The apoplastic enzyme, ascorbate oxidase (AO), is a blue copper oxidase that catalyses oxidation of ascorbate (AsA) to monodehydroascorbate (MDHA). In Arabidopsis thaliana, AO is encoded by three genes (At4g39830, At5g21105 and At5g21100) designated AO1, AO2, and AO3 respectively. Since AsA is the most abundant antioxidant in the apoplast and AO is active in this compartment, the regulation of apoplastic AsA redox status by AO and its role in development and environmental perturbations has become a subject of interest. Phylogenetic analysis showed that AO is present in higher plants, pteridophytes, mosses and green algae. Amino acid sequence analysis showed that AO2 and AO3 shared higher sequence identity than AO1. In silico analyses found that AO1 had a distinct expression pattern and subcellular localisation compared to AO2 and AO3, suggesting AO1 might be involved in alternative functions. Consistent with previous studies, AO activity was high in actively growing tissue of wild-type (WT) A. thaliana, supporting a possible role of AO in cell expansion. ao1, ao3 and ao1ao3 T-DNA insertion mutants were characterised. ao1 had similar level of AO activity to WT, while ao3 and ao1ao3 had 10-20% of WT AO activity. Compared with WT, these T-DNA insertion mutants did not show any phenotypic differences under unstressed or stressed (high light and drought) growth conditions. An artificial microRNA construct (amiR-AO) to silence all three AO genes was developed. Also, an overexpression plasmid (35S::AO3) harbouring AO3 gene was constructed. These constructs were used to transform A. thaliana. AO activity was undetectable in the amiR-AO line, while the 35S::AO3 line had 3-fold higher AO activity than the WT. Under unstressed normal growth conditions, the amiR-AO line had bigger rosette size, whereas the 35S::AO3 line exhibited early flowering and smaller number of rosette leaves. The amiR-AO line accumulated more anthocyanin and AsA than WT when acclimated to high light, whereas the 35S::AO3 line accumulated less anthocyanin than WT. In response to drought, the amiR-AO line did not show phenotypic differences compared to WT, while the 35::AO3 line had higher rate of leaf water loss and appeared to have greater sensitivity to drought. These results suggest that AO perturbation could, to some extent, affect the growth and stress response of A. thaliana although the effect is small.

Vitamin C (ascorbate or Asc) is an essential vitamin for humans. The transport of oxidized ascorbate occurs via Na^+-dependent vitamin C transporters (SVCTI/SVCT2). Ascorbate is a powerful antioxidant that may be beneficial in scavenging the reactive oxygen species associated with cardiovascular diseases. The objectives of this thesis were: to identify the SVCT isoform(s) expressed in pig coronary artery smooth muscle and endothelium, to determine if preloading cultured pig coronary artery smooth muscle cells with ascorbate protects them against oxidative stress, and to overexpress SVCT2 in these cells to see if an increase in ascorbate reserve helps protect the cells even more. Pig coronary artery smooth muscle tissue and cells cultured from the same tissue express SVCT2 and not SVCT1. Cultured pig coronary artery endothelial express SVCT2, however the limited amount of fresh endothelium isolated, restricted us from determining the isoform present in the fresh tissue. Ascorbate preloading (200 (mu)M overnight) did not decrease the damage caused by hydrogen peroxide as measured by oxidation of dichlorodihydrofluorescein diacetate or mitochondrial reductase activity. The mRNA and ^14C-Asc uptake was marginally greater in pig coronary artery smooth muscle cells stably transfected with a linear pcDNA3.1 SVCT2 plasmid than mock transfected controls. The ^14C-Asc uptake was 1.5 times greater than mock transfected cells after 60 min. A new SVCT2 plasmid, that contained SVCT2 coding region only, did not show greater ^14C-Asc accumulation compared to the plasmid that had the entire SVCT2 cDNA in transiently transfected HEK293T cells. This thesis is a beginning towards further study on the molecular and physiological role ascorbate plays in the coronary artery. / Thesis / Master of Science (MS)

Biochemical consequences of ascorbate deficiency were studied in the leaf tissue of Arabidopsis thaliana ascorbate-deficient vtc mutants with a view of understanding the relationship between ascorbate, stress response and metabolism. Ascorbate is an important antioxidant and is also a cofactor for 2-oxoglutarate-dependent dioxygenases, which are involved in the biosynthesis of a number of metabolites. The response of wild type (Col-0) and vtc1, vtc2-1, vtc2-2 and vtc3-1 mutants to high light intensity, wounding and salinity was investigated using a metabolomics and proteomics approach. Metabolite profiling and comparative proteomics were performed by liquid chromatography-quadrupole time of flight mass spectrometry (LC-QToF MS) and targeted analysis of plant hormones and flavonoids by liquid chromatography triple-quadrupole mass spectrometry (LC-QQQ-MS). These combined analyses revealed the effect of ascorbate deficiency and stress on metabolites and cell wall proteins. LC-QToF-MS based untargeted metabolite profiling methodologies were developed for analysis of metabolites on a large scale. Using this method about 3000-5000 metabolites (mass-retention time pairs) could be reproducibly detected in A. thaliana leaf extract and aligned between samples. Approximately 1000 metabolites were differentially expressed between WT and vtc mutants in different experiments. Of these, twenty eight compounds were confirmed to be differentially expressed by LC-QQQ-MS between WT and vtc mutants, and eight of these compounds were positively identified and validated with standards. The plant hormones abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) have all been implicated in plant stress responses and differences in their accumulation in some of the vtc mutants have been reported. A systematic study of the response to stress of these hormones in several vtc mutants was carried out using LC- QQQ- MS. While some of the mutants showed increased SA and SA-glycoside accumulation, stress-induced ABA and JA accumulation was generally unaffected. Methods for identifying the metabolites in a targeted manner by LC- QQQ-MS was developed and were shown that all vtc mutants were impaired in the accumulation of anthocyanin in response to HL treatment. In strong contrast to anthocyanin, flavonol glycosides were not affected by ascorbate deficiency. Therefore, ascorbate deficiency has a specific effect on the anthocyanin biosynthesis. Ascorbate occurs in the plant cell wall and isolation of apoplastic fluid showed that all vtc mutants have decreased apoplastic ascorbate compared to WT. Ionically-bound proteins were from the cell wall of A. thaliana leaves. Peroxidase specific activity in this fraction tended to be higher in vtc mutants than WT. High light intensity also increased peroxidase activity in WT and vtc mutants. To determine which peroxidase isoenzyme caused increased peroxidase activity, ionically-bound cell wall N-glycosylated proteins were isolated by Concanavalin A chromatography and analysed by LC-QToF-MS. Comparison of WT and vtc2-2 grown in low light and high light identified 937 peptides significantly different between WT and vtc2-2 and some are also affected by light intensity. Specifically, peroxidases 33 and 34 had increased abundance in vtc2-2. The results show that ascorbate deficiency causes a detectable change in the metabolome of A. thaliana leaves, with specific effects on anthocyanin accumulation being detected. Ascorbate deficiency also influences the expression of cell wall proteins. Peroxidase activity is increased, and this response could be related to the increased pathogen resistance reported in vtc mutants.

Myocardial ischemia-reperfusion (I/R) injury is an important health concern in myocardial infarction and situations such as angioplasty and cardiac surgeries. Therefore, patients and physicians need therapeutic interventions that are applicable at the time of surgery. Flavonoids and ascorbate (vitamin C) are known for their antioxidant activity and may be involved in the currently known health benefits of plant based foods and drinks. The objectives of this study were to 1) determine the extent to which ascorbate or catechin alone at levels which could be in blood after dietary supplementation, can protect myocardial tissue in the reperfusion phase of I/R injury, and 2) evaluate the possible cooperative or synergistic protective effect of ascorbate and catechin when given together. Isolated rat hearts (n=48) were perfused in the retrograde mode with modified Krebs-Henseleit buffer, and following the induction of 30 min global ischemia, ascorbate (150 µM) and/or catechin (5 µM) were added directly into the perfusate during 90 min reperfusion. To determine the histopathological features, hematoxylin and eosin (H&E) stain was used in one heart per condition; while to assess the biochemical analysis, the heart tissues were assessed for apoptosis (caspase-3 activity), oxidative stress (thiobarbituric acid reactive substances (TBARS) and total malondialdehyde (MDA) levels), and redox status (reduced and oxidized glutathione tissue levels). A comparison of IR hearts with two controls, sham (perfused for a 15 min stabilization period) and continuous perfusion (perfused for 135 min), showed in most but not all measurements that this was a suitable model of IR injury. The treatment experiments showed that 150 µM ascorbate protected the heart against lipid peroxidation and cell apoptosis by 100%, while 5 µM catechin protected by 67% and 90% respectively. No cooperative protective effect could be observed when ascorbate and catechin were used together. None of the treatments significantly affected either reduced or oxidized glutathione levels. In conclusion, this study showed strong protection by ascorbate, which could be used in clinically relevant situations, and is the first to report the protection by catechin at this dose under conditions of myocardial ischemia-reperfusion injury.

>Magister Scientiae - MSc / Food security has always been one of the priority concerns in Africa, and it is mostly threatened by drought stress due to climate change. Drought-induced stress is one of the serious limiting factors of plant production, and it is known to impose oxidative stress as a consequence of excessive reactive oxygen species (ROS) accumulation that lead to lipid peroxidation, which is manifested as increased cell death. Hence, this study investigated the influence of drought stress on two contrasting canola genotypes (Agamax and Garnet), by monitoring their physiological and molecular changes. The results showed that the plant growth and biomass of both genotypes were significantly affected by drought stress as a consequence of excessive ROS accumulation (manifested as H₂O₂ and OH· content). However, under drought stress conditions, the reduction in biomass and shoot length was more pronounced in the Garnet genotype when compared to that of the Agamax genotype. This was further supported by the increase in lipid peroxidation and cell death, which were shown to be significantly higher in the Garnet genotype when compared to the Agamax genotype under drought stress. ROS scavenging ability which prevents oxidative stress and ultimately ROS-induced cellular damage. Hence, given the higher levels of antioxidant activity coupled with the reduction in ROS accumulation that was observed in the Agamax genotype, we suggest that the Agamax genotype might be slightly less susceptible to drought stress, when compared to the Garnet genotype. Furthermore, understanding the proteomic responses of these two contrasting genotypes that showed a marked difference in response to drought stress might help in unlocking complex biological networks of proteins underlying drought stress tolerance. Hence we use two-dimensional (2D) gel electrophoresis coupled with Matrix assisted laser desorption/ionisation-time of flight/time of flight tandem mass spectrometry (MALDI TOFTOF MS) analysis for this part of the study, in order to detect and analyze those differentially expressed proteins or proteins whose abundance levels were influenced as a consequence of drought stress. To gain additional insight into the leaf proteomes of the two canola genotypes, a protamine sulphate precipitation (PSP) method was used to remove RuBisCo and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. A total of 55 well resolved protein spots were selected for mass spectrometry analysis of which 31 (56%) were positively identified using the selective criteria analysis (SCA). All positively identified proteins were then classified into functional categories including protein folding (3%), photosynthetic (29%), detoxification and protection (20%), and energy related proteins whereas 16% could not be classified into any functional category. Apart from spot 32 (Fe superoxide dismutase) and spot 34 (chloroplast beta-carbonic anhydrase), no further significant difference in protein expression/abundance was observed for all the identified proteins for both genotypes in response to drought stress. Both proteins (spots 32 and 34) have been shown to contain antioxidant activity properties which suggest that they might play a crucial role in improving drought stress tolerance in canola plants.

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L’ANS recombinante de Vitis vinifera (VvANS) a été exprimée chez E. coli, et purifiée par chromatographie d’affinité sur colonne Nickel. La production et purification de l'holoenzyme chargée en fer a été mise en point, afin d’éviter les réactions d'oxydation non-enzymatique incontrôlée en présence de sel de Fe(II). Un complexe VvANS-Fe(II) stable est formé en présence d'α- cétoglutarate et d'ascorbate, complexe qui est catalytiquement actif à PO2 ambiante en l'absence de sel de Fe(II). La transformation de la (+)-catéchine par VvANS a été étudiée avec ou sans ascorbate, en utilisant le complexe VvANSFe( II) ou l’holoenzyme co-incubée en présence de sulfate ferreux, afin d’étudier le rôle de l’ascorbate. Aucune activité enzymatique n’a été observée en l'absence d’ascorbate, ce qui indique qu'il s'agit d'un cofacteur indispensable de VvANS. Un adduit covalent ascorbate-cyanidine est produit in vitro, mais seulement en l'absence d'un autre réducteur nucléophile majeur, le glutathion GSH. Les deux stéréoisomères de la leucocyanidine (flavan-diols 3,4-cis et 3,4-trans), substrats potentiels de VvANS, mais non commerciaux, ont été synthétisés par réduction de la dihydroquercétine par NaBH4, puis identifiés par RMN du proton. L'analyse des deux stéréoisomères par spectrométrie de masse en tandem (MS/MS) montre que leurs voies de fragmentation MS/MS sont distinctes et peuvent être utilisées pour les distinguer lors de leur production. Les deux stéréoisomères sont stables en milieu aqueux congelé à -20°C. Douze flavonoïdes de quatre familles distinctes (flavanones, dihydroflavonols, flavan-3-ols et flavan-3,4-diols) ont été testés comme substrats potentiels. Tous les produits enzymatiques ont été purifiés par HPLC en phase inverse, puis identifiés par MS/MS, avec les résultats suivants: 1) Seuls les dihydroflavonols de configuration (2R,3R) sont acceptés comme substrats par VvANS dont l'activité diminue avec le nombre de groupements hydroxyles du cycle B. 2) Seuls les flavan-3-ols ou flavan-3,4-diols de configuration (2R,3S) ayant un catéchol ou trois OH phénoliques vicinaux sur le cycle B sont acceptés comme substrats. 3) La naringénine n'est pas substrat de VvANS, sans doute en raison de l'absence de groupement hydroxyle en C3. […] Le glutathion GSH est un puissant nucléophile, réducteur et piégeur de radicaux libres, qui est abondant dans la baie de raisin. Nous avons donc étudié son effet sur l'activité de VvANS avec tous les substrats identifiés. GSH n’a pas d'effet sur la transformation des dihydroflavonols et des flavan-3,4-diols, mais il modifie considérablement le mode de transformation de la (+)-catéchine et de la (+)-gallocatéchine. En présence de (+)-catéchine et de GSH, on observe deux produits majeurs, la cyanidine et un adduit thioéther cyanidine-glutathion, et le rendement de production est beaucoup plus élevé qu'en l'absence de GSH. De plus, l’adduit covalent ascorbate-cyanidine et le dimère issu de la (+)-catéchine obtenus lors de la réaction réalisée en l'absence de GSH ont disparu. Nos données suggèrent que l'adduit covalent cyanidine-glutathion est un thioéther en C4 qui fait l'objet d'un équilibre de tautomérisation céto-énolique en C3, et se décompose en cyanidine et GSH. En présence de (+)-gallocatéchine, un adduit thioéther similaire delphinidine-glutathion est aussi observé. Pour tester l'éventuelle spécificité de GSH, trois autres mercaptans (thiomalate, cystéine et cystéamine) ont été testés et aucun adduit similaire n’a été observé, ce qui suggère que GSH est un ligand spécifique, et pourrait être un coenzyme de VvANS. Nos résultats suggèrent que les anthocyanidines pourraient être produites in vivo à partir d'un substrat flavan-3-ol (catéchine ou gallocatéchine) via un intermédiaire thioéther de glutathion, alors que le stéréoisomère naturel (3,4-cis) de la leucocyanidine n'est pas transformé en cyanidine. / Recombinant anthocyanidin synthase from Vitis vinifera (VvANS) has been expressed in E. coli, and purified by nickel affinity chromatography. The production and purification of the iron-loaded enzyme has been developed in order to avoid uncontrolled nonenzymatic oxidation reactions in the presence of Fe(II) salt. A stable VvANS-Fe(II) complex is formed in the presence of 2-oxoglutarate and ascorbate, and this complex is catalytically active at ambient PO2 in the absence of Fe(II) salt. The transformation of (+)-catechin by VvANS has been studied with and without ascorbate, by using either the VvANSFe( II) complex or the holoenzyme co-incubated with ferrous sulfate, to investigate the role of ascorbate. No enzyme activity has been observed in the absence of ascorbate, which means that it is an essential enzyme cofactor. A covalent adduct ascorbate-cyanidin is produced in vitro, but only in the absence of glutathione (GSH), another major nucleophilic and reducing agent. The two stereoisomers of leucocyanidin (3,4-cis et 3,4-trans flavan-diols) which were expected to behave as substrates of VvANS, are not commercial and were synthesized by reduction of dihydroquercetin by NaBH4, and characterized by proton NMR. The analysis of the two stereoisomers by means of tandem mass spectrometry (MS/MS) shows that their fragmentation pathways are distinct and may be used to distinguish them during their production. The two stereoisomers are stable in frozen aqueous medium at -20°C. Twelve flavonoids of four distinct families (flavanones, dihydroflavonols, flavan-3-ols et flavan-3,4-diols) were tested as potential substrates of VvANS. All enzymatic products were purified by means of reverse-phase HPLC and characterized by MS/MS, with the following results: 1) Only dihydroflavonols of (2R,3R) configuration are accepted as substrates by VvANS, the activity decreasing with the number of hydroxyl groups of ring B. 2) Only flavan-3-ols or flavan-3,4-diols of (2R,3S) configuration having either a catechol or three vicinal phenolic OH on ring B are accepted as substrates. 3) Naringenin is not substrate of VvANS, most likely because a C3 hydroxyl group is missing. […] Glutathione GSH is a powerful nucleophilic and reducing agent as well as a free radical scavenger, which is abundant in grape berries. We therefore studied its effect on VvANS activity with all identified substrates. GSH has no effect on the transformation of dihydroflavonols and flavan-3,4-diols, but it considerably modifies the transformation pattern of (+)- catechin and (+)-gallocatechin. In the presence of (+)-catechin and GSH, we observe two major products, cyanidin and a cyanidin-glutathione thioether, with production yields which are much higher than in the absence of GSH. Moreover, the ascorbate-cyanidin covalent adduct and the (+)-catechin dimer that had been obtained in the absence of GSH have disappeared. Our data suggest that the cyanidin-glutathione adduct is a C4-thioether which is in equilibrium between the two keto-enolic tautomeric forms at C3, and decomposes into cyanidin and GSH. In the presence of (+)-gallocatechin, a similar delphinidin-glutathione thioether adduct is also observed. In order to test the possible specificity of GSH as a cofactor, three other mercaptans (thiomalate, cysteine and cysteamine) were tested, and no similar product was observed, which suggests that GSH is a specific ligand, and might be a coenzyme of VvANS. Our results suggest that anthocyanidins could be produced in vivo from a flavan-3-ol substrate (catechin or gallocatechin) via a glutathione thioether intermediate, whereas the natural 3,4-cis stereoisomer of leucocyanidin is not transformed into cyanidin by VvANS.

Le fer (Fe) est un micro-élément essentiel pour les plantes. Nous avons récemment montré que l'ascorbate joue un rôle central dans le transport de Fe dans lesgrainesen participant à la réduction de Fe3+ pour l'absorption de Fe2+. En outre, l'activité de l'efflux d’ascorbate à la surface de l'embryon est cruciale dans ce processus. Nous avons utilisé une stratégie de complémentation de levure pour isoler les transporteurs d'efflux d’ascorbate, en exprimant une banqued'ADNc d'Arabidopsis dans le mutant perte de fonction de la réductase ferrique, Δfre1, incapable de se développer en carence en Fe. L’expression de deux ADNc nommésMATEet GALa permis de restaurerla croissance de Δfre1 en catalysant l’efflux de l'ascorbate dans le milieu, permettant de rétablir l'activité de réduction ferrique. Sur la base de ces résultats très prometteurs, nous avons poursuivi l’étude du rôle de ces transporteurs d'efflux d’ascorbate putatifs, MATE et GAL, dans le transport et l'homéostasie du Fe chez Arabidopsis thaliana. Dans cette étude, nous avons identifié la protéine MATE comme un transporteur vacuolaire qui est potentiellement impliqués dans le chargement d’ascorbate vers la vacuole pour réduire le Feintra-vacuolaire. Cette activité de transport est cruciale pour la remobilisation de Fe au cours de la germination et pour répondre à la carence de Fe dans le reste de la plante. La protéine GAL est localisée à la membrane plasmique où elle pourrait catalyser l’efflux d’ascorbate pour remobiliser le pool de Fe apoplastique. En effet, des mutants perte de fonction de GAL sont très sensibles à la carence en fer et perturbés dans la perception du statut nutritionnel en Fe dans des conditions de suffisance en Fe. En conclusion, les deux transporteurs d'ascorbate putatifs identifiés dans cette étude semblent être impliqués dans l'homéostasie du fer en régulant la circulation des pools de Fe subcellulaires. Cette recherche a contribué à découvrir et à mettre en évidence le lien entre le transport de fer et le métabolisme de l’ascorbate. / Iron (Fe) is an essential microelement for plants. We have recently shown that ascorbate plays a central role in Fe transport to seeds by mediating Fe3+ reduction for the uptake of Fe2+. Moreover, the ascorbate efflux activity at the embryo surface was crucial in this process. We have used a yeast complementation strategy to isolate ascorbate efflux transporters, by expressing an Arabidopsis cDNA library in the Δfre1 mutant that lacks ferric reductase activity and is unable to grow in Fe limiting conditions. The expression of two cDNAs named MATE and GAL is able to rescue the growth defect of Δfre1by mediating efflux of ascorbate in the medium, reconstituting a ferric reduction activity. Therefore, we have studied the roles of putative ascorbate efflux transporters, MATE and GAL, in Fe transport and homeostasis in Arabidopsis thaliana. In this study, we have identified the MATE protein as a vacuolar transporter potentially involved in loading ascorbate to the vacuole to reduce intra-vacuolar Fe. This transport activity appears to be crucial to remobilize Fe during germination and to participate in the response to Fe deficiency in the rest of the plant, as revealed by the phenotypical analyses of knock out mutant plants. The GAL protein is localized to the plasma membrane where it could potentially catalyze the efflux of ascorbate toward the apoplast to remobilize apoplastic Fe pools. Indeed, GAL knock out mutants are highly sensitive to Fe deficiency and disturbed in the sensing of Fe nutritional status in Fe-replete conditions. In conclusion, the two putative ascorbate transporters identified in this study appear to be involved inthe iron homeostasis by regulating the movement of subcellular Fe pools. This research has contributed to discover and highlight the link between iron and ascorbate metabolism and transport.

Two aspects of the biological activity of N-nitrosamines were studied. First, the effect of ascorbate on the mutagenicity of N-nitrosopiperidines was studied in the Ames Salmanella/ mammalian microsome mutagenicity test. The addition of ascorbate significantly enhanced the mutagenicity of these compounds. This enhancement was selective for N-nitrosamines suggesting a possible role of ascorbate in N-nitrosamine induced carcinogenicity. Second, the technique of velocity sedimentation in alkaline sucrose density gradients was applied to the detection of N-nitrosamine induced DNA damage in Balb/c 3T3 cells. This technique detected N-nitrosamine induced DNA damage when the cells were made permeable before treatment. This technique compares favorably with other test systems used to evaluate N-nitrosamines and should be useful in further studies of N-nitrosamines.