The ratio of auxin to cytokinin determines de novo organogenesis in plants. Relatively little is known about the effect of genetically altered auxin signaling on in vitro organogenesis. Here, callusogenesis, shoot, and root formation were studied in loss- (LOF) and gain-of-function (GOF) alleles in two phylogenetically related Auxin Response Factors (ARFs), MONOPTEROS (MP/ARF5) and NON-PHOTOTROPHIC HYPOCOTYL 4 (NPH4/ARF7). Reduced MP activity greatly diminished shoot regeneration, and partially diminished callusogenesis and root formation. LOF in NPH4 strongly decreased callusogenesis, and mildly decreased shoot and root regeneration in particular categories of explants. By contrast, organogenesis responses were strongly increased in aerial explants carrying the GOF transgene dMP. Thus, both MP and NPH4 seem to act as positive regulators of certain organogenesis processes and the GOF dMP transgene may be of interest for stimulating organogenesis in plant species with poor regeneration properties. Also, organogenesis in vitro may reveal unknown developmental ARF functions.

The length of the primary root and the density of lateral roots determine the architecture of the root. In this thesis the effect of NAA, ABA and the NO donor SNP alone as well as the combination of ABA or NAA with SNP on lateral root development was investigated. The interaction between CPTIO, a NO scavenger, and NAA or SNP is also reported. Following preliminary experiments in which it was observed that the aerial part of the seedling influenced LR growth and that there was a possible inhibitory effect of light on cultured root tips, experiments were conducted with excised roots tips in the dark. NAA was shown to have the potential to initiate LRs across a wide concentration gradient with the total number of LRs and initiated lateral root primordia (LRP) remaining constant across the range of concentrations tested. Over the last decade, nitric oxide (NO), a bioactive molecule, has been reported to be involved in the regulation of many biological pathways. The presence of NO in the system provided via sodium nitroprusside (SNP), promoted LRP initiation based on the NAA concentration gradient; but without changing the total LR initiation, that is LRs plus primordia density remained constant along the concentration gradient of NAA. The absence of LR and LRP in the treatments of CPTIO (a NO scavenger) with SNP or NAA suggests that NO regulates LRP initiation triggered by NAA, which is in agreement with the recent paper published after the commencement of this study (Correa-Aragunde et al., 2006). In agreement with previous studies, ABA inhibited lateral root development by reducing LR density and the number of LRs. The experiments with fluridone, an ABA biosynthesis inhibitor, may indicate that endogenous ABA was at sufficient concentrations in the excised root tips to inhibit primordia initiation. In this study, evidence is presented for the first time to show that SNP can relieve the inhibitory effect of ABA on LR density and number of LRs suggesting the NO, released from SNP, acts downstream of ABA. Overall these data confirm a critical role for NO in LR initiation.

Au cours du développement des plantes, l'auxine contrôle de nombreux processus dont notamment la dominance apicale, le phototropisme, la phyllotaxie, la formation des racines latérales et le développement des fruits. Le métabolisme, la perception ainsi que la signalisation de l’auxine ont majoritairement été étudiés chez Arabidopsis. Afin d’élucider la fonction de cette hormone au cours du développement des fruits, nous avons utilisé la tomate comme plante modèle. En effet la tomate est à la fois une espèce référence pour la famille des Solanacées mais également pour les plantes à fruits charnus. Les gènes Aux/IAA jouent un rôle déterminant dans la voie de signalisation auxinique en formant notamment un complexe avec l’un des récepteurs de cette hormone et en réprimant l’activité des facteurs de transcriptions de type ARF. Au cours de ce travail, nous avons caractérisé la famille multigènique des Aux/IAA chez la tomate. Elle est composée de 25 membres que nous avons nommés en référence à ceux d’Arabidopsis. Le niveau d’expression des gènes Aux/IAA est variable en fonction du gène, de l’organe ou du tissu considéré. L’expression de plusieurs de ces gènes est régulée à la fois par l’auxine et l’éthylène, ce qui suggère que les Aux/IAA sont reliés aux voies de signalisation de ces deux hormones. L’élucidation de la fonction des Aux/IAA de tomate est réalisée à travers la caractérisation de plantes transgéniques avec une attention particulière portée aux lignées montrant des phénotypes affectant le développement et la qualité du fruit ou présentant un intérêt pour le dialogue entre l’auxine et l’éthylène. Parmi ces lignées, les plantes sous-exprimant le gène Sl-IAA27 présentent une altération du développement des fleurs et des fruits. De plus, la diminution de l’expression de Sl-IAA27entraine la sous-expression de gènes impliqués dans la voie de biosynthèse de la chlorophylle se traduisant par une diminution de la teneur en chlorophylle dans les feuilles. Ces résultats montrent la diversité fonctionnelle des Sl-IAA et souligne le rôle de régulateur joué par l’auxine au cours du développement du fruit. / The phytohormone auxin controls various developmental processes, including apical dominance, tropisms, vascular patterning and fruit set. Auxin metabolism, transport, perception and signaling are mainly studied in the plant model Arabidopsis. To understand the auxin regulation process of fruit development, the tomato plant which is a reference species for Solanaceae and fleshy fruit plants is a good model of study. Aux/IAA genes play a key role in auxin signaling pathway, through their participation to the receptor complex of the hormone and by repressing the activity of ARF type transcription factors. In this work the 25 Sl-IAA family members have been isolated and renamed according to their phylogeny relationship with AtIAAs. Sl-IAA genes display distinctive expression pattern in different tomato organs and tissues, and some of them display differential responses to auxin and ethylene, suggesting that Aux/IAAs may play a role in linking both hormone signaling pathways. To improve knowledge about Aux/IAA function, transgenic tomato plants have been generated. The involvement of Aux/IAAs in notably fruit development was addressed through the characterization of the Sl-IAA27 gene. Its down-regulation in plants lead to altered flower and fruit development with a modified shape of the fruits and reduced volume. Moreover, fertilization capacity was strongly altered by the silencing of Sl-IAA27 resulting in the formation of fruits with reduced seed number. In addition, the reduced expression of Sl- IAA27 leads to a down-regulation of genes involved in chlorophyll biosynthesis triggering reduced leaf chlorophyll accumulation content. These results showed a functional diversity among Sl-IAA family members and underlined the involvement of auxin notably in the regulation of fruit development.

Dentre os mecanismos relacionados à interação bactéria-planta, a biossíntese bacteriana de ácido indol acético (AIA) exerce um papel fundamental na promoção do crescimento vegetal, uma vez que é capaz de influenciar inúmeros processos fisiológicos nas plantas. Diferentes vias metabólicas são utilizadas pelas bactérias para a biossíntese do AIA, sendo a via do ácido indol-3-pirúvico (IPyA) a mais comumente descrita. Nesta via encontra-se o gene indol-3-piruvato descarboxilase (ipdC) com vital função na produção de AIA utilizando como precursor o aminoácido L-triptofano. Nesse contexto, estudos moleculares acerca das vias metabólicas e dos genes envolvidos nesse processo são preponderantes para o entendimento da inter-relação das vias regulatórias com a síntese do fitormônio. A rizobactéria Bacillus sp. (RZ2MS9) vem apresentando satisfatória atividade na promoção de crescimento vegetal. O sequenciamento do seu genoma apontou a presença de uma vasta gama de genes relacionados à promoção do crescimento, com destaque para genes codificadores de auxinas. Assim, o estudo teve por objetivo comprovar a função do gene ipdC na biossíntese do AIA pela via dpendente do L-triptofano através do nocaute sítio dirigido do gene ipdC na Rizobactéria Promotora do Crescimento em Plantas (RPCP) Bacillus sp. (RZ2MS9). Para tanto, foi realizado o nocaute sítio dirigido por meio da técnica de CRISPR-Cas9. O nocaute do gene ipdC foi eficiente, gerando mutantes disruptivos para o referido gene. A biossíntese do AIA pela linhagem ΔipdC apresentou reduções nas concentrações do fitormônio, de acordo com o tempo de crescimento, sendo 87,96% em 24 horas, 88,25% em 48 horas e 58,27% em 72 horas do crescimento em comparação à linhagem selvagem (WT). Além disso, a biossíntese do AIA na ausência do aminoácido L-triptofano também foi avaliada, não sendo constatada síntese do fitormônio em nenhum dos tempos crescimento, tanto na linhagem selvagem, quanto na linhagem ΔipdC. O presente estudo foi pioneiro no nocaute do gene ipdC em uma linhagem de Bacillus utilizando a técnica de CRISPR-Cas9. Os resultados obtidos contribuem para um melhor entendimento da influência do gene ipdC e da via IPyA na biossíntese do AIA pela linhagem RZ2MS9 e futuramente sera comprovado seu papel na promoção de crescimento vegetal. / Among the mechanisms related to the bacterium-plant interaction, the bacterial biosynthesis of indole acetic acid (AIA) plays a fundamental role in the promotion of plant growth, since it is capable of influencing innumerable physiological processes in plants. Different metabolic pathways are used by bacteria for the biosynthesis of IAA, with the indole-3-pyruvic acid (IPyA) pathway being the most commonly described. In this pathway, the indole-3-pyruvate decarboxylase (ipdC) gene has a vital role in the production of IAA using the amino acid L-tryptophan as a precursor. In this context, molecular studies about the metabolic pathways and the genes involved in this process are preponderant for the understanding of the interrelationship of the regulatory pathways with the phytormonium synthesis. The rhizobacterium Bacillus sp. (RZ2MS9) has been showing satisfactory activity in promoting plant growth. The sequencing of its genome pointed to the presence of a wide range of genes related to growth promotion, especially genes encoding auxins. Thus, the objective of the present study was to verify the function of the ipdC gene in the IAA biosynthesis L-tryptophan dependent through the knockout of the ipdC in the plant growth-promoting rhizobateria (PGPR) Bacillus sp. (RZ2MS9). Therefore, the knockout was realized using the CRISPR-Cas9. The knockout of the ipdC gene was efficient, generating disruptive mutants for the said gene. IAA biosynthesis by the ΔipdC strain showed reductions in phytormonium concentrations, according to the growth time, being 87.96% in 24 hours, 88.25% in 48 hours and 58.27% in 72 hours of growth compared to the Wild Type (WT). In addition, the biosynthesis of IAA in the absence of the amino acid L-tryptophan was also evaluated, with no phytormonium synthesis being observed at any growth time, both in the wild type and ΔipdC strain. The present study pioneered the knockout of the ipdC gene in a Bacillus strain using the CRISPR-Cas9. The results obtained contribute to a better understanding of the influence of the ipdC gene and the IPyA pathway in the IAA biosynthesis through the RZ2MS9 strain, and its role in plant growth promoting will be demonstrated in the future.

Dentre os mecanismos relacionados à interação bactéria-planta, a biossíntese bacteriana de ácido indol acético (AIA) exerce um papel fundamental na promoção do crescimento vegetal, uma vez que é capaz de influenciar inúmeros processos fisiológicos nas plantas. Diferentes vias metabólicas são utilizadas pelas bactérias para a biossíntese do AIA, sendo a via do ácido indol-3-pirúvico (IPyA) a mais comumente descrita. Nesta via encontra-se o gene indol-3-piruvato descarboxilase (ipdC) com vital função na produção de AIA utilizando como precursor o aminoácido L-triptofano. Nesse contexto, estudos moleculares acerca das vias metabólicas e dos genes envolvidos nesse processo são preponderantes para o entendimento da inter-relação das vias regulatórias com a síntese do fitormônio. A rizobactéria Bacillus sp. (RZ2MS9) vem apresentando satisfatória atividade na promoção de crescimento vegetal. O sequenciamento do seu genoma apontou a presença de uma vasta gama de genes relacionados à promoção do crescimento, com destaque para genes codificadores de auxinas. Assim, o estudo teve por objetivo comprovar a função do gene ipdC na biossíntese do AIA pela via dpendente do L-triptofano através do nocaute sítio dirigido do gene ipdC na Rizobactéria Promotora do Crescimento em Plantas (RPCP) Bacillus sp. (RZ2MS9). Para tanto, foi realizado o nocaute sítio dirigido por meio da técnica de CRISPR-Cas9. O nocaute do gene ipdC foi eficiente, gerando mutantes disruptivos para o referido gene. A biossíntese do AIA pela linhagem ΔipdC apresentou reduções nas concentrações do fitormônio, de acordo com o tempo de crescimento, sendo 87,96% em 24 horas, 88,25% em 48 horas e 58,27% em 72 horas do crescimento em comparação à linhagem selvagem (WT). Além disso, a biossíntese do AIA na ausência do aminoácido L-triptofano também foi avaliada, não sendo constatada síntese do fitormônio em nenhum dos tempos crescimento, tanto na linhagem selvagem, quanto na linhagem ΔipdC. O presente estudo foi pioneiro no nocaute do gene ipdC em uma linhagem de Bacillus utilizando a técnica de CRISPR-Cas9. Os resultados obtidos contribuem para um melhor entendimento da influência do gene ipdC e da via IPyA na biossíntese do AIA pela linhagem RZ2MS9 e futuramente sera comprovado seu papel na promoção de crescimento vegetal. / Among the mechanisms related to the bacterium-plant interaction, the bacterial biosynthesis of indole acetic acid (AIA) plays a fundamental role in the promotion of plant growth, since it is capable of influencing innumerable physiological processes in plants. Different metabolic pathways are used by bacteria for the biosynthesis of IAA, with the indole-3-pyruvic acid (IPyA) pathway being the most commonly described. In this pathway, the indole-3-pyruvate decarboxylase (ipdC) gene has a vital role in the production of IAA using the amino acid L-tryptophan as a precursor. In this context, molecular studies about the metabolic pathways and the genes involved in this process are preponderant for the understanding of the interrelationship of the regulatory pathways with the phytormonium synthesis. The rhizobacterium Bacillus sp. (RZ2MS9) has been showing satisfactory activity in promoting plant growth. The sequencing of its genome pointed to the presence of a wide range of genes related to growth promotion, especially genes encoding auxins. Thus, the objective of the present study was to verify the function of the ipdC gene in the IAA biosynthesis L-tryptophan dependent through the knockout of the ipdC in the plant growth-promoting rhizobateria (PGPR) Bacillus sp. (RZ2MS9). Therefore, the knockout was realized using the CRISPR-Cas9. The knockout of the ipdC gene was efficient, generating disruptive mutants for the said gene. IAA biosynthesis by the ΔipdC strain showed reductions in phytormonium concentrations, according to the growth time, being 87.96% in 24 hours, 88.25% in 48 hours and 58.27% in 72 hours of growth compared to the Wild Type (WT). In addition, the biosynthesis of IAA in the absence of the amino acid L-tryptophan was also evaluated, with no phytormonium synthesis being observed at any growth time, both in the wild type and ΔipdC strain. The present study pioneered the knockout of the ipdC gene in a Bacillus strain using the CRISPR-Cas9. The results obtained contribute to a better understanding of the influence of the ipdC gene and the IPyA pathway in the IAA biosynthesis through the RZ2MS9 strain, and its role in plant growth promoting will be demonstrated in the future.

This study seeks to address one of the simplest questions that can be asked about an interconnected system; what happens to one process in the absence of the other process? This is a more difficult task than it would appear at first, due to the absence of small molecule inhibitors that can inhibit splicing globally in yeast cells. The first results chapter describes the adaptation of a system called the auxin induced degron (AID) to the task of inhibiting pre-mRNA splicing. This system appears to have several advantages over previous methods of inhibiting splicing and has many potential applications. Another hurdle to understanding what happens to transcription in the absence of splicing is the differential stability of pre-mRNA versus mRNA. At steady state the vast majority of transcripts of a specified gene will be mRNA transcripts. This means that even if you could rapidly inhibit splicing it would be a long time before all the pre-existing mRNA would turn-over. If you waited until specified mRNAs turned over it is likely that the cells would be very sick making it difficult to separate primary and secondary effects. The second results chapter shows the use of a metabolic labelling technique using a uracil analogue called 4-thiouracil (4SU). 4SU is added for an extremely short amount of time (1.5 min, 2.5 min, and 5 min) and the RNA produced during the labelling time is isolated by affinity purification. This allows us to study the kinetics of pre-mRNA splicing in wild-type cells and to seek correlations between splicing kinetics and gene architecture. The third results chapter combines the methods used in the previous two chapters to give a new technique called AID4U-seq. AID4U-seq allows for rapid inhibition of splicing and then the ability to isolate only the transcripts that were created after this inhibition came into effect. This should allow for examination of the primary consequences of blocking pre-mRNA splicing at multiple stages during spliceosome assembly. Additionally AID4U-seq is immediately applicable to the study of other areas of RNA processing. Defining the effects on the transcriptome of inhibiting splicing at multiple stages of assembly is an ambitious aim likely to require many more years of research. Therefore this thesis chiefly seeks to illustrate a novel strategy to begin dissecting a complex issue in which splicing, transcription, degradation and the post-transcriptional modification of histones are all likely to have roles.

Typescript. / Digitized by Kansas Correctional Industries

O objetivo do presente estudo foi avaliar a ação do ácido indol-3-acético (AIA) combinado com a peroxidase de raiz forte (HRP), formando um sistema gerador de espécies reativas de oxigênio, sobre a viabilidade de Staphylococcus aureus. Para tal, avaliou-se a viabilidade do S. aureus através da contagem das unidades formadoras de colônias após crescimento em ágar manitol, potencial e integridade de membrana por citometria de fluxo e integridade do DNA através de eletroforese em gel de poliacrilamida. Para realização dos ensaios foram utilizadas cepas de S. aureus recuperadas de casos de mastites clínicas. As cepas foram cultivadas em meio BHI (brain-heart-infusion) a 37ºC \"overnight\". Nos ensaios, o microrganismo foi incubado na ausência (controle) e presença de AIA (1 mmol/L)/HRP (1 µmol/L) em diferentes tempos (0, 1,5, 3 e 6 horas) a 37ºC. Foram realizados também ensaios contendo o microrganismo incubado na presença de AIA ou de HRP. O sistema AIA/HRP inibiu em 96%, 98%, 99% a formação de colônias do microrganismo para os tempos de 1,5, 3 e 6 horas, respectivamente, em relação ao controle em cada tempo. Ocorreu uma redução na polarização da membrana do microrganismo em 38, 69 e 99% nos tempos 1,5, 3 e 6 horas, respectivamente e uma diminuição significativa do número de microrganismos com membrana integra de 17 e 22% quando estes foram incubados por 3 e 6 horas, respectivamente em relação ao controle nos respectivos tempos. A adição das enzimas antioxidantes catalase ou superóxido dismutase ao meio de incubação não alterou o efeito deletério promovido pelo sistema AIA/HRP avaliado pelas unidades formadoras de colônias, despolarização e integridade de membrana. O sistema AIA/HRP não induziu a fragmentação do DNA do S. aureus após 3 e 6 horas de incubação. No presente estudo, foi possível verificar que a oxidação do AIA pela HRP produz uma resposta citotóxica potente capaz de promover a inibição do crescimento de S. aureus em ágar manitol, provocar a despolarização e a perda da integridade da membrana do microrganismo, sugerindo a possibilidade da utilização do sistema AIA/HRP como uma possível terapia alternativa contra bactérias. / The objective of this study was to evaluate the action of the indole-3-acetic acid (IAA) in combination with horseradish peroxidase (HRP), forming a system generator of reactive oxygen species, on the viability of Staphylococcus aureus. To this end, was evaluated the of viability of S. aureus through the counting of the colony forming units after growth in mannitol agar, membrane potential and membrane integrity by flow cytometry and integrity of the DNA through the polyacrylamide gel electrophoresis. For the tests were used strains of S. aureus recovered from cases of clinical mastitis. The strains were grown in BHI medium (brain-heart-infusion) at 37°C overnight. In the tests, the microorganism was incubated in the absence (control) and presence of IAA (1 mmol/L)/HRP (1 µmol/L) at different times (0, 1.5, 3 and 6 hours) at 37°C. There were also conducted tests containing the microorganism incubated in the presence of IAA or HRP. The system IAA/HRP inhibited at 96%, 98%, 99% colony formation of microorganism to the times of 1.5, 3 and 6 hours, respectively, in relation to the control in every time. There was a decrease in polarization of the membrane of the microorganism on 38, 69 and 99% at times 1.5, 3 and 6 hours, respectively, and a significant decrease in the number of microorganisms with membrane integrity, 17 and 22% when they were incubated for 3 and 6 hours, respectively, in relation to the control in their time. The addition of the antioxidant enzymes catalase and superoxide dismutase in incubation medium did not alter the deleterious effect promoted by the system IAA/HRP assessed by colony forming units, membrane potential and membrane integrity. The system IAA/HRP did not induce the DNA fragmentation of S. aureus after 3 and 6 hours of incubation. In the present study, it was possible to verify that the oxidation of the IAA by HRP produces a potent cytotoxic response capable of promoting the inhibition of growth of S. aureus in mannitol agar, causing depolarization and the loss of integrity of the membrane of the microorganism, suggesting the possibility of using the system IAA/HRP as a possible alternative therapy against bacteria.

The developmental programs of maturing seed and fruit in pea (Pisum sativum L.) are tightly controlled by the interactions of several phytohormones, including gibberellins (GAs), auxins, and abscisic acid (ABA). To more fully understand these hormone networks and their roles in controlling development, transcription profiles of GA metabolism genes and metabolite profiles of key GAs, auxins, and ABA were determined in developing seeds, and histological studies were employed to correlate physiology and hormone metabolism. Data suggest that bioactive GA stimulates several aspects of seed growth, and ABA appears to promote bioactive GA1 synthesis in rapidly growing seed coats, and inhibit GA biosynthesis in the embryo axes of maturing embryos. Two putative auxin receptor genes were cloned, and their transcription profiles examined in developing seed and pericarp tissues. Pericarp PsAFB6A transcription was responsive to auxin and seed signals, indicating a potential role for the modulation of auxin sensitivity in fruit development.

The phytohormones auxin and cytokinin control plant development through a complex network of interactions which include synergistic, additive, and opposite effects whose mechanisms are unknown. The auxin-insensitive diageotropica (dgt) mutant provided a tool to dissect the relationship between auxin- and cytokinin-induced responses in tomato. Morphological, physiological, and molecular data support the proposal that auxin and cytokinins control a common set of developmental processes through separate signal transduction pathways which interact downstream from the DGT gene product. Morphological traits of dgt plants, such as reduced root and shoot growth, reduced leaf complexity, and reduced apical dominance were phenocopied by exogenous cytokinin application to wild-type plants, demonstrating that cytokinins and the DGT-mediated auxin response control a common set of phenotypic characteristics. Because the dgt mutation had no detectable effects on the levels of endogenous cytokinins or on the cytokinin sensitivity of whole plants, cytokinins were hypothesized to cause dgt-like effects on plant development through inhibiting auxin-induced responses. This hypothesis was supported by physiological experiments showing that auxin-induced elongation and ethylene synthesis were inhibited in cytokinin-treated wild-type and in untreated dgt hypocotyls. Differences between the effects of cytokinins and the dgt mutation on auxin responses became apparent at the molecular level. Experiments on the auxin-induced accumulation of transcripts for two ACC-synthase genes and one SAUR gene demonstrated that cytokinin treatment selectively reduced the auxin-induced expression of only one ACC-synthase gene, while the dgt mutation inhibited the auxin-inducibility of all three genes. The effects of the dgt mutation were thus more pleiotropic than the cytokinin effects, suggesting that cytokinins inhibit auxin-responses downstream from the DGT gene product. In vitro culture of dgt hypocotyl explants and calli demonstrated shared or interacting signal transduction pathways for auxin and cytokinin in the stimulation of cell division, and independent pathways for the control of organ regeneration and vascular differentiation. / Graduation date: 1997