Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves 116-131). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 126-136). Also available on the Internet.

Thesis (doctoral)--Swedish University of Agricultural Sciences, 2000. / Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2008. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 126-136). Also available on the Internet.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Biopolymers and bio-degradable polymers are of utmost importance to ensure a sustainable economy. Industry depends on raw material, which in many cases are derived from fossil fuels, but in light of looming energy crises and green revolutions attention is being directed at cellulose and starch biopolymers. This study was therefore set forth to investigate novel genetic key elements of cell wall metabolism in Arabidopsis thaliana and starch synthesis in an under-utilized southern African crop plant, Tylosema esculentum. In the first section of the study a cDNA library of good quality was constructed from regenerating A. thaliana protoplasts as it was expected to be enriching for genes involved in cell wall biosynthesis. Small scale EST sequencing of the library confirmed that a few sequences were similar to genes identified to be highly expressed during protoplast regeneration. The library was to be screened by expression in a microalgae as it is anticipated that cell wall metabolising genes would change the wall structure and visibly alter the colony morphology. An attempt was made at establishing a high-throughput transformation system in the unicellular algae Chlorella protothecoides in which the library was proposed to be screened. Conventional microalgal transformation techniques do not appear to be effective in this strain as the study produced no transgenic algae. Alternative studies into a screening system within another species could still lead to the identification of cell wall biosynthetic genes, which was the first objective in the study. The second objective in the study was to investigate the potential of the orphan crop T. esculentum as starch-producing cash-crop in developing southern African countries. In this section of the study a cDNA library of good quality was produced form the tuber of T. esculentum. The library was transferred to an expression vector and screened functionally in E. coli for the presence of sequences with starch synthase activity. No sequences have been identified yet and screening procedures are still on-going. The starch content in the tuber has also been determined for the first time. The relatively high starch content in combination with low agricultural inputs indicate the potential of the plant as an industrial starch source. Further investigations into the nature of the starch are proposed to identify prospective buyers within the industry. / AFRIKAANSE OPSOMMING: Biopolimere en bio-afbreekbare polimere is van kardinale belang om ‘n volhoubare ekonomie te ontwikkel. Industriële toepassings maak op die oomblik hoofsaaklik staat op fossielbrandstof verwante bronne, maar met die oog op ‘n groen revolusie en energie krissise wat dreig word meer belangstelling getoon in sellulose en stysel biopolimere. Hierdie studie is daarom onderneem om genetiese elemente te identifiseer wat betrokke is by die sintese van die selwand in Arabidopsis thaliana en stysel sintese in die suider Afrikaanse gewas Tylosema esculentum wat grotendeels onderbenut is. In die eerste deel van die studie is ‘n cDNA biblioteek, van goeie kwaliteit, geskep vanuit A. thaliana protoplaste wat besig was om hulle selwande te herbou. Dit word verwag dat die protoplaste gedurende die tydperk aktief besig sal wees om gene uit te druk wat betrokke is by selwandsintese. DNA volgordebepaling het bevestig dat ‘n klein aantal volgordes ooreengestem het met gene wat voorheen gevind was om in ‘n oormaat uitgedruk te word tydens die herbou van protoplas-selwande. Daar was beoog om die biblioteek in ‘n mikroalge uit te druk en sodoende die morfologie op kolonievlak waar te neem vir verandering wat in die selwand meegebring is. Om hierdie rede was die doel om ‘n hoë opbrengs transformasie sisteem te ontwikkel in die mikroalge Chlorella protothecoides. Algemene mikroalge transformasie tegnieke blyk om nie effektief in die spesie te wees nie aangesien geen transgeniese alge waargeneem is nie. Die ontwikkeling van ‘n soortgelyke proses in ‘n ander spesie kan steeds lei na die ontdekking van gene betrokke by selwandsintese in A. thaliana wat die eerste uitkoms van die projek as geheel was. Die tweede uitkoms van die projek was om te ondersoek wat die waarskynlikheid was om T. esculentum te kommersialiseer as ‘n stysel gewas en sodoende ‘n inkomste te skep vir arm boere in ontwikkelende lande in suider Afrika. In hierdie gedeelte van die projek was daar ‘n goeie cDNA biblioteek geskep uit die knol van T. esculentum. Die biblioteek is oorgedra na ‘n plasmied waarop dit aktief uitgedruk kon word in Escherischia coli G6MD2 en daar is gesoek na volgordes wat lei na die sintese van stysel in hierdie bakterieë. Tot op hede is geen sulke volgordes gevind nie, maar die ondersoek gaan steeds voort. Die styselinhoud van die knol is ook vir die eerste keer bepaal in hierdie ondersoek. ‘n Styselinhoud wat relatief hoog is en die lae moeite wat geverg word om die gewas te verbou toon dat die plant potensieel het as ‘n kommersiële bron van stysel. Verdere ondersoeke in die aard van die stysel word ook voorgestel om toekomstige industriële kopers te identifiseer.

Patógenos necrotróficos matam as células por meio de suas agressivas moléculas toxicas e enzimas líticas não específicas. Jasmonato (JA) é um importante hormônio produzido, para entre outras funções, regular positivamente genes envolvidos na defesa a necrotróficos. Um passo importante nesta rota é a produção do ácido linolênico, um ácido graxo de (18:3) que é dessaturado de (18:2) pelas enzimas FAD3, FAD7 e FAD8. Uma ferramenta muito utilizada hoje é o silenciamento gênico induzido por vírus (VIGS) que se utiliza de um mecanismo natural de defesa das plantas a vírus, para a descoberta da função gênica. O objetivo deste trabalho foi avaliar a interação entre A. thaliana e isolados do gênero Pythium sp., com os genes FAD3, FAD7 e FAD8 silenciados. Para alcançar tal objetivo um fragmento de 325 pb do gene FAD7 de A. thaliana foi isolado e clonado no vetor viral TRV2b. As plantas foram agro-infiltradas com o vetor TRV1 e TRV2b-GFP (controle) ou TRV2b-FAD7 (silenciada), após foi realizada a inoculação dos isolados de Pythium sp. e atribuídas notas de severidade, referente ao fenótipo e medido o comprimento das raízes. A agro-infiltração do vetor viral TRV2b-FAD7 ocasionou uma redução significativa na quantidade de mRNA dos genes FAD3 e FAD7, não interferindo na quantidade de mRNA do FAD8. Esta redução na expressão interferiu negativamente na resistência de A. thaliana a P. deliense, causando sintomas mais severos na parte aérea, sintomas que estão correlacionados significativamente com o comprimento de raiz. O silenciamento destes genes ocasionou a redução na expressão da defensina PDF1.2, induzida na infecção por patógenos necrotróficos. A resistência a P. deliense foi restituída pela adição de metil-jasmonato, pois a aplicação exógena deste, ocasionou a indução de PDF1.2, evidenciando a importância da produção de JA na indução desta defensina e na resistência a P. deliense. / Necrotrophic pathogens kill plant cells through its aggressive and nonspecific lytic enzymes and toxic molecules. Jasmonate (JA) is an important hormone that positively regulates genes involved in necrotrophic defense, among other functions. An important step in this pathway is the production of linolenic acid, a fatty acid (18:3) that is desaturated to (18:2) by FAD3, FAD7, and FAD8 enzymes. Virus induced gene silencing (VIGS) is a widely used tool today, which takes advantage of a natural defense mechanism of plants against viruses, for the discovery of gene function. The aim of this study was to evaluate the interaction between A. thaliana and isolates of the genus Pythium sp., after gene silencing of FAD3, FAD7, and FAD8. For this purpose, a 325 bp fragment of FAD7 from A. thaliana was isolated and cloned into a viral vector TRV2b. Plants were agro-infiltrated with the vector and TRV1 TRV2b-GFP (control) or TRV2b-FAD7 (silenced). Then, plants were inoculated with Pythium sp. isolates and disease severity was evaluated, based on phenotype and measures of root length. Agroinfiltration of TRV2b-FAD7 caused a significant reduction in the amount of mRNA from FAD3 and FAD7 but did not interfere in the amount of mRNA from FAD8. This down regulation had a negative influence on the resistance of A. thaliana to P. deliense, causing more severe symptoms in the shoot, symptoms that are significantly correlated with root length. Gene silencing led to a reduction in the expression of PDF1.2 defensin, which is induced in infections caused by necrotrophic pathogens. Resistance to P. deliense was restored by adding exogenous methyl jasmonate, which induced PDF1.2 expression. These results indicate the importance of JA in the induction of this defensin leading to resistance against P. deliense in A. thaliana.

Bioinformatics is a valuable tool to understand gene regulatory networks. Cis-regulatory elements (CREs) previously found in promoter regions are known to recruit transcription in signaling pathways. In this work it has been hypothesized to consider CREs as a family of related words that interact/bind to a family of related transcription factors, and thus have similar but distinct regulation patterns. A 1460 microarray gene expression collection was obtained via online databases to create a transcriptomic meta-dataset. A novel bioinformatic algorithm was applied to annotate all 65536 (64k) potential 8-letter CREs in the 500 bp upstream promoter region of all A. thaliana genes across the transcriptomic meta-dataset. Of the possible words, only 2,498 were significantly associated with a pattern of regulation in any of the 1,460 microarrays tested whereas the remaining motifs appeared not to be regulatory. Unique CREs were categorized into 4 regulatory types: inducer, suppressor, biregulator and insulator. A predicted protein protein interactome was created for an economically important plant Coffea canephora. Here, it has been hypothesized that evolutionary conservation of many core biological processes enable generation of predicted protein interactome for species with few resources other than sequenced genome. Of over 12,000 genes identified, 939 were predicted to have 4,587 interactions. Gene Ontology analysis revealed enrichment of processes conserved in all eukaryotes but depletion in unique plant processes. A third study was conducted to determine if homology modeling, evolutionary analysis, and structural evolution could determine key factors involved in function, and interaction specificity in Pus10 (EC 5.4.99.25) found in Archaea and Eukaryotes. Redundancy of Pus10 and the bacterial TrmA and TruB orthologs appear to have resulted in significant molecular evolution of Pus10 function. Neofunctionalization was identified in animal kingdom where thiouridine synthase, methylases and PSUSs (THUMP)-domain modification in early animal evolution coincides with appearance of TNF-related apoptosis-inducing ligand (TRAIL) apoptosis components. Subfunctionalization was identified for Thermococcales lineage of Archaea where a shorter forefinger-loop coincides with the loss of Ψ54 specificity as experimentally verified in P. furiosus. Absence of Pus10 was observed in Sulfolobus and higher fungi whereas in plant kingdom Pus10 function remains unknown with possible pseudogene in some lineages

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2017-06-08T16:49:11Z No. of bitstreams: 1 texto completo.pdf: 2323388 bytes, checksum: 0ed6ee0ac91d4f24d7fabdf607ee71c4 (MD5) / Made available in DSpace on 2017-06-08T16:49:11Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2323388 bytes, checksum: 0ed6ee0ac91d4f24d7fabdf607ee71c4 (MD5) Previous issue date: 2016-03-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O flúor (F), elemento mais eletronegativo da tabela periódica, é liberado para atmosfera por diversas atividades industriais, sendo facilmente absorvido pelas plantas através das folhas. A sua fácil absorção e alta reatividade com biomoléculas torna o F um dos poluentes atmosféricos mais fitotóxicos, pois gera danos oxidativos e altera o desenvolvimento celular. Embora existam muitas informações sobre os efeitos do F na fisiologia e estrutura das plantas, ainda é necessário compreender melhor os mecanismos de tolerância desenvolvidas pelas plantas em resposta ao estresse, e isso se torna mais fácil em uma espécie modelo. Além disso, conhecer os efeitos do F sobre uma espécie modelo de ensaios moleculares é extremamente importante para que análises posteriores de transcriptoma, proteoma e metaboloma sejam realizadas afim de elucidar os mecanismos de ação do poluente. Arabidopsis thaliana é uma planta modelo muito utilizada em trabalhos moleculares e ecotoxicológicos e, apesar das inúmeras vantagens de se trabalhar com essa espécie, são raros os estudos sobre os efeitos do F na mesma. Desta forma, objetivou-se compreender os efeitos do F em A. thaliana, e os mecanismos de defesa dessa espécie envolvidos no combate à fitotoxidez causada por este elemento. Para isso, plantas de A. thaliana foram submetidas à nevoeiros com 0, 20, 40 e 80 mg F L-1 por dez dias consecutivos, aplicando-se 15 mL de solução duas vezes ao dia. Ao término do experimento foram feitas coletas para a determinação do teor de F, e para avaliações das alterações morfoanatômicas, micromorfológicas, e fisiológicas. A. thaliana acumulou altíssimas concentrações de F nas folhas, mesmo nas plantas expostas a 20 mg F L-1, as quais não desenvolveram sintomas visuais de fitotoxidez, o que a caracteriza como espécie hiperacumuladora e tolerante. Nos tratamentos com 40 e 80 mg F L-1, necroses foliares iniciaram com 96 e 48 h, respectivamente, após a primeira aplicação dos nevoeiros, ocorrendo como pequenas manchas acinzentadas distribuídos pela lâmina foliar, incluindo margem e ápices. Foi observada redução na produção de biomassa e murcha foliar em função do aumento de F nos nevoeiros. Os danos na superfície das folhas foram caracterizados pelo aspecto plasmolisado das células, erosão das ceras epicuticulares, deformações das cristas estomáticas e da base dos tricomas, além da ruptura da parede periclinal externa das células epidérmicas. Estruturalmente, foram observadas deformações nas nervuras medianas das folhas, redução do tamanho dos feixes vasculares, e estreitamento da lâmina foliar nas regiões onde as células do parênquima lacunoso colapsaram. Entretanto, nos locais com ocorrência de hipertrofia celular e aumento do número de células no mesofilo houve um aumento da espessura. A face abaxial da folha foi mais afetada pelo F do que a adaxial, sendo observadas reentrâncias, redução do tamanho das células e ruptura da epiderme nessa face, enquanto que a epiderme da face adaxial se manteve intacta. Foi verificado aumento significativo na área dos elementos de vasos na nervura mediana em folhas expostas ao poluente mas sem injúria aparente. O F aumentou os níveis de aldeído malônico, indicando a ocorrência de estresse oxidativo, o qual contribuiu para a redução da assimilação interna de carbono (A), pigmentos fotossintetizantes (por degradação) e respiração (Rd). Além disso, o F elevou a concentração interna de carbono (Ci) e redução da condutância estomática (gs), ocasionada pela obliteração dos ostíolos por fragmentos de ceras epicuticulares, e perda da turgidez pelas células-guarda. Esses resultados sugerem aparentes limitações bioquímicas à fotossíntese, com possíveis reduções da atividade de enzimas do ciclo de Calvin, tais como a RuBisCO. A. thaliana demonstrou elevada tolerância ao F, já que a menor concentração utilizada neste trabalho (correspondente ao dobro da detectada em regiões poluídas) não promoveu danos severos e ainda resultou no elevado acúmulo do poluente nos tecidos. A. thaliana é uma espécie promissora em estudos sobre os mecanismos moleculares de combate ao estresse oxidativo causado pelo F. / The fluorine (F), most electronegative element of the periodic table, is released to atmosphere from various industrial activities, being easily absorbed by the plants through the leaves. Its easy absorption and high reactivity with biomolecules makes the F one of the most phytotoxic air pollutants because it generates oxidative damage and changes the cellular development. Although there are a lot of information about the effects of F in physiology and structure of plants, it is still necessary to better understand the mechanisms of tolerance developed by plants in response to stress, and it becomes easier in a kind model. Also, knowing the effects of F on a kind model of molecular assays is extremely important for further analysis of transcriptome, proteome and metabolome are carried out in order to elucidate the mechanisms of action pollutant. Arabidopsis thaliana is a model plant widely used in molecular and ecotoxicological studies and, despite the many advantages of working with this kind are few studies on the effects of F in it. Thus, this study aimed to understand the effects of F in A. thaliana, and the defense mechanisms of this kind involved in combating toxicity symptoms caused by this element. Thus, plants of A. thaliana were submitted to fog with 0, 20, 40 and 80 mg F L-1 for ten consecutive days, applying 15 ml of solution twice daily. At the end of experiment were made collections for determining the F content, and evaluations of morphoanatomic, Micromorphological, and physiological. A. thaliana accumulated extremely high fluoride concentrations in leaves, even in plants exposed to 20 mg F L-1, which did not develop visible symptoms of phytotoxicity, which characterized as tolerant hyperaccumulator species. In the treatments with 40 and 80 mg F L-1, foliar necrosis started with 96 and 48 h, respectively, after the first application of fogs, occurring as small gray spots distributed by leaf blade, including margin and apexes. There was a reduction in biomass production and leaf wilt due to the increase of F in the fogs. Damage to the leaf surface were characterized by the appearance of cells plasmolisado, erosion of the waxes, deformations of stomatal crests and the base of trichomes, beyond the rupture of the outer wall paraclinal of epidermal cells. Structurally, the median veins of the leaves deformations were observed, reducing the size of vascular bundles, and narrowing of the leaf blade in the regions where the cells of the spongy parenchyma collapsed. However, in areas with occurrence of cellular hypertrophy and an increase in the number of mesophyll cells was increased thickness. The abaxial side of the leaf was more affected by the adaxial F, recesses being observed, reduction of cell size and epidermis break this surface, while the adaxial epidermal remained intact. significant increase was seen in the area of vessel elements in the midrib in leaves exposed to the pollutant but no apparent injury. F increased malondialdehyde levels, indicating the occurrence of oxidative stress, which contributed to the reduction of carbon internal assimilation (A), photosynthetic pigments (degradation) and respiration (Rd). Furthermore, the F increased internal concentration of carbon (C) and reduced stomatal conductance (gs) caused by obstruction of ostia fragments by epicuticular waxes, and loss of turgor the guard cells. These results suggest apparent biochemical limitations to photosynthesis, possible reduction of the enzyme activity of the Calvin cycle, such as RuBisCO. A. thaliana showed high tolerance to F, since the lower concentrations used in this study (corresponding to double the detected in polluted areas) did not cause severe damage and even resulted in increased accumulation of pollutant tissue. A. thaliana is a promising species in studies of the molecular mechanisms of combating oxidative stress caused by F.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2017-08-14T17:47:20Z No. of bitstreams: 1 texto completo.pdf: 967714 bytes, checksum: c92f6485b9f379f10f5616747d0f04f5 (MD5) / Made available in DSpace on 2017-08-14T17:47:20Z (GMT). No. of bitstreams: 1 texto completo.pdf: 967714 bytes, checksum: c92f6485b9f379f10f5616747d0f04f5 (MD5) Previous issue date: 2016-07-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A oxidação de carboidratos na mitocôndria é a principal fonte de energia para metabolismo celular. Contudo, em condições de limitação energética, substratos alternativos são necessários para a manutenção da respiração. A oxidação de aminoácidos tem papel fundamental nesse processo gerando elétrons que podem ser transferidos para cadeia de transporte de elétrons mitocondrial através do sistema flavoproteína de transferência de elétrons/ flavoproteína de transferência de elétrons oxidoredutase da ubiquinona (ETF/ETFQO). A associação entre autofagia e o fornecimento de substratos alternativos para geração de energia tem sido relatada, porém pouco se sabe acerca do papel da autofagia no metabolismo primário para a manutenção do processo respiratório. Com intuito de se investigar a importância metabólica da autofagia durante o desenvolvimento e em condições de senescência induzida pela escuro, plantas mutantes de Arabidopsis thaliana com comprometimento do processo autofágico foram utilizadas. Mutantes atg apresentaram redução no crescimento e na produção de sementes. Sob escuro prolongado, fenótipos de senescência antecipada assim como redução no conteúdo de clorofila e na eficiência fotoquímica máxima do FSII (Fv/Fm) foram observados nos mutantes atg. A análise do perfil metabólico revelou uma extensa reprogramação metabólica em que o aumento do conteúdo de aminoácidos foi parcialmente comprometido, limitando seu uso como substrato para suprir a respiração nos mutantes atg. Adicionalmente, níveis de transcritos de genes envolvidos em vias de catabolismo de aminoácidos e degradação do cloroplasto (CV) foram induzidos nesses genótipos. Em conjunto, os resultados obtidos demonstram uma potencial função compensatória de CV como processo de degradação de proteínas em condições de limitação de carbono, particularmente quando o processo autofágico é comprometido. / The oxidation of carbohydrate in mitochondria is the primary energy source for cellular metabolism. However, during energy-limited conditions alternative substrates are required to support respiration. The oxidation of amino acids plays a key role in this process by generating electrons that can be transferred to mitochondrial electron transport chain via the electron transfer flavoprotein/ ubiquinone oxireductase (ETF/ETFQO) system. Compelling evidence has demonstrated the close association of autophagy in providing alternative substrates for power generation under carbohydrate-limited conditions; however, how and to which extent autophagy and primary metabolism interact to support respiration remains unclear. To obtain a comprehensive picture of the metabolic importance of autophagy during development and extended darkness Arabidopsis thaliana mutants with impairments in autophagy were used. atg mutants showed reduction of growth and seed production. Following extended darkness atg mutants were characterized by early signs of senescence as well as decreased chlorophyll content and maximum photochemical efficiency of PSII (Fv/Fm). Metabolite profile of dark-treated leaves revealed an extensive metabolic reprogramming in which increases in amino acids contents were partially compromised and thus limiting their utilization as substrate to sustain respiration in atg mutants. Additionally, transcript levels of genes involved in alternative pathways of respiration, amino acid catabolism, and chloroplast vesiculation (CV) were up-regulated in atg mutants. Our results thus suggest that autophagy contributes to energy availability by supplying amino acids for alternative pathways of respiration. Furthermore, our finding demonstrated the potential role of CV as a compensatory protein degradation pathway under C-limiting conditions when autophagy is impaired .