Thesis (M.S.)--Ohio University, June, 2005. / Includes bibliographical references (p. 73-81)

Thesis (M.S.)--Ohio University, June, 2005. / Title from PDF t.p. Includes bibliographical references (p. 73-81)

Made available in DSpace on 2016-08-12T19:15:37Z (GMT). No. of bitstreams: 1 JoseGOS_DISSERT.pdf: 750943 bytes, checksum: a7db17eb67ceb1187e14a357b382b729 (MD5) Previous issue date: 2016-02-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The introduction of exotic species in natural communities is usually mediated by human activity. One example is Cryptostegia madagascariensis, species considered invasive in almost every country in which it was introduced. In Brazil, it is invading wetlands in the Northeast and is known as the Carnaubeiras (Copernicia prunifera) killer. The species is highly tolerant to flooding, but it also has drought tolerance. It is necessary to understand how adaptation to drought occurs, to identify the capacity of this plant to invade new areas. The objective of this work is to understand the adaptation of C. madagascariensis to low water availability. This study was conducted in the seedling nursery of the Department of Plant Sciences at the Federal Rural University of the Semi-Arid (UFERSA). It was adopted a randomized blocks design in subdivided plots, with four blocks. In the plots, the water treatments were placed (irrigated and stressed). The subplots were the plant sages (4, 6 and 8 months). When the plants showed leaf senescence, irrigation was resumed and leaves were selected for biochemical analyzes of photosynthetic pigments, total soluble sugars and total free amino acids were made. At 5 days of water deprivation, the plants had reduced photosynthesis, stomatal conductance and transpiration. At that point, there was a statistical difference for the water potential at noon, pre-dawn, free amino acids and soluble sugars in plants that were subjected to stress. The behavior exhibited by Cryptostegia madagascariensis indicates that this species has a conservative profile, reducing their leaf area and promoting stomatal closure to conserve water / A introdução de espécies exóticas em comunidades naturais é, geralmente, mediada pela atividade humana. Um exemplo disso é a Cryptostegia madagascariensis, que se tornou invasora em quase todos os países nos quais foi introduzida. No Brasil, está invadindo áreas alagáveis do Nordeste, sendo conhecida como assassina das carnaubeiras (Copernicia prunifera). A espécie é altamente tolerante ao alagamento, mas também tem tolerância à seca. É necessário entender como ocorre a adaptação à seca para identificar a capacidade dessa planta para invadir novas áreas. Assim, o objetivo desse trabalho foi compreender a adaptação de C. madagascariensis a condições de baixa disponibilidade de água. O presente estudo foi conduzido em viveiro de mudas no delineamento em blocos casualizados no esquema de parcelas subdivididas, com quatro blocos. Nas parcelas, foram avaliados os regimes hídricos (irrigadas e estressadas) e as idades (4, 6 e 8 meses). Quando as plantas apresentaram senescência foliar, foi retomada a irrigação e selecionadas as folhas para serem feitas as análises bioquímicas. Foram feitas as análises de pigmentos fotossintéticos, açúcares solúveis totais e aminoácidos livres totais. Com cinco dias de estresse, as plantas apresentaram redução da fotossíntese, condutância estomática e transpiração. Nesse ponto, houve diferença estatística para o potencial hídrico ao meio dia, no pre-dawn, aminoácidos livres e açúcares solúveis nas plantas que foram submetidas ao estresse. O comportamento apresentado pela Cryptostegia madagascariensis indica que a espécie possui um perfil conservador, reduzindo sua área foliar e promovendo o fechamento estomático para conservar água no seu interior

Made available in DSpace on 2016-08-12T19:18:54Z (GMT). No. of bitstreams: 1 JoseGOS_DISSERT.pdf: 750943 bytes, checksum: a7db17eb67ceb1187e14a357b382b729 (MD5) Previous issue date: 2016-02-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The introduction of exotic species in natural communities is usually mediated by human activity. One example is Cryptostegia madagascariensis, species considered invasive in almost every country in which it was introduced. In Brazil, it is invading wetlands in the Northeast and is known as the Carnaubeiras (Copernicia prunifera) killer. The species is highly tolerant to flooding, but it also has drought tolerance. It is necessary to understand how adaptation to drought occurs, to identify the capacity of this plant to invade new areas. The objective of this work is to understand the adaptation of C. madagascariensis to low water availability. This study was conducted in the seedling nursery of the Department of Plant Sciences at the Federal Rural University of the Semi-Arid (UFERSA). It was adopted a randomized blocks design in subdivided plots, with four blocks. In the plots, the water treatments were placed (irrigated and stressed). The subplots were the plant sages (4, 6 and 8 months). When the plants showed leaf senescence, irrigation was resumed and leaves were selected for biochemical analyzes of photosynthetic pigments, total soluble sugars and total free amino acids were made. At 5 days of water deprivation, the plants had reduced photosynthesis, stomatal conductance and transpiration. At that point, there was a statistical difference for the water potential at noon, pre-dawn, free amino acids and soluble sugars in plants that were subjected to stress. The behavior exhibited by Cryptostegia madagascariensis indicates that this species has a conservative profile, reducing their leaf area and promoting stomatal closure to conserve water / A introdução de espécies exóticas em comunidades naturais é, geralmente, mediada pela atividade humana. Um exemplo disso é a Cryptostegia madagascariensis, que se tornou invasora em quase todos os países nos quais foi introduzida. No Brasil, está invadindo áreas alagáveis do Nordeste, sendo conhecida como assassina das carnaubeiras (Copernicia prunifera). A espécie é altamente tolerante ao alagamento, mas também tem tolerância à seca. É necessário entender como ocorre a adaptação à seca para identificar a capacidade dessa planta para invadir novas áreas. Assim, o objetivo desse trabalho foi compreender a adaptação de C. madagascariensis a condições de baixa disponibilidade de água. O presente estudo foi conduzido em viveiro de mudas no delineamento em blocos casualizados no esquema de parcelas subdivididas, com quatro blocos. Nas parcelas, foram avaliados os regimes hídricos (irrigadas e estressadas) e as idades (4, 6 e 8 meses). Quando as plantas apresentaram senescência foliar, foi retomada a irrigação e selecionadas as folhas para serem feitas as análises bioquímicas. Foram feitas as análises de pigmentos fotossintéticos, açúcares solúveis totais e aminoácidos livres totais. Com cinco dias de estresse, as plantas apresentaram redução da fotossíntese, condutância estomática e transpiração. Nesse ponto, houve diferença estatística para o potencial hídrico ao meio dia, no pre-dawn, aminoácidos livres e açúcares solúveis nas plantas que foram submetidas ao estresse. O comportamento apresentado pela Cryptostegia madagascariensis indica que a espécie possui um perfil conservador, reduzindo sua área foliar e promovendo o fechamento estomático para conservar água no seu interior

As part of the study, photosynthetic system constructs based on BF2-chelated dipyrromethene (BODIPY), BF2-chelated azadipyrromethene (AzaBODIPY), porphyrin, phthalocyanine, oxasmaragdyrin, polythiophene, fullerene (C60), single-walled carbon nanotube and graphene are investigated. Antenna systems of BODIPY dyads and oligomers having BODIPY as an excitation energy donor connected to different acceptors including BODIPY, azaBODIPY, oxasmaragdyrin and aluminum porphyrin are studied. Different synthetic methodologies are used to afford donor-acceptor systems either directly linked with no spacer or with short spacers of varying length and orientation. The effect of donor orientation, donor optical gap as well as nature of donor-acceptor coupling on the donor-acceptor spectral overlap and hence the rate of excitation energy transfer is investigated. In all these systems, an ultrafast energy transfer followed by electron transfer is observed. In particular, in a directly connected BODIPY-azaBODIPY dyad an unusually ultrafast energy transfer (~ 150−200 f) via Förster mechanism is observed. The observation of energy transfer via Förster instead of Dexter mechanism in such closely coupled donor-acceptor systems shows the balance between spatial and electronic coupling achieved in the donor-acceptor system. Moreover, in donor-acceptor systems involving semiconducting 1D and 2D materials, covalently functionalized single-walled carbon nanotubes via charge stabilizing (TPA)3ZnP and noncovalently hybridized exfoliated graphene via polythiophene chromophores are studied for their charge transportation functions. In both cases, not only an ultrafast charge transfer in the range of (~ 2−5 p) is observed but also the charge-separated states were long lived implying the potential of these functionalized materials as efficient charge transporting substrates with organic chromophores for photovoltaic and optoelectronic applications where ultrafast intercomponent charge transfer is vital. In addition, as a final part of this dissertation, the mechanisms of electron injection and back electron transfer in heterogeneous systems involving supramolecularly anchored high potential chromophores on TiO2 film are studied by femtosecond transient absorption spectroscopy. In this study, not only are important insights gained on the utilization of supramolecular anchoring of chromophores such as porphyrins, phthalocyanines, and their perflorinated high potential analogues, chromophores currently showing promise as highly efficient sensitizers in dye sensitized solar cells, on TiO2 film but also on the effect of anchor length and sensitizer orientation on the rates of electron injection and back electron transfer at the sensitizer-TiO2 interface.

No description available.

The making and breaking of O-O bonds has implications ranging from artificial photosynthesis and water oxidation to the use of O₂ as a selective, green oxidant for transformations of small molecules. Oxidative generation of O₂ from coupling of two H₂O molecules remains challenging, and well defined systems that catalytically evolve O₂ are exceedingly rare. Recent theoretical studies have invoked metal oxyl radicals (L[subscript n]M=O*) containing a singly occupied M-O π-type orbital as precursors to O-O bond forming events in both biological and synthetic water oxidation catalysts. However, the lack of stable metal oxyl complexes makes it difficult to explore and understand this hypothesis. The activation of dioxygen (breaking of O-O bonds) to produce terminal metal oxos also remains a challenge. There is an inherent kinetic barrier to the spin-forbidden reactions of triplet dioxygen, and features that engender selective O₂ reduction are not easily transferable from system to system. The primary thrust of this thesis work has been to elaborate new methods to generate well-defined metal oxyl radicals for studies of their reactions in radical bond-forming reactions similar to the radical coupling hypothesis of water oxidation. A library of >20 5- and 6-coordinate high-valent oxorhenium complexes containing redox-inert and redox-active ligands has been prepared. The complexes containing redox-active ligands have shown the ability for ligand-mediated radical coupling reactions. Mechanistic studies of bimetallic O₂ homolysis (the microscopic reverse of water oxidation) and nitroxyl radical deoxygenation at five-coordinate oxorhenium(V) reveal that, in both net 2e⁻ reactions, coupling to a redox-active ligand lowers the kinetic barrier to the reaction by facilitating formation and stabilization of 1e⁻ oxidized intermediates. Coordinatively unsaturated high-valent oxorhenium complexes containing redox-active ligands direct bond-forming reactions towards the metal center. This is undesirable towards the goal of forming O-O bonds. To address this problem coordinatively saturated Re(V) and Re(VII) complexes were prepared. Oxidation of these species by chemical oxidants allowed for the isolation of "masked" oxyl species. These complexes showed reactivity towards Si and trityl radicals to produce new Si-O and C-O bonds, whereas their closed-shell congeners were inert. We have successfully developed a method for the preparation and isolation of "masked" oxyl radicals and shown their utility in ligand-mediated radical coupling reactions.

Call number: LD2668 .T4 1984 L43 / Master of Science

Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / Dissertation presented for the degree of Doctor of Philosophy Stellenbosch University. / ENGLISH ABSTRACT: Phosphorus (P) is essential for the survival of all living organisms and forms part of several key biological molecules and processes. The basic biological function of all cells depends on the availability of P as structural element in phospholipids and nucleic acids. P plays a central role in the energy metabolism of the cell by activating metabolic intermediates of carbohydrate metabolism and by acting as an energy currency in the form of adenosine tri-phosphate (ATP). ATP is produced during photosynthesis from the energy derived from sunlight, probably the most important biological process on earth. The balance of P supply and demand is of critical importance here. Plants assimilate P in the form of orthophosphate (Pi) via its roots and utilises complex mechanisms to redistribute and balance the Pi concentrations throughout the plant. These processes are collectively known as phosphate homeostasis and in this study we utilised molecular techniques to study some key aspects of this complex network of mechanisms in the plant Arabidopsis thaliana. When the role of the PHT1;5 Pi transporter was investigated in photosynthesis under Pi limitation a new mechanism utilised by plants to supply Pi for the production of ATP in the chloroplast was discovered. During periods of adequate Pi supply plants make use of the triose phosphate / phosphate translocator (TPT) to exchange Pi for phosphorylated carbon intermediates. This transporter does, however, not function at the low Pi concentrations present during Pi limitation and the plant therefore express an alternative transporter i.e. PHT1;5. Together with this transporter several genes were identified that was expressed to allow the export of carbon intermediates from the chloroplast via starch turnover. Amongst these, several alternative isoforms of the enzymes responsible for starch turnover are expressed during Pi limiting conditions. It is therefore suggested that the products of starch degradation, e.g. glucose and maltose are the potential candidates for carbon export from chloroplasts under Pi limiting conditions. In an attempt to perturb the Pi concentrations in the Arabidopsis vacuole we expressed the three genes of a newly discovered polyphosphate (PolyP) polymerase from the yeast Sacharomyces cerevisiae in Arabidopsis. This enzyme complex accumulates PolyP in the yeast vacuole and since the plant vacuole is playing a key role in buffering Pi concentrations we anticipated some observable effects that could lead to the elucidation of the mechanisms involved. Production of PolyP was conclusively shown in plant callus, but it was only at very low concentrations with no detectable perturbing effect and undetectable in whole plants. With the aim to apply this technology to the PolyP and PHT1;5 lines developed in the other parts of this study, newly developed fluorescent indicator protein nanosensors (FLIPPi) were evaluated as a method for detecting and monitoring in vivo Pi concentrations in multicellular plant organs. This technique is capable of detecting changes in metabolite concentrations in real-time and it was applied to the roots of Arabidopsis seedlings subjected to Pi limitation. We specifically looked at changes in the cytosol, but our results revealed no detectable changes occurring in the Pi concentrations in this compartment. This was interpreted to indicate lower levels of Pi in this compartment as was previously expected. / AFRIKAANSE OPSOMMING: Fosfaat (P) is essensieël vir die oorlewing van alle organismes en maak deel uit van etlike kern biologiese prosesse en molekules. Die basiese biologiese funksionering van alle selle hang direk af van die beskikbaarheid van P as strukturele element van fosfolipiede en nuklëinsure. Fosfaat speel 'n sentrale rol in die energie metabolisme van 'n sel deur metaboliese intermediante te aktiveer en deur op te tree as die geld eenheid van sellulere energie in die vorm van adenosien tri-fosfaat (ATP). ATP word gegenereer gedurende fotosintese vanaf die energie wat van sonlig vasgevang word, dit is waarskeinlik die belangrikste biologiese proses op aarde. Dit is van kritiese belang dat die fosfaat vraag en aanbod hier fyn gebalanseer word. Plante assimileer P in die vorm van ortofosfaat (Pi) deur hulle wortels en maak gebruik van komplekse meganismes om Pi deur die plant te versprei en konsentrasies te balanseer. Hierdie prosesse staan gesamentlik bekend as fosfaat homeostase en in die huidige studie het ons gebruik gemaak van molekulêre tegnieke om 'n paar belangrike aspekte van hierdie komplekse netwerk van prosesse in die plant Arabidopsis thaliana te bestudeer. Toe die rol van die PHT1;5 Pi transporter in fotosintese onder toestande van Pi tekort bestudeer is, is 'n nuwe meganisme ontdek waarmee plante Pi verskaf aan chloroplaste vir die proses van fotosintese onder toestande van Pi tekort. Gedurende periodes wat die plant genoegsame Pi tot sy beskikking het, word van die triose fosfaat / fosfaat uitruiler (TPT) gebruik gemaak om Pi uit te ruil vir gefosforileerde koolstof metaboliete. Hierdie transporter kan egter nie onder die lae Pi konsentrasies wat voorkom in die sitoplasma onder Pi tekort toestande funksioneer nie, en gevolglik moet die plant van 'n alternatiewe transporter naamlik PHT1;5 uitdruk. Verskeie ander gene is ook geidentifiseer wat saam met hierdie transporter onder toestande van Pi tekort uitgedruk word en die plant toelaat om koolstof tussengangers uit die chloroplaste uit te vervoer via die proses van stysel produksie en afbraak. Onderandere is verskeie alternatiewe isoforme van die gene wat verandwoordelik is vir stysel produksie en afbraak identifiseer wat uitgedruk word onder toestande van Pi tekort. In 'n poging om die Pi konsentrasies in die Arabidopsis vakuool te versteur is drie gene van die nuut ontdekte polifosfaat (PolyP) polimerase kompleks van die gis Sacharomyces cerevisiae in Arabidopsis uitgedruk. Hierdie ensiem kompleks is verandwoordelik vir die akkumulasie van PolyP in die gis vakuool en siende die plant vakuool 'n kern rol speel in die buffering van Pi konsentrasies in die plant, het ons sekere waarneembare gevolge verwag wat kon lei tot die ontrafeling van die meganismes hierby betrokke. Die produksie van PolyP in plant kallus is duidelik gedemonstreer, maar dit was slegs teen baie lae konsentrasies met geen waarneembare versteuringseffek nie, en kon glad nie in heel plante waargeneem word nie. Met die oog daarop om hierdie tegnologie toe te pas op die bestudering van die PolyP en PHT1;5 lyne wat in die ander dele van hierdie studie ontwikkel is, is 'n nuut ontwikkelde fluoresente indikator protein nanosensor (FLIPPi) tegnologie evalueer as 'n metode om Pi konsentrasies in vivo in multisellulere plant organe waar te neem en te monitor. Hierdie tegniek is in staat daartoe om veranderinge in Pi konsentrasies in selle direk te monitor en is gevolglik op die wortels van Arabidopsis saailinge onder Pi tekort toestande toegepas. Daar is spesifiek na veranderinge in die sitosol gekyk, maar ons resultate kon geen waarneembare veranderinge in Pi konsentrasies in hierdie kompartement uitwys nie. Hierdie resultaat beteken waarkeinlik dat die Pi konsentrasies in hierdie kompartement waarskeinlik baie laer is as wat voorheen verwag is.

Thesis (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Plants cannot avoid stress and must therefore be capable of rapidly responding to extreme environmental changes. An inability to control and regulate the photosynthetic process during stress conditions will lead to the formation of highly reactive oxygen species that concomitantly causes photo-oxidative damage to the pigments and proteins of the photosynthetic apparatus. Since light is the primary source of energy for the photosynthetic process, it is clear that plants are continuously required to balance the light energy absorbed for the photochemical reactions against photoprotection in a dynamic way in order to survive. Carotenoids are precursors of abscisic acid, but more importantly structural components of the photosynthetic apparatus. During photosynthesis carotenoids function as accessory light-harvesting pigments, and also fulfil a photoprotective function by quenching the reactive molecules formed during conditions that saturate the photosynthetic process. Due to the importance of carotenoids to plant fitness and human health (as Vitamin A precursors) this study has attempted to isolate and characterise genes that are directly, or indirectly involved in carotenoid biosynthesis in Vitis vinifera. In total eleven full-Iength- and eight partial genes have been isolated, cloned and sequenced. These genes can be grouped into the following pathways: (i) the 1- deoxy-D-xylulose 5-phosphate (DOXP)/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (i.e. the plastidic isopentenyl diphosphate biosynthetic pathway); (ii) the mevalonate pathway (i.e. the cytosolic/mitochondrial IPP biosynthetic pathway); (iii) the carotenoid biosynthetic pathway; (iv) the abscisic acid biosynthetic pathway (as a degradation product of carotenoids); and general isoprenoid biosynthetic pathways (as precursors of carotenoids). The full-length genes (i.e. from the putative ATG to the STOP codon) of DOXP synthase (DXS), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (lytB), IPP isomerase (IPI), 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS), phytoene synthase (PSY), Iycopene ~-cyclase (LBCY), ~-carotene hydroxylase (BCH), zeaxanthin epoxidase (lEP), 9-cis-epoxy carotenoid dioxygenase (NCED), farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) have been isolated from cDNA. In addition, the full-length genomic copy and putative promoters of DXS, PSY, LBCY, BCH, NCED and lEP have also been isolated from genomic DNA by the construction and screening of sub-genomic libraries. Alignments of the genomic copies of these genes to the corresponding cDNA sequences have provided useful information regarding the genomic organisation of these genes, including the intron-exon junction sites in V. vinifera. The copy number of the DXS, PSY, LBCY, BCH, NCED and lEP encoding genes in the Vitis genome have been determined. DXS, PSY, BCH and lEP are single copy genes, whereas LBCY and NCED have two and three copies, respectively. The transcriptional activity of the putative promoters of six of the isolated genes (i.e. DXS, PSY, LBCY, BCH, lEP and NCED) were tested with a transient reporter gene assay. None of the putative promoters tested showed any transcriptional activity of the reporter gene. The transcription of these genes, has however been shown using northern blot analysis and/or RT-PCR. Preliminary expression profiles for PSY, LBCY, BCH, and lEP were determined in different plant organs and the expression of these genes was generally higher in photosynthetically active tissues. The expression of these genes following different treatments (abscisic acid, NaCI and wounding) was also assayed. The functionality of five of the isolated full-length genes (IPI, GGPS, PSY, LBCY and BCH) has been shown in a bacterial colour complementation assay. In silica analysis of the predicted protein sequences of all eleven isolated genes revealed that they are conserved and share a high degree of homology to the corresponding proteins in other plant species. The sequences were further analysed for conserved domains in the protein sequences, and these proteins typically demonstrated similar domain profiles to homologues in other species (plant, bacteria and algae). The predicted protein sequences were further analysed for transit peptides, the presence of which would provide evidence for the sub-cellular localisation of the mature peptides. Since these genes are involved in biosynthetic pathways that are active in discrete organelles, the sub-cellular localisation of most of these proteins is known. The carotenoid biosynthetic genes (PSY, LBCY, BCH and ZEP), the abscisic acid biosynthetic gene, NCED, as well as the DOXP/MEP pathway genes (DXS, lytB and IPI) were all localised to the chloroplast. The mevalonate pathway gene, HMGS, was localised to both the cytosol and the mitochondria, and the general isoprenoid precursor genes, FPS and GGPS, were localised to the cytosol and the chloroplast, respectively. All these results are in agreement with the localisation of the respective pathways. In order to increase our understanding of carotenoid biosynthesis and functions in plants, we constitutively overexpressed one of the isolated genes (BCH) in the model plant, Nicotiana tabacum. Plants expressing the BCH gene in the sense orientation maintained a healthy photosynthetic rate under stress conditions that typically caused photoinhibition and photodamage in the untransformed control plants. This result was inferred using chlorophyll fluorescence and confirmed using CO2 assimilation rates and stomatal conductance. Chlorophyll fluorescence measurements indicated that the photo protective non-photochemical quenching ability of the BCH-expressing plants increased, enabling the plants to maintain photosynthesis under conditions that elicited a stress response in the untransformed control plants. An integral photosynthetic protein component, the D1 protein, was specifically protected by the additional zeaxanthin in the BCH sense plants. Plants expressing an antisense BCH proved the converse, i.e. lower levels of BCH resulted in decreased zeaxanthin levels and made the transgenic plants more susceptible to high-light induced stress. These results have shown the crucial role of carotenoids (specifically the xanthophylls) in the photoprotective mechanism in plants. The increased photoprotection provided by the BCH expressing plants suggests that the scenario in plants is not optimal and can be improved. Any improvement in the photoprotective ability of a plant will affect both the fitness and productivity of the plant as a whole and will therefore find application in a number of crop plants on a global scale. This study has resulted in the successful isolation and characterisation of genes involved in the direct, or indirect, carotenoid biosynthetic pathways. The further study and manipulation of these genes in model plants will provide useful insights into the physiological role of specific carotenoids in photosynthesis and in plants as a whole. / AFRIKAANSE OPSOMMING: Plante het nie die vermoë om stres te ontwyk nie en moet dus vinnig op veranderinge in hulomgewingstoestande kan reageer. Indien hulle nie die fotosinteseproses kan kontroleer en reguleer tydens streskondisies nie, sal dit tot die vorming van hoogs reaktiewe suurstofspesies lei, wat beide die pigmente en proteiene van die fotosintetiese apparaat sal beskadig. Lig is die primêre energiebron vir fotosintese en daarom is dit noodsaaklik dat plante deurgaans 'n dinamiese balans tussen fotosintese en fotobeskerming moet handhaaf. Karotenoiëde is voorlopers vir die vorming van absisiensuur, maar meer belangrik vir die plant, ook integrale komponente van die fotosintetiese apparaat. Tydens fotosintese word karotenoiëde vir die opneem van lig benodig, terwyl dit ook die fotosintetiese apparaat beskerm wanneer lig 'n versadigingspunt bereik vir fotosintese. Weens die belang van karotenoiëde vir plant- en menslike gesondheid (as Vitamiene A voorlopers), het hierdie studie beoog om gene te isoleer en karakteriseer wat direk of indirek 'n rol in karoteenbiosintese in Vitis vinifera speel. Elf vollengte- en agt gedeeltelike gene is geïsoleer, gekloneer, en gekarakteriseer. Hierdie gene kan in die volgende biosintetiese paaie gegroepeer word: (i) die 1- deoksi-D-xilulose 5-fosfaat (DOXP)/2-C-metiel-D-eritritol-4-fosfaat (MEP) pad (d.w.s. die plastiediese isopenteniel difosfaat biosintetiese pad); (ii) die mevalonaat pad (d.w.s. the sitosoliese/mitokondriale IPP biosintetiese pad); (iii) die karotenoiëd biosintetiese pad; (iv) die absisiensuur biosintetiese pad (as 'n afbraak produk van karotenoiëde) en die algemene isoprenoïed bisintetiese paaie (as voorlopers van karotenoiëde ). Die vollengte gene (d.w.s. vanaf die geskatte ATG tot die STOP kodon) van DOXP-sintase (DXS), 4-hidroksi-3-metielbut-2-eniel difosfaatreduktase (lytB), IPPisomerase (IPI), 3-hidroksi-3-metielglutariel koensiem A sintase (HMGS), fitoeën sintase (PSY), likopeen p-siklase (LBCY), p-karoteen hidroksilase (BCH), zeaxantien oksidase (ZEP), 9-cis-epoksi karotenoiëd dioksigenase (NCED), farnesiel difosfaat sintase (FPS)en geranielgeraniel difosfaat sintase (GGPS) is met behulp van. RTPKR vanaf eDNA geïsoleer. Die vollengte genomiese kopieë en die verwagte promotors van die DXS, PSY, LBCY, BCH, NCED and ZEP gene is ook geïsoleer d.m.v. die opstel en sifting van subgenomiese biblioteke. Vergelykende analises van die genoom- en eDNA kopieë het insiggewende data oor die genomiese rangskikking van die gene, insluitende die intron-ekson setels in V. vinifera gelewer. Die kopiegetalle van DXS, PSY, LBCY, BCH, NCED en ZEP is bepaal. DXS, PSY, BCH en ZEP is in die Vitis-genoom as enkel kopieë teenwoordig, terwyl LBCYen NCED twee en drie kopieë, repektiewelik, beslaan. Die transkipsionele aktiwiteit van die verwagte promotors van ses van die geïsoleerde gene (naamlik DXS, PSY, LBCY, BCH, ZEP en NCED) is d.m.v. 'n tydelike verklikkergeentoets ondersoek. Geeneen van die promotors het die transkripsie van die verklikkergeen bemiddel nie. Die transkripsie van die gene is egter wel bewys deur van northernhibridisasies en/of RT-PKR gebruik te maak. Die promotors van hierdie gene kan dus as transkipsioneel aktief beskou word. Voorlopige uitdrukkingsprofiele van PSY, LBCY, BCH, en ZEP is in verskillende plantorgane bepaal; die profiele was deurgaans hoër in fotosinteties aktiewe weefsels. Die uitdrukkingsprofiele van die gene is verder ook in reaksie op verskillende induktiewe behandelings (absisiensuur, NaCI en beskadiging) bepaal. Vyf van die vollengte gene (IPI, GGPS, PSY, LBCYen BCH) is funksioneel bewys in 'n bakteriese funksionele kleurkomplementasiesisteem. In silico analises van die afgeleide proteïene van al elf geïsoleerde gene het 'n hoë vlak van homologie met ooreenstemende proteiene van ander plantspesies getoon. Gekonserveerde domeine is ook in die proteïensekwense van die geïsoleerde gene teenwoordig. Hierdie proteïene het deurgaans dieselfde domeinprofiele vertoontoon as homoloë in ander spesies (bakterieë, alge en plante). Die sub-sellulêre teikening van die gene kon voorspel word deur die seinpeptiede in die proteiensekwense te eien. Aangesien hierdie gene betrokke is by biosintetiese paaie wat in diskrete kompartemente plaasvind; is die sub-selluiêre lokalisering van hierdie proteïene voorspelbaar. Die karotenoïed biosintetiese gene (PSY, LBCY, BCH en ZEP), die absisiensuur biosintetiese geen, NCED, sowel as die DOXP/MEP pad se gene (DXS, lytB en IPI) kom almal in die chloroplast voor. Die mevalonaatpadgeen, HMGS, word na beide die sitosol en die mitokondria geteiken, terwyl die algemene isoprenoïed voorlopergene, FPS en GGPS, onderskeidelik na die sitosol en die chloroplast geteiken word. Die verkreë voorspellings stem met die lokalisering van die biosintetiese paaie in die selooreen. Om ons kennis rakende karotenoïed biosintese en veral hulle funksie(s) in plante te verbreed, het ons een van die geïsoleerde gene, BCH, in die model plant, Nicotiana tabacum, konstitutief ooruitgedruk. Plante wat die BCH geen in die "sense" orientasie uitgedruk het, kon normale fotosintetiese aktiwiteit handhaaf onder kondisies wat foto-inhibisie en foto-osidatiewe skade in die ongetransformeerde kontrole plante veroorsaak het. Hierdie resultaat is met chlorofil fluoresensie analises aangetoon terwyl dit met CO2 assimilasie- en huidmondjie geleidingseksperimente bevestig is. Chlorofil fluoresensie metings het aangetoon dat die beskermingsvermoë van die transgeniese plante verhoog is, en dit dan die plante in staat stelom fotosintetese te handhaaf onder streskondisies van hoë lig. Proteïen analises het aangetoon dat 'n integrale fotosintetiese proteien, die 01 proteïen, word veral deur die verhoogde zeaxantien vlakke in die BCH transgeniese plante beskerm. Plante wat verminderde zeaxantien vlakke gehad het, weens die konstitutiewe ooruitdrukking van die BCH geen in die anti-"sense" orientasie, het die teenoorgestelde bewys. Met ander woorde. laer BCH vlakke (en dus laer zeaxantien vlakke) het tot plante wat meer vatbaar was vir hoë lig geïnduseerde stress gelei. Hierdie resultate het die essensiële beskermende rol wat karotenoiede tydens fotosintese speel, uitgelig. Die vermoë om hierdie beskermende meganisme te manipuleer in transgenies plante het aangetoon dat die sisteem in plante, alhoewel effektief, nie optimaal is nie. Enige verbetering in 'n plant se inherente vermoë om streskondisies te weerstaan sal die plant se algemene gesondheid en dus produktiwiteit beïnvloed. As sulks sal hierdie in meeste gewasspesies toepassing vind. Hierdie studie beskryf die isolering en karakterisering van gene wat direk, of indirek, by karotenoïedbiosintese betrokke is. Verdere studies, en veral die manipulering van hierdie gene in model plante, sal die fisiologiese rol van spesifieke karotenoïeede in fotosintese, en die plant as 'n geheel, ontrafel.