Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Vitamin C (ascorbate or AsA) is a secondary metabolite produced in many eukaryotes including yeasts, plants and animals. It plays essential roles as an anti-oxidant and enzyme cofactor, functions as an electron donor and -acceptor and is involved in various developmental processes. This study was initiated with the aim of increasing vitamin C production in tomato. Three genes, namely GDP-mannose pyrophosphorylase (GMPase) from Saccharomyces cerevisiae, arabinono-1,4-lactone oxidase (ALO) from Saccharomyces cerevisiae and myo-inositol oxygenase 2 (MIOX2) from Arabidopsis thaliana were ectopically expressed in the tomato cultivar Money Maker. GMPase converts D-mannose-6-P to GDP-D-mannose. This reaction forms part of the well characterized, “Smirnoff-Wheeler” pathway. ALO catalyzes the terminal step in erythroascorbate synthesis in yeast. In situ it also metabolizes the plant and animal substrates for ascorbate manufacture. Myo-inositol (MI) is converted into D-glucuronate by the activity of MIOX. D-Glucuronate is a precursor to L-guluno-1,4-lactone synthesis which is the precursor to AsA in animals and thought to be present in plants. The genes were independently introduced with the aid of Agrobacterium tumefaciens mediated transformation and expressed under the control of the CaMV 35S promoter. Plants with increased GMPase activity consistently showed increased L-ascorbate levels in leaves and fruit of between 20- and 70% compared to the wild-type. Plants transcribing the ALO gene exhibited small increases in L-ascorbate in green fruit (p < 0.1). Leaf tissue from MIOX plants displayed significant activity increases (p < 0.05), and substantial decreases in MI. In green fruit two MIOX lines had increases in activity, cell wall uronic acids and AsA levels. Marginal increases in L-ascorbate would not warrant industrial application, but follow-up research with over-expression of other enzymes of the “Smirnoff-Wheeler” pathway should be explored. / AFRIKAANSE OPSOMMING: Vitamien C (askorbiensuur of AsA) is ʼn sekondêre metaboliet wat in baie eukariote, insluitend gis, plante en diere geproduseer word. Dit speel ʼn noodsaaklike rol as ʼn anti-oksidant en ensiem kofaktor, funktioneer as ʼn elekronskenker en aanvaarder en is betrokke in verskillende ontwikkelings prosesse. Hierdie studie was geїnisieer met die doel om vitamien C produksie in tamatie te vermeerder. Drie gene, naamlik GDP-mannose pirofosforilase (GMPase) van Saccharomyces cerevisiae, arabinono-1,4-laktoon oksidase (ALO) van Saccharomyces cerevisiae en mio-inositol oksigenase 2 (MIOX2) van Arabidopsis thaliana was ektopies uitgedruk in the tamatie kultivar, Money Maker. GMPase skakel D-mannose-6-P om na GDP-D-mannose. Hierdie reaksie is deel van die goed gekenmerkde “Smirnoff Wheeler” baan. ALO kataliseer the terminale stap in eritroaskorbiensuur sintese in gis. In situ metaboliseer dit ook die plant en dier substrate om askorbiensuur te vervaardig. Mio-inositol (MI) is omgeskakel na D-glukuronsuur deur die aktiwiteit van MIOX. D-glukuronsuur is ʼn voorloper in L-guluno-1,4-laktoon sintese wat dan ʼn voorloper is van AsA in diere en word ook verdink om in plante teenwoordig te wees. Die gene was onafhanklik ingestel met die hulp van Agrobakterium tumefaciens gemedїeerde transformasie en uitgedruk onder die beheer van die CaMV 35S promotor. Plante met verhoogde GMPase aktiwiteit het in blare en vrugte konsekwente toename in L-askorbiensuur vlakke met tussen 20 – 70% gewys in vergelyking met wilde-tipe. Plante wat ALO getranskribeer het, het klein stygings in L-askorbiensuur in groen vrugte gewys (p < 0.1). Blaarweefsel van MIOX plante wat verhoogde aktiwiteit vertoon het, (p < 0.05), het ook aansienlike dalings in MI gehad. In groen vrugte van MIOX het twee lyne verhoogte aktiwiteit, selwand uronsuur en AsA vlakke gehad. Klein toename in L-askorbiensuur is nie gepas vir industriële toepassing nie, maar opvolg navorsing moet ondersoek word met die oor-uitdrukking van ander “Smirnoff-Wheeler” baan ensieme.

Magister Scientiae - MSc (Biotechnology) / Brassica napus is a plant that is used for human and animal consumption. This plant is also used for phytoremediation due to its relatively higher level of heavy metal tolerance. In South Africa, mining is one of the main drivers of the economy. One of the major negative environmental impacts of mining is heavy metal contamination. Soil metal content can rise to levels that are quite high and can even have a negative impact on the yields of B. napus crop. The glucosinolate-myrosinase system of B. napus is a system that is used as defence against biotic stressors. Indole glucosinolate breakdown products have been proven to enhance the antioxidant capacity of plants. Some have also shown growth promoting properties in plants. We studied the effect of exogenous DIM on B. napus and it role in Zr induced heavy metal stress. Germination percentages revealed that DIM increased germination, Zr application decreased germination and the DIMZr treatment reversed the negative impact of Zr application on B. napus. The effect of treatments on the biomass of B. napus was assessed by determining the dry weights. Results show that exogenous DIM improves biomass. Zr application decreased biomass and DIM-Zr treatment ameliorated the effect of Zr application. / 2020-08-31

Made available in DSpace on 2015-04-11T13:55:49Z (GMT). No. of bitstreams: 1 Thiago Mendes de Lima.pdf: 1095985 bytes, checksum: af3d342d9c71734320c0b651931b5083 (MD5) Previous issue date: 2014-02-21 / Fundação de Amparo à Pesquisa do Estado do Amazonas / A utilização de agentes antioxidantes tem sido proposta para melhorar a resistência adesiva após clareamento dental. O presente estudo in vitro avaliou o efeito do hidrogel de ascorbato de sódio a 10% (AS), aplicado após o clareamento com peróxido de hidrogênio a 35% (PH), na resistência adesiva em esmalte de uma resina composta (RC). Foram selecionados 50 dentes bovinos e aleatoriamente divididos em cinco grupos experimentais (n=10) subdividindo-os em grupos testados tardiamente (7 dias) e após envelhecimento (3 meses): Grupo 1 (G1: 7 dias/ G1A: 3 meses) - sem clareamento (grupo controle) + RC; G2/G2A PH + RC; G3/G3A PH + 14 dias + RC; G4/G4A PH 35% + AS 10% (30 min) + RC; G5/G5A PH + 14 dias + AS (30 min) + RC. Foram obtidos palitos de resina-adesivo-esmalte (1mm x1mm) e submetidos ao tese de resistência à microtração em uma máquina de ensaio universal EMIC DL2000 (100N a 0,5mm/min). Os tipos de falhas foram analisados em microscopia digital (40x) e eletrônica de varredura, classificado-as como coesiva, adesiva, mista. Os valores (Mpa) de resistência adesiva à microtração foram avaliados estatisticamente através da Análise de Variância a dois critérios (ANOVA) e do teste de Tukey para comparações das médias, com significância 1%. Os resultados foram: G1: 20,09Aab ± 5,33 / G1A: 24,09Abc ± 5,12; G2: 17,14Aa ± 4,94 / G2A: 20,84Aab ± 5,21; G3: 20,36Aab ± 5,30 / G3A: 22,76Abc ± 5,40; G4: 22,18Aabc ± 5,30 / G4A: 30,22Ad ± 8,05; G5: 21,92Aab ± 5,90 / G5A: 27,25Ac ± 7,12. Pode-se concluir que, o uso do ascorbato de sódio a 10% interfere positivamente na resistência adesiva de restaurações envelhecidas em 3 meses, aumentando os valores de resistência adesiva.

L'ozone troposphérique est un gaz à effet de serre et un puissant oxydant qui impacte la santé et la production végétale. Afin d'analyser son impact sur le fonctionnement de la végétation il est nécessaire d'effectuer une partition entre le dépôt stomatique, qui pénètre à l'intérieur des feuilles, et le dépôt non stomatique, qui résulte de la destruction chimique de l'ozone sur les cuticules foliaires, le sol ou dans l'air à l'intérieur du couvert. L'objectif central de ce travail de thèse est d'améliorer la compréhension et la modélisation des processus impliqués dans le dépôt non stomatique d'ozone et plus particulièrement dans le dépôt sur les films d'eau à la surface des feuilles. Le modèle de transfert de masse et d'énergie sol-végétation-atmosphère MuSICA, capable d'estimer les quantités d'eau sur le feuillage à différents niveaux dans le couvert, a permis de modéliser la solubilisation puis la diffusion et réaction de l'ozone dans l'eau sur les feuilles. Cette voie de dépôt a permis d'expliquer les flux d'ozone observés sur blé en conditions humides en ajustant les valeurs du taux de réaction chimique de l'ozone dans le film d'eau de 103 s 1 en début de saison à 105 s 1 pendant la sénescence. Ce taux semble varier entre les espèces végétales (blé, hêtre et pin maritime) mais la tendance avec la sénescence persiste. Ces résultats ont été confortés par un dispositif expérimental original en laboratoire, sur blé, hêtre, pin blanc et chêne pédonculé. Des échanges de composés chimiques entre la feuille et le film d'eau réactifs avec l'ozone pourraient expliquer les ordres de grandeur obtenus et l'augmentation du taux de réaction en période de senescence. / Tropospheric ozone is a greenhouse gas but also a powerful oxidant that impacts vegetation health and productivity. In order to analyse the impact of ozone on vegetation functioning it is necessary to separate stomatal ozone deposition that penetrates inside the leaf interiors from non-stomatal deposition that result from ozone chemical destruction on leaf cuticles, in the soil or in the air inside the canopy. The central objective of this PhD work is to improve our understanding and modelling of the processes involved in non-stomatal ozone deposition and more specifically in ozone deposition on water films on leaf surfaces. For that purpose I used the soil-vegetation-atmosphere transfer model of mass and energy MuSICA. The choice of this model was motivated by its ability to estimate the quantities of water on foliage at different levels inside the canopy. This allowed me to model ozone dissolution, diffusion and reaction in the water film on wet leaves. This new deposition pathway could explain observed ozone fluxes over wheat in wet conditions by adjusting the chemical reaction rate of ozone in the water film from 103 s 1 at the beginning of the growing season, up to 105 s 1 during senescence. This reaction rate seemed to vary between species (wheat, beech, maritime pine) but the trend with the senescence remained. These results were then confirmed by lab experiments using an original setup, on wheat and beech, but also white pine and pedunculate oak. Exchanges of chemical compounds between the leaf and the water film that would react with ozone could explain the order of magnitude of the reaction rate and its increase during senescence.

With new insights on mechanism, there is renewed interest in the use of pharmacological ascorbate (AscH-) in cancer therapy. The generation of H2O2 with AscH- acting as an electron donor to O2 is central to AscH- -induced cytotoxicity. We hypothesized that catalytic manganoporphyrins (MnPs) would increase the rate of oxidation of AscH- thereby increasing the flux of H2O2, resulting in increased cytotoxicity. We tested three different MnPs: MnTBAP, MnT2EPyP, and MnT4MPyP, which represent a range of physicochemical and thermodynamic properties. Of the MnPs tested, MnT4MPyP had the greatest effect on increasing the rate of oxidation of AscH-, as seen by the concentration of ascorbate radical [Asc*-] and rate of oxygen consumption. MnPs and AscH-, when combined at concentrations that had minimal effects alone, synergistically increased the cytotoxicity as seen by decreased clonogenic survival in human pancreatic cancer cell lines. Catalase, but not superoxide dismutase, reversed the cytotoxicity of AscH- and MnP, consistent with an H2O2-mediated mechanism. In addition, there was a marked increase in the steady-state concentration of ascorbate radical upon the addition of MnPs to whole blood ex vivo from mice infused with ascorbate as well as from patients treated with pharmacologic ascorbate. The combination of MnT4MPyP with ascorbate inhibited in vivo tumor growth. We conclude that MnPs can increase the rate of oxidation of AscH-, leading to an increased flux of H2O2 resulting in increased ascorbate-induced cytotoxicity

High-dose pharmacologic ascorbate has promise as an adjuvant to traditional therapies for cancer. It is hypothesized that the peak plasma concentration is a key determinant in treatment efficacy. From the Phase I clinical trails on the use of pharmacological ascorbate as an adjuvant to Gemcitabine in the treatment of stage IV pancreatic cancer at the University of Iowa Hospitals and Clinics, we found that monitoring plasma ascorbate concentration [AscH-]pl with each infusion is both very time consuming and expensive for large scale implementation. A method to determine the amount and protocol to infuse ascorbate to achieve a desired patient [AscH-]pl would be of great benefit. Current models lack flexibility for various infusion proto- cols. Additionally, constructing a model of ascorbate pharmacokinetics would allow investigation of an optimal dosing regime to maintain constant plasma ascorbate levels. A mechanistic model and an empirical model were developed and validated. The mechanistic model suitably replicated the results obtained in the clinical trial but contained too many variables to be useful in a clinical setting. The empirical model showed good results in replicating the trial results and requires only a few easily measured variables to generate predictions High dose ascorbate has been shown to produce hydrogen peroxide. In furthering the studies of how ascorbate affects tumor cells, the action of glucose-6-phosphate dehydrogenase (G6PD) is considered because it supplies NADPH to several peroxide removal pathways. To this end, the kinetics of G6PD were studied using kinetic simulations. G6PD exhibits a reserve capacity, which is the difference between the activity when all intracellular NADP is oxidized to the rate at which is operates when intracellular NADP is at the physiologic 90 % reduced to 10 % oxidized ratio. These simulations yielded an interesting pattern which is also seen by evolutionary biologists. G6PD exhibits a response capacity, which is the difference between the maximum G6PD activity exhibited when there is no demand for NADPH greater than normal cell functions and the activity exhibited when all cellular NADP is oxidized.

Vitamin C (ascorbic acid, AsA), an important primary metabolite of plants, functions as an antioxidant, an enzyme cofactor, and a cell-signaling modulator in a wide array of crucial physiological processes including biosynthesis of the cell wall, secondary metabolites and phytohormones, stress resistance, photoprotection, cell division, senescence, and growth. To identify genes that may regulate vitamin C levels in plants, about 3000 activation-tagged Arabidopsis lines were treated with ozone, which is a power oxidizing agent. Two mutants were selected for identification of potential genes involved in the regulation of vitamin C synthesis. A putative F-box gene, VCF1, and a purple acid phosphatase, AtPAP15, were identified for further characterization. Two homozygous SALK T-DNA knockouts in the open reading frame (ORF) of VCF1 exhibited high tolerance to ozone when treated with 450 ppb for 3 hours and the AsA levels of these mutants were 2 to 3 fold higher than wild-type (wt) plants. Developmental studies, using RT-PCR, indicated that foliar expression of the VCF1 gene increased with plant age from 1 to 5 weeks, whereas AsA decreased during this same period. The expression of VCF1 was higher under a low-light condition in which AsA was reduced considerably. The AsA levels in two VCF1 overexpressing lines were only 50 to 70% of wt plants. These results suggested that the putative F-box gene functions as a negative regulator of leaf ascorbate content. Overexpression of AtPAP15 with the CaMV 35S promoter resulted in up to 3-fold higher AsA levels than wt plants, where two independent SALK T-DNA insertion mutants in AtPAP15 had 50% less AsA than wt plants. Enzyme activity of bacterially expressed GST:AtPAP15 was greatest with phytate as a substrate indicating that AtPAP15 is a phytase. Phytase catalyzes hydrolysis of phytate (myo-inositol hexakisphosphate) to yield myo-inositol and free phosphate. Thus, AtPAP15 may regulate AsA levels by controlling the input of myo-inositol into this branch of AsA biosynthesis in Arabidopsis. AtPAP15 was expressed in all tested organs in wt plants and suggests that the enzyme may have functions other than phytate degradation during seed germination. / Ph. D.

Pseudoperonospora cubensis (Bert. et Curt) Rost. is the causal agent of cucurbit downy mildew (CDM). It is the most damaging cucumber pathogen on the Eastern Shore of Virginia and eastern parts of the United States. Pseudoperonospora cubensis is an obligate oomycete pathogen, infecting crops within the Cucurbitaceae family. The disease is characterized by angular chlorotic lesions and a downy or felt-like appearance on the abaxial side of the leaf. Control of this pathogen includes use of resistant cucumber cultivars and costly fungicide programs. Continuous use has led to resistance to commonly used fungicides. This has become a major concern and in response, seed companies have developed cucumber cultivars which claim downy mildew resistance.  This study evaluates different cucumber cultivars and assesses their level of resistance to CDM. The results indicate that an integrated management approach of reduced fungicide application and the use of resistant cultivars can suppress levels of CDM and yield a cucumber crop. Additionally, a molecular study was conducted, comparing the relative expression of genes encoding a basic PR-1 protein, a cytosolic ascorbate peroxidase protein and three resistance (R) gene proteins, in nineteen cultivars. All of the selected genes were analyzed using real-time PCR. The relative expression levels of the R-genes varied between cultivars. The basic PR-1 protein decreased expression in the majority of the cultivars, suggesting no involvement in the first twenty-four hours. Cytosolic ascorbate peroxidase relative expression levels suggest an increase in susceptible cultivars and a decrease in tolerant cultivars. / Master of Science

Water stress is the most critical environmental factor limiting crop production in the US Piedmont. The presence of humic substances in composted organic amendments may increase crop tolerance to water stress through their hormone-like effects on plant metabolism. The objectives of this study were to calculate N mineralization rates of composted and non-composted organic materials used in this long-term field study, and to determine differences in soil physiochemical properties, corn and soybean leaf physical and biochemical properties yield and seed quality between organically amended and inorganically fertilized treatments. Nitrogen mineralization rates were greatest in the poultry litter (21%) and Panorama yard waste compost (4.5%) amended plots. Nitrogen uptake (120 mg/pot, 133 mg/pot, respectively) in these treatments were greater than that in the control (0N) (91.3 mg/pot) treatment. Wolf Creek biosolids compost and Huck's Hen Blend yard waste compost induced N immobilization (-5.0% and 0.18%, respectively), and had N uptake values similar to the control (92.6 mg/pot and 95.7 mg/pot). Rivanna biosolids compost immobilized N (-14.8%) but N uptake (136 mg/pot) was greater than that in the control due to the relatively high inorganic N content in the amendment. The total N concentration and C:N values were less reliable variables in predicting N mineralization when a significant portion of the total N was in the inorganic form. The annual application of poultry litter, Rivanna biosolids compost, and Panorama yard waste compost at 100% agronomic nitrogen and 30 % agronomic nitrogen rates in the field study improved soil fertility and increased total organic and humified carbon contents relative to the inorganically fertilized and control treatments. The amended treatments had slightly greater plant available water contents (average 10.0 cm/15 cm) than the control (8.38 cm/15 cm). Leaf water potential measurements revealed that neither crop experienced water stress during the sampling season. Treatment differences in leaf antioxidant activity were only observed in corn. All corn plants that were fertilized with amendments supplying the crop's nitrogen needs, regardless of the source, had greater leaf nitrogen (+29%), chlorophyll (+33%), and protein contents (+37%), lower superoxide dismutase (-29%) and ascorbate peroxidase (-17%) activities, and lower malondialdehyde (-33%) contents relative to the control and low nitrogen treatments. There were no observed differences in catalase activity, which was likely due to the evolutionary advantage of C4 metabolism. Yield was strongly related to midseason leaf nitrogen contents (R2=0.87, p<0.0001) and not soil humified carbon (R2=0.02, p=0.0543). There were no observed treatment differences in soybean leaf physiology and metabolism. Differences, however, were observed over time. As the leaves senesced, leaf chlorophyll, protein, superoxide dismutase and catalase activities decreased, and the malondialdehyde content increased. Ascorbate peroxidase activity slightly increased with time. Catalase activity in soybean was primarily driven by the oxidation of glycolate, a product of photorespiration, and not the formation of reactive oxygen species in the chloroplasts. The organically amended treatments had higher yields (9-21% increase), greater protein contents (4-9% increase), and seed weights (5-14% increase) relative to the fertilizer and control treatments. It was concluded that differences in soybean yield and seed quality were due to non-nutritive benefits of the organic amendments and not available water or plant nutrition. / Master of Science

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