts prepared from the leaves have traditionally been used for the treatment of various diseases. Reports have also indicated that S. frutescens provides certain health benefits to cancer and HIV/AIDS patients. Analysis of extracts indicated the presence of several compounds (bitter triterpenoid glycosides, several flavonoids, amino acids, small amounts of saponins (no alkaloids though), asparagine, Larginine, canavanine, gamma-aminobutyric acid (GABA) and pinitol) which contribute to the medicinal properties of this plant. The first part of this study involved testing the effect of six treatments (light, dark, soaking of seeds, physical scarification, chemical scarification and flaming of seeds) on the in vitro germination of Sutherlandia seeds to elucidate the factors which control seed germination. Those treatments which removed the seed coat were most successful for germination with physical scarification being the most efficient method, resulting in 98.6% of the seeds germinating after 21 days. Although the organogenesis of Sutherlandia explants (cotyledons and hypocotyls) in vitro were investigated (results not included in this thesis), omitting plant growth regulators (PGR) in the cultivation medium was best for shoot multiplication. However, this PGR-free system successfully provided a continuous supply of plant material for further studies. It would be possible to successfully adopt it for commercial production of plants to assist with cultivation of Sutherlandia as a field crop. Another advantage of this system is spontaneous rooting with 85% of the in vitro microshoots rooting in PGR-free medium. These rooted plants were acclimated in the glasshouse using vented lids to harden off the shoots and this method resulted in 100% survival of plants. The second part of this study investigated the induction of hairy root cultures of S. frutescens using Agrobacterium-mediated transformation. The efficiency of three Agrobacterium strains (A4T, LBA9402 and C58C1) to transform different S. frutescens explants (cotyledons and hypocotyls) was analyzed. All three strains were equally efficient at inducing hairy roots in both hypocotyls and cotyledons. However, transformation of S. frutescens was dependent on the type of explant used with the hypocotyls being more efficiently transformed than the cotyledons. Overall the transformation of both the hypocotyl (93%) and cotyledon (47%) was highest when the strain A4T was used. Four hairy root clones were selected and their cultivation in a liquid system was optimized by investigating their growth in four different types of media (Gamborg B5 (Gamborg et al., 1968), White’s (White, 1934; White, 1954), MS (Murashige and Skoog, 1962) and half strength MS medium). All the growth of hairy root clones was best in the B5 and MS medium, with White’s medium being the least effective cultivation medium. Molecular analysis of hairy roots was used to prove the transgenic status of these four putative transgenic clones. This was achieved using polymerase chain reaction (PCR) amplification of rol A (320 bp), B (780 bp) and C (600 bp) genes to determine the presence of the TL-DNA in the plant genome. During Southern hybridization a radioactively labeled rol A probe was used to determine the copy number of the rol A gene. The three rol genes were present in all four hairy root clones. The third part of this study focused on the effect of three abiotic stress factors (nitrogen availability, salinity and drought) on the synthesis of four metabolites (gamma-aminobutyric acid (GABA), asparagine, arginine and canavanine). The effect of nitrogen availability on metabolite synthesis and the morphology was determined using in vitro shoot cultures as well as the hairy root clone C58C1-g. Nitrogen availability studies were conducted by cultivating the microshoots or root tips on modified MS medium. The MS medium contained either the normal amount of nitrogen (1.9 g L-1 KNO3 and 1.65 g L-1 NH4NO3) in the MS medium (1x nitrogen), half the normal nitrogen concentration in MS medium (0.5x nitrogen) or twice the normal nitrogen concentration in MS medium (2x nitrogen). The arginine and asparagine levels in the roots and shoots and the canavanine level in the shoots were directly correlated with the amount of nitrogen in the medium (as the nitrogen level increased, the metabolite levels increased). The GABA level in the shoots was inversely correlated with the amount of nitrogen in the medium. Several reasons may explain these metabolic changes including the assimilation of extra nitrogen into asparagine, canavanine and arginine in the shoots. The reduced GABA levels may indicate the preferential flux of the free GABA into other nitrogen assimilatory pathways such as protein synthesis as well as its rapid utilization to replenish the tricarboxilic acid cycle intermediates. The effect of water (induced by including 3% (w/v) PEG in the medium) and salt stress (induced by including either 50 or 100 mM NaCl in the medium) was only investigated in the shoot cultures as the root cultures lacked the synthesis of canavanine. Water stress did not significantly alter the metabolite levels, but resulted in a significant decrease in the growth (fresh weight and total shoot length) and the rooting response of these microshoots. Salt stress only resulted in a significant increase in arginine levels with increasing salinity and also caused a reduction in the rooting and growth response. Lowered plant vigour may be the first visual sign of water stress. Addition of NaCl may lead to ion toxicity and requires osmotic adjustment resulting in changes at the metabolic level concomitant to physiological growth changes. Finally, the anti-bacterial activity and the phytochemistry of transgenic root cultures and untransformed in vitro and ex vitro plant material was examined. Only the extracts prepared from the wild harvested leaf material exhibited moderate anti-bacterial activity (1.25 mg ml-1) against all the bacteria (Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis and Staphylococcus aureus) tested. Changes to the secondary metabolism of hairy roots were investigated using TLC and LC-MS analysis. Several of the compounds in the hairy root extracts were present in higher levels than in the control root extracts. Transformation also increased the complexity of the phytochemical pattern of the hairy roots, either due the synthesis of novel compounds or upregulated synthesis of existing metabolic pathways. The production of hairy roots and the establishment in a liquid system during this study was an important step towards upscaling these cultures to a bioreactor. In future these roots can assist in developing cultures which produce a high yield of the desired metabolites. / AFRIKAANSE OPSOMMING: Sutherlandia frutescens (L.) R. Br., ook bekend as Lessertia frutescens is ‘n peulagtige meerjarige struik, inheems tot Suid Afrika. Ekstrakte wat van die blare voorberei word, is tradisioneel gebruik vir die behandeling van verskeie siektes. Berigte het ook daarop gedui, dat S. frutescens sekere gesondheidsvoordele vir kanker en HIV/VIGS pasiënte inhou. ‘n Ontleding van die ekstrakte, dui op die teenwoordigheid van verskeie verbindings (bitter triterpenoïed glikosiede, verskeie flavonoïede, aminosure, klein hoeveelhede saponiene (alhoewel geen alkaloïede), asparagien, L-arginien, canavanien, gamma-aminobottersuur (GABS) en pinitol) wat tot die medisinale eienskappe van hierdie plant bydrae. Die eerste deel van die studie het die effek van ses behandelings (lig, donker, week van sade, fisiese skarifikasie, chemiese skarifikasie en die vlam van sade) op die in vitro ontkieming van Sutherlandia sade getoets met die doel om die faktore wat saadontkieming beheer, te identifiseer. Die beste behandeling vir saadontkieming was dié behandelings wat die saadhuid verwyder het. Die mees effektiewe metode van saadhuidverwydering was die fisiese skarifikasie van sade, wat gelei het tot ‘n 98.6% ontkieming van sade na 21 dae. Alhoewel in vitro organogenese van Sutherlandia eksplante (kotiel en hipokotiel) ondersoek was (resultate nie ingesluit in die tesis nie), was plant groei reguleerders (PGR) uitgesluit in die groeimedium om stingelvermeerdering te bevorder. Nie te min was die PGR-vrye sisteem suksesvol om ‘n voortdurende bron van plant material vir verder studies te verskaf. Dit sou egter moontlik wees om die PGR-vrye sisteem suksesvol te kon aanpas vir die kommersiële produksie van plante met die doel om Sutherlandia as ‘n landbougewas te bevorder. ‘n Verdere voordeel van dié sisteem, is die spontane wortelvorming, met 85% van die in vitro mikrostingels wat wortels in die PGR-vrye medium produseer het. Hierdie bewortelde plante was in die glashuis geakklimatiseer met behulp van geventileerde deksels (vir stingel afharding) en het tot ‘n 100% oorlewing gelei. Die tweede deel van die studie het die induksie van S. frutescens harige wortelkulture met behulp van Agrobacterium-bemiddelde transformasie ondersoek. Die effektiwiteit van drie Agrobacterium stamme (A4T, C58C1 en LBA9402) om verskillende S. frutescens eksplante (kotiel en hipokotiele) te transformeer, was geanaliseer. Al drie stamme was ewe effektief om harige wortels op beide hipokotiel en kotiele te induseer. S. frutescens transformasie blyk egter tog van die tipe eksplant afhanklik te wees, aangesien die hipokotiele meer effektief as die kotiele getransformeer kon word. Met inagneming van beide die hipokotiel (93%) en kotiel vii (47%), was transformasie optimaal met die gebruik van die A4T stam. Vier harige wortelklone was geselekteer en hulle produksie in ‘n vloeibare sisteem was geoptimiseer deur hulle groei in vier verskillende tipe media (Gamborg B5 (Gamborg et al., 1968), White’s (White, 1934; White, 1954), MS (Murashige and Skoog, 1962) en half-sterkte MS medium) te ondersoek. B5 en MS medium was beskou as die beste vir alle die harige wortelklone se groei, terwyl White’s medium die minste doeltreffende groeimedium was. Molekulêre analise van die harige wortels was gebruik ten einde die transgeniese status van die vier vermoedelike transgeniese klone te bewys. Dit was behaal deur polimerase kettingreaksie amplifisering (PKR) van die rol A, B en C gene ten einde die teenwoordigheid van die TL-DNS in die plant genoom aan te toon. Tydens Southern hibridisasie was ‘n radioaktief gemerkte peiler gebruik om die aantal rol A geen kopieë te bepaal. Die drie rol gene was teenwoordig in al vier harige wortelklone. Die derde deel van die studie het gefokus op die effek van drie abiotiese stress faktore (stikstof beskikbaarheid, sout- en droogte stres) op die produksie van vier metaboliete (GABS, asparagien, canavanien en arginien). Die effek van stikstof beskikbaarheid op die metaboliet produksie asook die morfologie was bestudeer deur gebruik te maak van in vitro mikrostingels asook die harige wortel kloon C58C1-g. Stikstof beskikbaarheidstudies was uitgevoer deur die mikrostingels of wortelpunte in ‘n gewysigde MS medium te groei. Die MS medium was aangepas om die normale hoeveelheid stikstof (1.9 g L-1 KNO3 en 1.65 g L-1 NH4NO3) in MS medium (1x stikstof), of die helfte van die normale stikstof konsentrasie (0.5x stikstof) of twee keer die normale stikstof konsentrasie in MS medium (2x stikstof) te bevat. Die arginien en asparagien vlakke in die wortels en stingels, asook die canavanien vlak in die stingels was positief gekorreleerd aan die stikstof konsentrasie in die medium. Die GABS vlak in die stingels was egter omgekeerd eweredig aan die stikstof konsentrasie in die medium. Verskeie redes kan aangevoer word om die metaboliet veranderinge te verduidelik, insluitende die assimilasie van addisionele stikstof in asparagien, canavanien en arginien in die stingels. Die verlaagde GABS vlakke kan dui op die voorkeur van vrye GABS vloei na ander stikstofassimilerende metaboliese paaie soos proteïen sintese, asook die snelle benutting van GABS ten einde die Trikarboksielsuursiklus intermediêre produkte aan te vul. Die effek van droogte (geïnduseer deur die byvoeging van 3% (m/v) PEG tot die medium) en sout stres (geïnduseer deur 50 of 100 mM NaCl byvoeging tot die medium) was slegs in die stingel kulture ondersoek weens die afwesigheid van canavanien produksie in die wortel kulture. Water stres het nie ‘n betekenisvolle verandering in die metaboliet vlakke meegebring nie, maar dit het wel tot ‘n beduidende afname in groei (vars massa en totale stingel lengte) en bewortelingsreaksie in die mikrostingels gelei. Sout stres het slegs tot ‘n betekenisvolle viii toename in arginien vlakke asook ‘n afname in die wortelvorming en groeireaksie tydens die toenemende sout vlakke gelei. ‘n Verlaging in plant groeikragtigheid mag ‘n eerste visuele teken van water stres wees. Die toevoeging van NaCl tot die medium kan tot ioontoksisiteit lei en plante reageer deur middel van osmotiese aanpassing wat tot veranderinge in die metaboliet vlakke asook veranderinge in fisiologiese groei, lei. Die finale deel van die studie het die anti-bakteriële aktiwiteit en die fitochemie van die transgeniese wortel kulture asook die ongetransformeerde in vitro en ex vitro plant materiaal ondersoek. Slegs die ekstrakte verkry vanaf blaar materiaal geoes uit die natuur, het matige anti-bakteriële aktiwiteit (1.25 mg ml-1) teen al die bakterië (Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis en Staphylococcus aureus) wat ondersoek is, getoon. Aanpassings in die sekondêre metabolisme van die harige wortels is deur middel van dunlaag chromatografie (DLC) en vloeibare chromatografie-massa spektroskopiese (VC-MS) analise ondersoek. Verskeie verbindings was in hoër vlakke in die harige wortels teenwoordig, as in die kontrole wortel ekstrakte. Transformasie het ook die kompleksiteit van die harige wortels se fitochemiese patroon verhoog, moontlik weens die produksie van nuwe verbindings of weens die opregulasie van bestaande metaboliese paaie. Die produksie van harige wortels en die vestiging daarvan in ‘n vloeibare sisteem tydens hierdie studie word beskou as ‘n belangrike stap na die opskalering van die kulture na bioreaktore. Hierdie wortels kan toekomstig tot die ontwikkeling van kulture met ‘n hoë produksie van gewenste metaboliete lei.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2010. / Includes bibliography. / ENGLISH ABSTRACT: The study of plants on a sub-cellular level is an important, yet challenging area and its application allows for novel insight into the understanding of metabolism and its regulation. In this study I describe the development of a reverse phase liquid chromatography mass spectrometry (RPLC-MS) technique in which 29 phosphorylated and nucleotide sugars could be detected and quantified. The method was validated with the use of authentic standards and the system displayed very good linearity (Rª > 0.95), while the recovery of the standards added to the plant material before extraction was between 65 and 125%. Further, Arabidopsis thaliana wild type (Col-0) and adenylate kinase (adk1) mutant leaf discs were fed 13C labeled glucose over a period of 24 hours and harvested at defined time intervals. Non aqueous fractionation, and metabolite profiling via the above mentioned rpLC-MS method in conjunction with gas chromatography mass spectrometry (GC-MS) allowed for the detection and quantification of primary metabolites on a sub-cellular level as well as the determination of their relative isotopic label enrichments through primary carbon metabolism. Finally, a yeast complementation system was designed for the identification of tonoplast bound sucrose import proteins. The screening system identified 22 unique sequences from an Arabidopsis thaliana cDNA library. Four unknown sequences were identified, one of which displayed tonoplast membrane association upon in silico analysis. Three ATP-binding proteins were also identified as well as a sub-unit from the exocyst gene family. Further studies will include the functional characterization of the latter, as well as the development of additional cDNA libraries more suited for screening of sequences that encode sucrose importer proteins. / AFRIKAANSE OPSOMMING: Die studie van plante op a sub-sellulere vlak is ‘n belangrike maar uitdagende navorsingsarea en die toepassing daarvan dra by tot unieke insig tot ‘n beter begrip van metabolise regulasie. In die studie bespreek ek die ontwikkeling van ‘n teenoorgestelde fase vloeistof kromatografie massa spektrometrie (RPLC-MS) tegniek waarin 29 gefosforileerde en nukleotied suikers gevind en gekwantifiseer kon word. Geldigverklaring van die metode is bewerkstelling met die gebruik van oorspronklike standaarde and die systeem het baie goeie liniariteit (Rª > 0.95) getoon, terwyl die herstelbaarheid van standaarde wat bygevoeg is by die plant material voor ekstraksie tussen 65% en 125% was. Arabidopsis thaliana wilde type (Col-O) en die adenaliet kinase (adk1) mutant blaar dele is met 13C gemerkte glukose gevoed oor ‘n tydperk van 24 uur en geoes by spesifieke tydstippe. Nie-vloeibare fraksionering en metaboliet uitleg is vermag vanaf die genoemde RPLC-MS metode met behulp van gas kromotografie massa spektrometrie (GC-MS) wat die bepaling en kwantifikasie van primere metaboliete op n sub-sellulere vlak sowel as die bepaling van hul relatiewe isotropiese merker verrykers deur primere metabolisme toelaat. Verder is n gis komplementere systeem ontwerp vir die identifikasie van tonoplas gebinde sukrose invoer proteine. Die verkenningsysteem het 22 unieke volgordes opgelewer vanaf ‘n Arabidopsis thaliana kDNA biblioteek. Vier onbekende volgordes is geidentifiseer, een wat tonoplas membraan assosiasie toon met in silico analise. Drie ATP-bindings proteine is ook geidentifiseer asook ‘n sub-eenheid van die eksosyst geen familie. Verdere studies sal die funksionele karakterisering van die laaste protein insluit, asook die ontwikkeling van additionele kDNA biblioteke meer gepas vir verkenning sodiende identifiseer van volgordes wat sukrose invoer proteine vertaal.

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.

The main aim of the work presented in this dissertation was to explore the possibility to genetically manipulate the sucrose storing crops, sugarcane and sweet sorghum, to convert their sucrose reserves into higher-value alternatives. For the purpose of this study we focussed on fructans as alternative sucrose-based high-value carbohydrates, since these fructose polymers are of significant commercial interest. To investigate the technical feasibility of transforming sugarcane and sweet sorghum to produce this novel carbohydrate, we proposed to transfer the fructosyltransferase genes from Cynara scolymus into these plants by means of particle bombardment. In order to apply this technology to sweet sorghum, an in vitro culture system suitable for transformation had to be established. For this purpose an extensive screening process with different combinations of variables were conducted. Though the relationships between these variables proved to be complex, it was concluded that immature zygotic embryos could be used to initiate a genotype-independent totipotent regeneration system with a 65% callus induction rate, provided that initiation takes place during summer. Stable transformation and regeneration of these calli were however not successful and will have to be optimised to allow future applications. By introducing fructosyltransferase genes into sugarcane, we succeeded in transforming sugarcane into a crop that produces a variety of fructans of the inulintype. Low molecular weight (LMW) inulins were found to accumulate in the mature internodes of 42% of the transgenic sugarcane plants expressing the sucrose:sucrose 1-fructosyltransferase (1-SST) gene, and in 77% of the plants that incorporated both 1-SST and fructan:fructan 1-fructosyltransferase (1-FFT), while only 8% of these plants accumulated high molecular weight (HMW) inulins. Our results demonstrated that sugarcane could be manipulated to synthesise and accumulate fructans without the induction of phenotypical irregularities. Inulins with a degree of polymerisation up to 60 were found in sugarcane storage tissue. In these HMW inulin-producing plants, up to 78% of the endogenous sucrose in the mature sugarcane culm was converted to inulin. This enabled inulin accumulation up to 165.3 mg g-1 fresh weight (FW), which is comparable to that found in native plants. These transgenic sugarcane plants, therefore exhibit great potential as a future industrial inulin source. Fructan production was detected in all the sugarcane plant tissue tested, predominantly as 1-kestose. In contrast with the fact that fructan accumulation in leaves did not affect the endogenous sucrose concentrations in these organs, the sucrose content of mature internodes that accumulated high levels of 1-kestose was severely reduced. However, increases in total sugar content, in some instances up to 63% higher than control plants, were observed. This phenomenon was investigated with the use of radio-labelled-isotopes. An increase in the allocation of incoming carbon towards sucrose storage, resulting in higher carbon partitioning into both 1- kestose and sucrose, were detected in the culms of transgenic compared to control lines. This modification therefore established an extra carbohydrate sink in the vacuoles that affected photosynthate partitioning and increased total soluble sugar content. The data suggests that sucrose sensing is the main regulatory mechanism responsible for adapting carbon flow in the cells to maintain sucrose concentration.

Thesis (PhD(Agric) (Plant Biotechnology))--University of Stellenbosch, 2010. / Includes bibliography. / ENGLISH ABSTRACT: Salinity is one of the major limiting abiotic stresses on legume plant yield, leading to early senescence of root nodules. This occurs because of accumulation of reactive oxygen species (ROS) in plant cells under salinity stress. Concurrent with the increase in cellular reactive oxygen species levels is the increase in cellular antioxidants and corresponding antioxidant enzymes. This feature is observed mostly in the shoots and roots of more tolerant genotypes compared to the susceptible genotypes. It is accepted that the mechanism of plant tolerance to stress is dependent upon the response of the antioxidant systems. Most studies carried out on shoot tissues suggest that scavenging of ROS by the plant antioxidant system is modulated by nitric oxide (NO). However, the pathways by which NO mediates such antioxidant responses are not fully understood. For legumes, salinity stress has adverse effects on yield and this is in part due to inhibition of nitrogen fixation in the root nodules of the legumes, which causes severe nitrogen starvation in nitrogen-deficient soils. Nodules are specialized organs comprising of both the rhizobia and the plant tissue, hence the physiological aspects may vary from the findings from the leaves. It was therefore deemed necessary to establish the role of NO on the nodule antioxidant system in the absence and presence of salinity stress. For the purposes of this study, the effect of both exogenously applied NO and endogenous NO on superoxide dismutase, glutathione peroxidase and glutathione content was determined. The studies involved the use of nitric oxide donors like sodium nitroprusside (SNP) and diethylenetriamine/nitric oxide adduct (DETA/NO), their respective fixed controls potassium ferricyanide and diethylenetriamine (DETA), plus a nitric oxide synthase inhibitor (to inhibit nitric oxide production by the enzyme nitric oxide synthase) on nodulated roots. The data obtained in this work points out specifically at roles played by nitric oxide in regulating superoxide dismutases, glutathione peroxidase and glutathione during salinity stress and proposes a link between nitric oxide-mediated changes in these antioxidant systems and salinity stress tolerance. Both the exogenously applied and endogenous nitric oxide increases the enzyme activities of superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione reductase (GR). However, there is both time dependency and nitric oxide concentration dependency on the enzyme activities. The total SOD enzyme activity increases upon nitric oxide exposure and with time of exposure. The individual SOD isoforms identified and studied in the root nodules all contribute to this increase in SOD activity upon nitric oxide treatment except for MnSOD I. This increase in activity is regulated at transcriptional level as the RT-PCR results targeting the individual isoforms reveals an increase in transcript levels after 6 hours of nitric oxide treatment. However, the CuZn SOD I isoform transcripts are reduced upon nitric oxide treatment. A similar response was also observed in GPX enzyme activity in which nitric oxide increased the GPX activity above all the controls. The GR enzyme activity exhibits an opposite response because the activity decreases with time of exposure to NO and concentration of NO. In order to determine the effect of NO under saline conditions, an experiment was set up that involved incubation of nodulated roots in solutions containing 150 mM NaCl. The stressed nodules exhibited generally higher levels of enzyme activities than the non-stressed nodules. Furthermore, exposure to nitric oxide donor in combination with NaCl induced even higher activities of SOD and GPX than NaCl or nitric oxide donor alone. There were also higher levels of reduced glutathione and total glutathione recorded under stress compared to optimal conditions. Nitric oxide increased the concentration of these forms of glutathione, suggesting an improved redox status based on the GSH/GSSG ratios under salinity stress in the presence of nitric oxide. Attenuation of nitric oxide synthesis with L-Nω-Nitroarginine methyl ester (L-NAME) reverses all the recorded effects of nitric oxide on antioxidant enzymes and glutathione pool. This was observed in salinity stressed nodules and non-stressed nodules. This work further establishes that NO plays a pivotal role in modulating the enzymatic activities through a pathway that is mediated by guanosine 3,5-cyclic monophosphate (cGMP). The experiment involving the inhibition of soluble guanylyl cyclase (sCG) (an enzyme that catalyzes the biosynthesis of cGMP), cell-permeable cGMP anaologue and L-NAME revealed that GPx activity is modulated through a cGMP-dependent pathway and NO is positioned up-stream of cGMP in the pathway leading to improved GPX activity. Cyclic GMP also modulates the GPX activity in a concentration dependent manner. NO improves the redox status of the cell under both saline conditions and non-saline conditions and this effect is modulated through a cGMP-dependent pathway. It is thus rational to conclude that; in the root nodules of legumes, like in other plant tissues, the increased accumulation of antioxidants and the increased activity of their corresponding enzymes, as modulated through the cGMP-dependent pathway by nitric oxide, confer root nodule tolerance to salinity. This concept directly points out at an attractive strategy for developing legumes that are genetically improved for enhanced root nodule tolerance to salinity; via differential regulation of antioxidants and antioxidant enzyme genes in the root nodules under abiotic stress. Towards attaining the goal for such genetic improvement, experiments involving construction of an abiotic stress-responsive and nodule-specific chimeric promoter were carried out. By fusing the 5-untranslated (5-UTR) region of the LEA gene that contains an abiotic stress-responsive cis-acting element (from theGmPM9 promoter) to the nodulin N23 promoter bearing the highly functional cluster of motifs for nodule specificity, the candidate nodule specific promoter that is abiotic stress responsive (ASREF/NSP) was constructed. The construct harbouring this ASREF/NSP chimeric promoter was fused to the -glucuronidase (GUS) reporter gene so as to study the functionality of the promoter in Medigaco truncatula plants. The construct was delivered into the Medicago plants through Agrobacterium rhyzogenes mediated transformation to produce composite Medicago plants. The transgenic roots have been cultured for futher manipulation and to confirm the functionality of the promoter. Furthermore several strategies can be deployed via the use of this chimeric promoter so as to enhance the nodular antioxidant system. This would involve either gene regulator-chimeric promoter fusion or the use of a single gene approach. As part of this work, the MtNOA gene homologous to AtNOAs, has been cloned from Medicago trancatula and put as ASREF/NSP fusion in a binary vector pBINPLUS and delivered into Medicago trancatula for nodule-specific and abiotic stress-induced nitric oxide synthesis. Since there is no plant NOS identified to date, the possibility of the use of a regulatory gene in this aspect is still limited. There are other options involving the use of the chimeric promoter with the individual genes encoding the antioxidant enzyme genes such as genes encoding SOD, GPX and the glutathione synthatase to enhance the plant antioxidant system during abiotic stress. / AFRIKAANSE OPSOMMMING: Geen opsomming was ingedien met die tesis

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Despite numerous attempts involving a variety of target genes, the successful transgenic manipulation of sucrose accumulation in sugarcane remains elusive. It is becoming increasingly apparent that enhancing sucrose storage in the culm by molecular means may depend on the modification of the activity of a novel gene target. One possible approach to identify target genes playing crucial coarse regulatory roles in sucrose accumulation is to assess gene expression during the developmental transition of the culm from active growth to maturation. This study has resulted in the successful optimisation of a mRNA hybridisation technique to characterise the expression of 90 carbohydrate metabolism-related genes in three developmentally distinct regions of sugarcane culm. A further goal of this work was to extend the limited knowledge of the regulation of sucrose metabolism in sugarcane, as well as to complement existing data from physiological and biochemical studies. Three mRNA populations derived from the different culm regions were assayed and their hybridisation intensities to the immobilised gene sequences statistically evaluated. The relative mRNA transcript abundance of 74 genes from three differing regions of culm maturity was documented. Genes exhibiting high relative expression in the culm included aldolase, hexokinase, cellulase, alcohol dehydrogenase and soluble acid invertase. Several genes (15) were demonstrated to have significantly different expression levels in the culm regions assessed. These included UDP-glucose pyrophosphorylase and UDP-glucose dehydrogenase, which were down-regulated between immature and mature internodes. Conversely, sucrose phosphate synthase, sucrose synthase and neutral invertase exhibited up-regulation in maturing internodal tissue. A variety of sugar transporters were also found to be up-regulated in mature culm, indicating a possible control point of flux into mature stem sink tissues. Combined with knowledge of the levels of key metabolites and metabolic intermediates this gene expression data will contribute to identifying key control points of sucrose accumulation in sugarcane and assist in the identification of gene targets for future manipulation by transgenic approaches. / AFRIKAANSE OPSOMMING: Ondanks verskeie pogings, waartydens verskeie gene geteiken is, is daar nog weinig sukses behaal om sukrose-akkumulering te verhoog. Toenemend wil dit voorkom asof suksesvolle genetiese manipulering van sukroseberging in die stingel van die verandering van ‘n nuwe geen afhanklik sal wees. Een van die moontlike benaderings wat gevolg kan word om potensiële teiken gene wat ‘n belangrike rol in die beheer van sukrose-opberging speel te identifiseer, is om geen uitdrukkingspatrone in die stingel tydens die omskakeling van aktiewe groei tot volwassenheid te karakteriseer. In hierdie studie is ‘n metode gebaseer op die hibridisering van mRNA geoptimiseer en suksesvol aangewend om die uitdrukkingspatrone van 90 verskillende geselekteerde gene, wat vir sleutelensieme in die beheer van koolhidraatmetabolisme kodeer, te bestudeer. Die doel met die ondersoek was om die beperkte kennis oor die regulering van koolhidraatmetabolisme uit te brei en om die bestaande inligting afgelei van fisiologiese en biochemiese-studies aan te vul. Drie verskillende mRNApopulasies, verkry uit verskillende dele van die stingel, is ontleed deur verskillende peilers te gebruik. Die gegewens is statisties ontleed en dit het afleidings oor die verandering in uitdrukking van hierdie gene moontlik gemaak. Die relatiewe konsentrasies van 74 verskillende gene is gedokumenteer. Gene wat sterk uitgedruk word het aldolase, heksokinase, sellulase, alkoholdehidrogenase en ongebonde suurinvertase ingesluit. Die uitdrukkingspatrone van 15 gene het tussen die verkillende weefsels gevarieer. Gene waarvan die uitdrukking tydens die oorgang na volwassenheid verlaag sluit in UDP-glukose pirofosforilase en UDP-glukose dehidrogenase en waarvan die uitdrukking verhoog sukrosefosfaatsintase, sukrosesintase en neutrale invertase in. Die uitdrukking van verskeie suikertransporter gene verhoog tydens volwassewording. Hierdie inligting te same met die huidige kennis oor heersende metabolietvlakke sal bydrae tot die identifisering van geenteikens vir toekomstige genetiese manupulering.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Nitric oxide (NO) has previously been shown to play a vital role in plants that are undergoing oxidative stress arising from abiotic stress. To better understand the role of NO on the antioxidative pathway, the effect of NO on Superoxide Dismutase (SOD) activity was studied during salt stress on soybean nodules. The enzymatic activity of specific MnSOD and FeSOD isoforms increased upon 1 week of exposure of nodules to NO or salt stress, the activity of CuZnSOD isoforms however increased in response to salt stress only. Furthermore, 4 putative FeSOD and MnSOD transcripts were identified and shown to increase in response to NO and salt stress. The promoter sequences of these NO-responsive putative SOD genes were analysed alongside a cystatin (AtCYS-1) which is also NO-inducible. Putative NO-responsive cis-acting elements as well as abiotic stress-responsive cis-acting elements were studied amongst these promoter sequences. The MYCL element and the AtMYB4 binding site were found to occur in all four NO-inducible SOD promoter sequences as well as in the AtCYS-1 promoter sequence. This suggests that NO acts via MYCL and/or AtMYB4 to up-regulate specific FeSODs and MnSODs, causing an increase in the activity of these SOD isoforms, thus reducing oxidative stress and cell death in soybean nodules. Furthermore, NO may also be up-regulating cystatins to inhibit cysteine proteases, thus preventing the onset of programmed cell death (PCD) and subsequently reducing salt stress-induced cell death. / AFRIKAANSE OPSOMMING: Geen opsomming

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Inorganic Phosphate (Pi) is one of the key nutrients required by all living organisms on earth. This nutrient is of vital importance to higher plants but it is not readily available for uptake from the soil, implying constant stress on plants. During photosynthetic dark and light reactions, phosphate is a prerequisite for all reactions to occur and to ensure plant survival. This statement implies that a careful homeostatic control of this nutrient is necessary in order to maintain a balanced carbon flow in all sub-cellular plant compartments. Phosphate limitation is a threat to plant survival and one way of addressing this nutritional hurdle is by feeding plants with fertilizer. This method of crop development and general plant maintenance by humans has a devastating effect on the environment, as phosphate causes eutrophication and various other consequences which are detrimental to animal life. Plants, however, are naturally equipped with Pi transporters which are activated conditionally depending on the external Pi availability. These transporters are present in most sub-cellular compartments and some of them have been identified and characterised, while others remain to be a prediction. If these transporters are characterised accordingly it might eventually mean that the use of fertilizers may no longer be necessary. In order to contribute to successful Pi-efficient crop development, a clearer understanding of P-dynamics in the soil and its recycling ability inside the plant itself is necessary. During this study it was attempted to characterise a putative high affinity Pi transporter, PHT1;5, from Arabidopsis thaliana via a Escherichia coli and yeast heterologous expression system and its Km value predicted in order to verify/hypothesise whether it is a high or low affinity transporter. This transporter is expressed in leaves and could be a promising tool for future carbon partitioning studies during phosphate limitation. / AFRIKAANSE OPSOMMING: Anorganiese fosfaat (Pi) word beskou as een van die belangrikste nutriente benodig vir alle lewe op aarde. Dit vervul ‘n hoof rol in talle noodsaaklike prosesse in hoër plante en is veral ‘n voorvereiste vir fotosintetiese reaksies om plaas te vind. In ‘n plant se natuurlike omgewing is anorganiese fosfaat nie geredelik bekskikbaar in grond nie en dus word daar vermoed dat plante onder konstante fosfaat stres gevind word. Omdat fosfaat so ‘n belangrike rol speel tydens fotosintese is dit noodsaaklik dat daar ‘n balans op sellulêre vlak gehandhaaf word, veral wat die verspreiding van koolhidrate tussen die verskillende kompartemente van die sel betref. Plante se oorlewing word bedreig deur ‘n tekort aan fosfaat in die omgewing en die enigste onmiddelike oplossing daarvoor is deur die toediening van bemestingstowwe. Hierdie metode van landery ontwikkeling en algemene instandhouding van plante deur die mensdom het ’n baie negatiewe effek op die omgewing. ‘n Oormaat fosfaat lei tot eutrifikasie en het verkeie ander negatiewe nagevolge wat dodelik is vir die dierelewe. Plante beskik ook oor natuurlike interne fosfaat transporters om hierdie tekort te oorkom. Hierdie transporters word op grond van eksterne fosfaat beskikbaarheid ge-aktiveer of ge-deaktifeer. Die transporters is teenwoordig in meeste sub-sellulêre kompartemente en sommige is al ge-identifiseer en gekarakteriseer, terwyl ander slegs ‘n voorspelling bly. Gedurende hierdie studie was ‘n poging aangewend om ‘n anorganiese fosfaat transporter van Arabidopsis thaliana, PHT1;5, te karakteriseer met behulp van mikro-organismes soos Escherichia coli en gis. Die poging het ingesluit om ‘n Km waarde vir hierdie transporter te voorspel en sodoende ‘n hipotese daar te stel van of dit hoë of lae affiniteit het vir fosfaat. Die transporter word groot en deels aangetref in blare en kan dus dien as ‘n belowende apparaat vir toekomstige koolhidraat uitruiling studies gedurende fosfaat tekort.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2007. / Sugarcane is cultivated because of the high levels of sucrose it stores in its internodes. Starch metabolism has been a neglected aspect of sugarcane research despite the problems caused by it during sugarcane processing. Currently there is no information available on the starch content in different South African commercial sugarcane varieties. This project had two main aims of which the first was to determine the starch content in the internodal tissues of six commercial sugarcane varieties. The activities of ADP-Glucose Pyrophosphorylase (AGPase) and β- amylase were also determined. The second aim of the project was to manipulate starch metabolism in sugarcane using transgenesis. To achieve this, transformation vectors for the down-regulation of AGPase activity and over-expression of β-amylase activity were designed. These vectors were then used to transform sugarcane calli and the results were analysed in suspension cultures. Starch levels in sugarcane internodal tissue increased more than 4 times from young to mature internodes. There were also large differences between varieties. When mature tissues of different varieties were compared, their starch concentration varied between 0.18 and 0.51 mg g-1 FW, with the majority of the varieties having a starch concentration between 0.26 and 0.32 mg g-1 FW. NCo376’s starch concentration was much lower than the rest at 0.18 mg g-1 FW and N19’s was much higher at 0.51 mg. g-1 FW. There was also a very strong correlation between starch and sucrose concentration (R2 = 0.53, p ≤ 0.01) which could be due to the fact that these metabolites are synthesized from the same hexose-phosphate pool. No correlation was evident between starch concentration and AGPase activity. This was true for correlations based on either tissue maturity or variety. β-amylase activity expressed on a protein basis was almost 5 times higher in the young internodes compared to mature internodes, suggesting that carbon might be cycled through starch in these internodes. AGPase activity in the transgenic suspension cultures was reduced by between 0.14 and 0.54 of the activity of the wild type control. This reduction led to a reduction in starch concentration of between 0.38 and 0.47 times that of the wild type control. There was a significant correlation between the reduction in AGPase activity and the reduction in starch (R2 = 0.58, p ≤ 0.05). β-amylase activity in the transgenic suspension cultures was increased to 1.5-2 times that of the wild type control. This led to a reduction in starch concentration of between 0.1 and 0.4 times that of the wild type control. Once again the increase in β-amylase activity could be correlated to the reduction in starch concentration of the transgenic suspension cultures (R2 = 0.68, p ≤ 0.01). In both experiments there was no significant effect on sucrose concentration.

Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2009. / Dichrostachys cinerea, Carpobrotus dimidiatus, Capparis tomentosa and Leonotis leonurus are all plants that are indigenous to South Africa. These plants are used in traditional medicine to treat various ailments. However, there is little or no scientific data to justify these traditional uses. Furthermore, it is difficult to reconcile traditional knowledge with scientific evidence because of the overwhelming targeting of signal-responsive systems by plant defensive compounds, multiple sites of action and the connectedness of the signaling pathways, which provide many cures and have pleiotropic effects. In order to evaluate the action spectrum of these plants, and validate its widespread use, this research evaluated the antibacterial, antioxidant, anti-inflammatory, anti-mosquito and immunomodulatory properties of these plants. Antimicrobial activity of the extract was determined by evaluating the bactericidal and fungicidal action using the agar disc diffusion assay. Anti-oxidative properties of the extracts were tested using the DPPH photometric assay. Anti-inflammatory properties were carried out using the 5-lipoxygenase assay. The larvicidal, repellency and insecticidal assay was determined against A.arabiensis. The safe use of these plant extracts was determined by evaluating toxicity, a brine shrimp lethality assay and an in vitro cell culture system using human myelogenous leukemia cell line. Potential carcinogenic activity was evaluated using the Ames Salmonella Mutagenecity assay. The immunomodulatory activity of the extracts on human peripheral blood mononuclear cells 6 was evaluated on freshly harvested lymphocytes using the MTT assay. Cytokine response was evaluated by measuring the secretion of interferon-gamma and interleukin-10. Elucidation of the B cells, T cells, activated T cells, CD 4+, CD 8+ and NK cells was performed by flow cytometry. The extracts showed anti-microbial activity against Escherichia coli, Pseudomonas aeruginosa, Klebsiella oxytoca, Salmonella typhimurium, Serratia marcescens, Bacillus cereus and Tricoderm sp. The highest activity was shown by methanolic and aqueous extracts of L. leonurus leaves followed by methanolic and aqueous extracts of D. cinerea. Extracts of C. tomentosa and D.cinerea demonstrated a higher degree of free radical scavenging than rutin, which was used as a standard indicating that these plants have strong antioxidant properties. None of the plants showed significant anti-inflammatory activity when compared to NDGA. In the anti-mosquito assays, the extracts showed strong repellency and insecticidal activity. L. leonurus extracts demonstrated the highest insecticidal and repellency activity against the mosquito, and was also found to cause ‗knockdown‘ and mortality. The extracts display no toxicity, cytotoxicity and mutagenicity. The immunological studies for immune modulation showed that the methanol extracts of these plants induce a Th1- predominant immune response because they significantly suppressed the secretion of IL-10 and augment IFN-γ production, which are hallmarks used to indicate a stimulation of the innate immune response. This study also provides new information, with respect to the potential use of these plants in producing a mosquito repellent and an immunostimulant. / National Research Foundation