The brevipedicellus (bp) mutant, caused by a loss-of-function mutation in the KNAT1 homeobox gene, is known to affect the stem morphogenesis. BP and ERECTA (ER) genes are required to promote internode and pedicel development and delineate nodal boundaries to maintain the radial symmetry of stems and pedicels. My research aims to identify genes acting on the BP pathway by utilizing a forward genetics approach. The suppressor4 mutant, identified from the bp er mutant screen, exhibits moderate length and perpendicularly-oriented pedicels with partially formed distal pedicel bulges, absent in the bp mutant. The kinky mutant, identified from the bp mutant screen, develops severe bends at the floral nodes and enhanced achlorophyllous stripes. These results suggest the SUPPRESSOR4 gene contribution in inhibiting the development of distal pedicel bulge and influencing both pedicel angle and length whereas the KINKY gene might act with BP in regulating proper inflorescence development.

Dans cette thèse nous nous intéressons à la manière dont la structure des plantes émerge du fonctionnement de leur méristème apical. Pour cela, nous étudions la structure du méristème apical d'Arabidopsis thaliana à différentes échelles. La thèse commence par étudier les plantes à l'échelle macroscopique dont la phyllotaxie a été perturbée et par le développement d'outils mathématiques pour quantifier et analyser ces perturbations. Ensuite, nous étudions à une échelle plus microscopiques quelles peuvent être les raisons de telles perturbations. Pour cela, nous avons testé une version étendue d'un modèle proposé par Douady et Couder (1996) dans lequel plusieurs paramètres clés sont modifiés par différentes sources de bruit. Cette étude de modélisation suggère que la stabilité de la taille de la zone de la zone centrale peut être un facteur clé dans la robustesse phyllotaxie. Alors que des modèles 3D réalistes des champs d'inhibition autour des primordia ont été développés récemment, une telle étude est toujours manquante pour les tissus réalistes en 3D dans le cas de la zone centrale. Cela nous conduit finalement à analyser en profondeur le réseau de régulation génétique qui contrôle la taille de la zone centrale dans le méristème. Nous avons implémenté une version 3D d'un modèle de la littérature de la zone centrale et testé ce modèle sur des méristèmes 3D obtenues à partir des images 3D de la microscopie laser.

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.

M.Sc. (Biochemistry) / During microbial invasion, a variety of defense responses are induced in host plants. In order for host plants to combat potential diseases induced by microbes, they must be equipped with pattern recognition receptors (PRRs) localized at the cell surface, since such receptors enable the perception of conserved microbial epitopes termed microbe/pathogen-associated molecular patterns (M/PAMPs), thereby resulting in the activation of plant innate immunity via M/PAMP-triggered immunity (P/MTI). Lipopolysaccharide (LPS) is the major component of the outer leaflet of the external membrane of Gram-negative bacteria. This thermo-stable lipoglycan is exposed towards the external environment and plays an important role in bacterial adaptation to external surroundings. LPS is recognized as a major M/PAMP in plants, and thus potentiates or elicits defense-related responses such as the production of antimicrobial compounds and the expression of immune response genes. One of the most widely investigated effects of LPS on plants is its ability to prevent and/or suppress the hypersensitive response (HR) induced by an array of bacteria. The HR is a programmed cell death response which ends in a local necrosis of plant tissue, thereby resulting in a reduced number of viable bacteria that can further promote disease progression in the host.

M.Sc. (Biochemistry) / Plant innate immunity occurs in two interconnected branches, the first being the recognition of pathogen conserved surface structures known as pathogen- or microbe-associated molecular patterns (P/MAMPs) by the plant plasma membrane pathogen recognition receptors (PRRs), leading to activation of P/MAMP-triggered immunity (P/MTI). The second branch involves the recognition of pathogen avirulence (Avr) genes by the corresponding plant disease resistance (R) genes, known as the ‘gene-for-gene‘ interaction, and results in a more efficient or stronger defence response, namely effector-triggered immunity (ETI). Lipopolysaccharide (LPS) acts as a P/MAMP that induces an innate immune response in both plants and animals. LPS, especially the lipid A component, has been shown to play a vital role in activating immune responses in animals. Other LPS components such as lipooligosaccharide (LOS) and the core-oligosaccharide have also been shown to trigger an immune response in plants such as Arabidopsis thaliana. In mammalian cells, LPS binds to the LPS-binding protein (LBP) forming a LPS-LBP complex, which binds to a Toll-like receptor 4/myeloid differentiation-2 (TLR4/MD-2) complex together with the co-receptor CD14, a glycosylphosphatidylinositol (GPI)-linked protein, and activates an immune response. To date, there is still no knowledge about the LPS receptor(s) in plants.....

Invertasen sind Schlüsselenzyme in der Kohlenhydratverteilung und haben möglicherweise auch während einer Pathogeninfektion eine zentrale Bedeutung. In vorliegender Arbeit wurde zunächst die Regulation verschiedener Stoffwechselwege in Arabidopsis thaliana nach Infektionen mit einem virulenten oder avirulenten Stamm von Pseudomonas syringae untersucht. Mit Hilfe der Chlorophyllfluoreszenz-Bildgebung konnten räumliche und zeitliche Veränderungen der Photosynthese verfolgt werden. Verschiedene Parameter waren unterschiedlich reguliert. In beiden Interaktionen waren Effekte nur lokal um die Infektionsstellen erkennbar und qualitativ ähnlich. Unterschiede waren im zeitlichen Eintreten und Verlauf sichtbar. Die Methode schien geeignet für die sensitive, nicht-invasive Pathogenfrüherkennung vor dem Auftreten sichtbarer Symptome. Die Regulation verschiedener Gene innerhalb von Source-Sink-Übergängen und die Aktivität von Invertasen war in den beiden Interaktionen qualitativ unterschiedlich. Die Infektion mit virulenten Bakterien resultierte in einer Repression photosynthetischer Gene. Die Aktivität vakuolärer Invertasen stieg vorübergehend nach Infektion mit virulenten Bakterien an, während sie nach Infektion mit avirulenten Bakterien sank. Die Aktivität extrazellulärer Invertasen war in beiden Interaktionen reprimiert. Die erfolgreiche Generierung verschiedener Bakterienstämme von P. syringae, die das grün fluoreszierende Protein exprimieren, kann bei der weiteren Charakterisierung von Pflanze-Pathogen-Interaktionen helfen. Die Regulation von Invertasen erfolgt auf transkriptioneller und posttranslationaler Ebene. Um die Funktion von Invertasen in Pflanze-Pathogen-Interaktionen zu verstehen, wurde zunächst die Regulation von Invertasen durch endogene proteinogene Invertaseinhibitoren untersucht. In Übereinstimmung mit in silico Expressionsdaten konnte durch Untersuchung von Reportergenlinien und in Northern Blot Analysen eine starke Expression von Invertaseinhibitoren in Blättern von A. thaliana festgestellt werden. Nach Applikation biotischer und abiotischer Stressfaktoren wurde diese Expression nahezu vollständig reprimiert. Die indirekte Bestimmung der Invertaseinhibitoraktivität durch Messung der Invertaseaktivität in Mischextrakten zeigte, dass diese nach einer Pathogeninfektion vollständig reprimiert war. In funktionellen Ansätzen wurden transgene Pflanzen generiert, die Invertaseinhibitoren unter Kontrolle induzierbarer Promotoren exprimieren. Die Induktion der Invertaseinhibitorexpression änderte die Sensitivität gegenüber verschiedenen Pathogenen nicht signifikant. In einem pharmakologischen Ansatz wurde der chemische Inhibitor Acarbose zur Hemmung der Invertaseaktivität in A. thaliana verwendet. Eine Behandlung von Blättern bei gleichzeitiger Infektion mit Bakterien verursachte eine erhöhte Sensitivität der Pflanzen gegenüber der Infektion, eine stärkere Repression verschiedener Chlorophyllfluoreszenzparameter sowie ein erhöhtes Bakterienwachstum im Vergleich zu einer Infektion mit den Bakterien allein. Keine Effekte wurden auf transkriptioneller Ebene bei der Untersuchung von Genen verschiedener Stoffwechselwege gefunden. Die Invertaseaktivität nach zusätzlicher Behandlung mit Acarbose war tendenziell niedriger als die Aktivität nach einer Pathogeninfektion alleine. Acarbose erhöhte die Spiegel an Salicylsäure unabhängig von einer Pathogeninfektion. Da das Bakterienwachstum in Mutanten des Salicylsäure-vermittelten Abwehrweges bei zusätzlicher Behandlung mit Acarbose ebenfalls erhöht war, kann eine Beteiligung dieses Abwehrweges am Acarboseeffekt bisher ausgeschlossen werden. Invertasen sind neben ihrer Beteiligung an der Abwehr für die Regulation von Entwicklungsprozessen wichtig. In einem funktionellen Ansatz mit Pflanzen, die Invertaseinhibitoren induzierbar produzieren, wurde die Funktion von Invertasen getestet. Zur Generierung spezifischer Effekte wurden die Inhibitoren unter Kontrolle synthetischer Promotoren in A. thaliana exprimiert. Unerwarteterweise war das Wachstum putativ transgener Keimlinge jedoch im 4-Blatt-Stadium arretiert. Eine Analyse der Aktivität der ß-Glucuronidase in den entsprechenden Reporterlinien zeigte eine Korrelation zwischen der Wachstumsarretierung und einer hohen Aktivität dieser Promotoren unter verschiedenen in vitro Bedingungen. Dieser negative Effekt der Invertaseinhibition auf das Keimlingswachstum wurde in transgenen Tabakpflanzen bekräftigt, die Invertaseinhibitoren unter Kontrolle eines Tetracyclin-induzierbaren Promotors exprimierten. Eine erfolgreiche Induktion des Promotors resultierte in einer Reduktion des Frischgewichtes der Keimlinge. Mittels in silico Expressionsdaten und Northern Blot Analysen konnte für A. thaliana eine spezifische und starke Expression verschiedener Invertaseisoformen in Keimlingen nachgewiesen werden. Diese komplementären Ergebnisse zeigen die Notwendigkeit der Invertaseaktivität für eine normale Keimlingsentwicklung. / Invertases are key metabolic enzymes in carbohydrate partitioning which may also play an important role during pathogen infection. In this study, the regulation of different metabolic pathways in Arabidopsis thaliana plants after infection by a virulent and an avirulent strain of Pseudomonas syringae was investigated. With help of chlorophyll fluorescence imaging spatio-temporal changes in photosynthesis were monitored. The monitored chlorophyll fluorescence parameters were showing differential regulation. The effects were restricted to the vicinity of the infection site and did not spread to uninfected areas of the leaf. Qualitatively similar changes in photosynthetic parameters were observed in both interactions. Major differences between the responses to both strains were evident in the onset and time course of changes. Changes could be detected by chlorophyll fluorescence imaging before symptoms were visible by eye. In contrast to photosynthesis, the regulation of marker genes for source/sink relations and the activities of invertase isoenzymes showed qualitative differences between both interactions. Inoculation of the virulent but not the avirulent strain resulted in downregulation of photosynthetic genes and upregulation of vacuolar invertases. The activity of vacuolar invertases transiently increased upon infection with the virulent strain but decreased with the avirulent strain while extracellular invertase activity was downregulated in both interactions. As an advanced tool for the characterization of the interaction between A. thaliana and P. syringae bacteria expressing the green fluorescing protein were generated. Invertases are regulated on transcriptional and posttranscriptional level. To understand the function of invertases in plant-pathogen-interactions, the regulation of endogenous proteinaceous invertase inhibitors was investigated. According to expression profiling the analysis of reporter gene lines and Northern Blot analyses revealed a strong expression of invertase inhibitors in source leaves of A. thaliana. After application of biotic and abiotic stress factors this expression was completely repressed. Indirect determination of invertase inhibitor activity by measurement of invertase activity in mixed extracts revealed a complete repression of inhibitor activity after pathogen infection. In functional approaches transgenic plants were generated, expressing invertase inhibitor proteins under control of inducible promoters. However, no effect of the induction of inhibitor expression on the sensitivity to different pathogens could be observed so far. In a pharmacological approach acarbose war used as an invertase inhibitor. Simultaneous treatment of plants with acarbose and bacteria revealed an increased sensitivity of the plant towards the bacterial infection and a stronger repression of chlorophyll fluorescence parameters compared to plants treated with bacteria alone. No effects on photosynthesis, carbohydrate metabolism and defence could be observed on transcriptional level. A tendency of lowered invertase activity could be observed after treatment with acarbose and bacteria compared to bacterial treatment alone. Acarbose treatment enhanced the levels of salicylic acid independent of bacterial infection. The acarbose-mediated increase in sensitivity was also detectable in sid2 and cpr6 mutants indicating that the effect of acarbose is independent of the salicylic acid mediated defence pathway. Invertases are important enzymes in higher plants, which are involved in regulating developmental processes and responses to external factors. In a functional approach the role of invertases was investigated using transgenic plants ectopically expressing inhibitor proteins to decrease invertase activity. For generating specific effects, these inhibitor proteins were expressed in A. thaliana under the control of synthetic promoters consisting of tetramers of pathogeninducible elements, which were reported to yield low constitutive expression. Unexpectedly, seedling growth of putative transgenic plants was arrested at the four-leaf stage. Analysis of ß-glucuronidase activity of corresponding reporter gene lines showed a correlation of the growth arrest with high activity of these promoters in seedlings grown under tissue culture conditions. The negative effect of invertase inhibition on seedling growth was substantiated by transgenic tobacco plants expressing an invertase inhibitor under control of a tetracycline inducible promoter. Ectopic induction of the invertase inhibitor during early seedling development resulted in a reduced fresh weight of seedlings. Expression profiling and Northern Blot analyses further supported the importance of invertase in Arabidopsis thaliana seedling development. Different invertases were specifically and strongly expressed. These complementing results show that invertase activity is required for normal seedling development.

Miao Yansong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 78-83). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.ii / Statement --- p.iii / 摘要 --- p.vi / Acknowledgements --- p.vii / List of Tables --- p.xi / List of Figures --- p.xii / List of Abbreviations --- p.xiv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1. --- The plant secretory pathway --- p.2 / Chapter 2. --- Two different types of vacuoles in plant cells --- p.2 / Chapter 3. --- Vacuolar sorting receptor (VSR) proteins --- p.3 / Chapter 4. --- BP-80 and prevacuolar compartment --- p.6 / Chapter 5. --- The Arabidopsis VSR proteins --- p.7 / Chapter 6. --- Research objectives --- p.8 / Chapter Chapter 2 --- Development of Transgenic Tobacco BY-2 Cell Lines Expressing GFP-AtVSR Fusions --- p.10 / Chapter 1. --- Introduction --- p.11 / Chapter 2. --- Materials and Methods --- p.12 / Chapter 2.1 --- Structure of Golgi marker and PVC marker --- p.12 / Chapter 2.2 --- Construction of GFP-VSR reporters --- p.14 / Chapter 2.3 --- Agrobacterium electroporation --- p.24 / Chapter 2.4 --- Transformation of tobacco BY-2 cells --- p.24 / Chapter 2.5 --- Screening of transgenic BY-2 cells expressing GFP-VSR fusions --- p.25 / Chapter 2.6 --- Chemicals --- p.27 / Chapter 3. --- Result.s --- p.28 / Chapter 3.1 --- Chimeric GFP reporters as tools to study subcellular localization of Arabidopsis vacuolar sorting receptor proteins in transgenic BY-2 cells --- p.28 / Chapter 3.2 --- Establishment of transgenic tobacco (Nicotiana tabacum) BY-2 cell lines stably expressing seven GFP-AtVSR reporters --- p.29 / Chapter 4. --- Conclusion --- p.38 / Chapter Chapter 3 --- Subcellular Localization of the Seven GFP-AtVSR Fusions to Prevacuolar Compartments in Transgenic Tobacco BY-2 Cells --- p.39 / Chapter 1. --- Introduction --- p.40 / Chapter 2. --- Materials and Methods --- p.41 / Chapter 2.1 --- Confocal immunofluorescence studies --- p.41 / Chapter 2.2 --- Antibodies for immunolabeling --- p.42 / Chapter 2.3 --- Wortmannin and brefeldin A treatment --- p.42 / Chapter 2.4 --- Electron microscopy of resin-embedded cells --- p.43 / Chapter 3. --- Results --- p.44 / Chapter 3.1 --- Vacuolar sorting receptor proteins in plants --- p.44 / Chapter 3.2 --- PVC localization of GFP-AtVSR fusions in transgenic tobacco BY-2 cells --- p.47 / Chapter 3.3 --- The spacer sequences did not affect PVC localization of GFP-AtVSR7 --- p.56 / Chapter 3.4 --- Wortmannin-induced vacuolated PVCs contained VSRs in tobacco BY-2 cells --- p.58 / Chapter 3.5 --- Wortmannin-induced vacuolation of PVCs is a general response in plant cells --- p.62 / Chapter 4. --- Conclusion --- p.65 / Chapter Chapter 4 --- Discussion and Future Perspectives --- p.66 / Chapter 1. --- The hypothesis in this study --- p.67 / Chapter 2. --- GFP and BY-2 cells --- p.67 / Chapter 3. --- A reporter system to study subcellular localization of VSR proteins in transgenic tobacco BY-2 cells --- p.68 / Chapter 4. --- PVC localization of the seven GFP-AtVSR fusions in transgenic BY-2 cells --- p.69 / Chapter 5. --- VSR spacer sequences did not affect PVC localization of GFP-AtVSR fusions in transgenic tobacco BY-2 cells --- p.71 / Chapter 6. --- PVC localization of GFP-PV72 and GFP-AtVSR 1 fusions in transgenic tobacco BY-2 cells --- p.73 / Chapter 7. --- Wortmannin-induced vacuolation of PVC is a general response in plant cells --- p.75 / Chapter 8. --- Future perspectives --- p.75 / References --- p.78

Genomic imprinting is the epigenetic modification of loci, primarily by DNA methylation, which results in parent-of-origin-specific monoallelic expression of a small subset of genes. In plants, imprinting occurs during endosperm development and a balance of maternally- and paternally-expressed imprinted genes is essential for normal seed development. Dependence on DNA methylation for imprinting highlights the potential to manipulate seed development, and consequently seed size, by altering DNA methyltransferase activity. DNA METHYLTRANSFERASE 1 (MET1) is the primary plant maintenance DNA methyltransferase and plays a significant role in imprinting. However, no evaluation of the potential role for other MET1 family members in genomic imprinting has been reported. The current model for the control of imprinting in plants suggests that maintenance DNA methyltransferases are required throughout development, yet the tissue-specific requirement of these enzymes is unconfirmed as analysis has relied solely on constitutive DNA methyltransferase mutants. To address these problems and to evaluate the potential to alter seed size, the work reported in this thesis investigated the potential involvement of putative maintenance DNA methyltransferases MET2a, MET2b and MET3 and the tissue-specific role of MET1 in imprinting. Imprinting was not significantly altered in met2a-1, met2b-1 and met3-1 mutants, indicating that MET1 is the sole DNA methyltransferase required for imprinting. Transcriptional analysis suggested MET1 is expressed throughout floral organ development and in the male and female gametophyte generation indicating that MET1 is potentially available to maintain imprinting-dependent methylation in these tissues. Tools to suppress MET1 tissuespecifically were developed to investigate the tissue-specific requirement of MET1 for imprinting. Analysis indicates that such tools could also be used to alter seed size by manipulating imprinting in commercially important species. Further work is needed to validate this approach.

fósforo (P) e o nitrogênio (N) são nutrientes geralmente limitantes ao crescimento e desenvolvimento vegetal. Respostas aclimatativas à sua limitação referem-se às alterações do desenvolvimento radicular e mobilização, transporte, assimilação e metabolismo destes nutrientes. Para que este processo seja desencadeado, é necessária expressão de genes intimamente relacionados com a percepção e transdução do sinal de deficiência de P e N. Os genes envolvidos neste processo ainda são pouco conhecidos. Neste estudo objetivou-se averiguar o papel dos genes mutados nas rotas de aclimatação à limitação de Pi e assimilação de N através da identificação das modificações em nível de expressão gênica nos mutantes p9, p23 e p37, bem como através da avaliação da resposta dos mutantes p9 e p37 ao etileno. Os três mutantes são complementares e ineficientes em utilizar organofosfatos como fonte de P e p23 e p37 são resgatados quando o N é retirado do meio. Há interação P-N em relação à expressão de genes das rotas de aclimatação à limitação de P (AtACP5 e AtPT2), de assimilação de N (NRT1.1, NIA1 e NIA2) e na modulação das raízes laterais (ARF8). Os mutantes têm alteração na expressão dos genes que codificam transportadores de N e P de alta afinidade, assimilação de N, especialmente NIA2, e no caso de p9 e p23, ainda, ARF8. Na ausência de Pi e/ou N, houve rápida exaustão do centro quiescente nas raízes primárias em p23 e p37, assim, os genes mutados devem fazer parte de uma rota que medie as respostas do crescimento radicular em função da disponibilidade de Pi e N. Os mutantes p9 e p37 apresentam deficiência na sinalização ao etileno, sendo possível que p9 possua alteração na homeostase hormonal, e que modificação nos níveis de auxinas e citocininas afetem a síntese de etileno. / Phosphorus (P) and nitrogen (N) are nutrients highly required by plants, and limit plant growth and development. The main acclimation responses to P and N starvation include changes in root development, mobilization, transport, assimilation and metabolism of these nutrients. For properly acclimation, the expression of genes closely related to the perception and signal transduction of P and N deficiencies must work accurately. Which genes are involved in this process is still unclear, therefore, this study aimed to identify changes at the expression level in the p9, p23 and p37 mutants in an attempt to identify the role of the mutated genes in the acclimation pathway to Pi starvation. Furthermore, we aimed to verify the P-N interaction and the response of the mutants p9 and p37 to ethylene. The three mutants are inneficient in using organophosphates as the only source of P, and p9 and p23 recover the COL phenotype in the absence of N. Interaction between P and N was observed in the expression of genes involved in Pdeficiency acclimation, namely AtACP5 and AtPT2, as well as in the N assimilation, NRT1.1, NIA1 and NIA2, and ARF8, involved in lateral root modulation. It was observed that the mutated gene in p9, p23 and p37 affects genes that encode high affinity N transporters, genes involved in N assimilation, especially NIA2. And p9 and p23 also has the regulatory circuit that acts on the modulation of lateral roots affected. A rapid depletion of QC in primary roots of p23 and p37 was observed in the absence of Pi and/or N. It suggests that the mutated genes are involved in a pathway mediating the root growth in response to Pi and N availability. The mutants, p9 and p37, have some kind of deficiency in ethylene signaling. It is also possible that p9 is affected in its hormonal homeostasis, and changes in auxin and cytokinin levels affect the ethylene synthesis.

As plantas expressam diferentes mecanismos de defesa em resposta a patógenos. Em geral, ocorre o reconhecimento de padrões moleculares associados ao patógeno (PAMPs) por receptores específicos das plantas, desencadeando as respostas de defesa através das vias de sinalização mediadas por jasmonato (JA), etileno (ET) e ácido salicílico (AS). Além disso, a descoberta de sinais endógenos como AtPep1 que regulam a expressão de genes de defesa em Arabidopsis thaliana tem auxiliado na compreensão dos mecanismos de defesa. No entanto, os mecanismos de defesa de plantas a patógenos necrotróficos como Pythium spp. são pouco conhecidos. Desta forma, o desenvolvimento de estratégias para identificar e caracterizar genes de resistência à Pythium spp. pode auxiliar na compreensão dos mecanismos de defesa, possibilitando a obtenção de cultivares resistentes. Uma das estratégias empregadas para a identificação de genes de resistência a patógenos do gênero Pythium consistiu na construção de uma biblioteca de cDNAs diferencialmente expressos em A. thaliana susperexpressando AtPROPEP1, que são mais resistentes à P. irregulare e a P. deliense. Paralelamente, foi realizada a busca de um gene ortólogo ao AtPROPEP1 de A. thaliana em diferentes espécies de solanáceas. Outra estratégia utilizada envolveu a avaliação do efeito da aplicação dos hormônios JA e AS e do regulador de crescimento etefon na resistência de plantas de tomate à P. dissotocum. Posteriormente foi realizado o silenciamento dos genes CTR1, ERF1 e SlPROPEP em plantas de tomate a fim de verificar o papel dos mesmos na resistência das plantas à P. dissotocum. Os resultados obtidos na biblioteca subtrativa indicam que A. thaliana superexpressando AtPROPEP1 pode induzir a expressão não somente da defensina PDF1.2, mas também de GRP3, outro gene envolvido na defesa de plantas contra patógenos. Através da busca de ortólogos de AtPROPEP1 em diferentes solanáceas foi possível identificar um ortólogo em Solanum lycopersicum - SlPROPEP. O tratamento com etefon aumentou a resitência das plantas à P. dissotocum comparado com as plantas não tratadas. Este tratamento também induziu a expressão relativa de SlPROPEP, PR-1, PR-5 e da defensina DEF2. Assim como o tratamento com etefon o silenciamento do gene CTR1, que atua como um regulador negativo da via de sinalização de defesa por ET aumentou a resistência das plantas à P. dissotocum. Enquanto que as plantas silenciadas para os genes ERF1 e SlPROPEP foram mais suscetíveis. O silenciamento de SlPROPEP reprimiu a expressão relativa dos genes PR-1, PR-5, ERF1, LOX-D e DEF2 envolvidos na defesa de plantas à patógenos. Estes resultados, em conjunto com o aumento da resistência nas plantas tratadas com etefon, sugerem que a via de sinalização de ET atua na resistência de tomate à P. dissotocum sendo que esta resistência possivelmente é mediada por SlPROPEP. / Plants express different defense mechanisms in response to pathogens. In general, specific receptors in plants reconize pathogen-associated molecular patterns (PAMPs), triggering the response defenses by signaling pathways mediated by jasmonate (JA), ethylene (ET) and salicylic acid (SA). Furthermore, the discovery of endogenous signals, as AtPep1, which regulate defense genes expression in Arabidopsis thaliana, has aided the understanding of the defense mechanisms. However, the defense mechanisms of plants to necrotrophic pathogens such as Pythium spp. are poorly known. Thus, development of strategies to identify and characterize the resistance genes to Pythium spp. can help the understand of defense mechanisms, allowing the obtainance of resistant cultivars. One of the strategies used for the identification of resistance genes to pathogens within the genus Pythium consisted to construct a cDNA library of differentially expressed genes in plants overexpressing AtPROPEP1, which are more resistant to P. irregulare and P. deliense. Simultaneously, a search for orthologous genes to AtPROPEP1 from A. thaliana in different species of Solanaceae was performed. Another strategy used involved the evaluation of application of JA and AS and of the growth regulator ethephon on the resistance of tomato plants to P. dissotocum. Then, the silencing of the genes CTR1, ERF1 and SlPROPEP was performed in tomato plants in order to check the role of the tomato resistance to P. dissotocum. The results obtained from subtractive cDNA library indicate that A. thaliana plants overexpressing AtPROPEP1 may induce expression not only of the PDF1.2 defensin but also the GRP3 expression, another gene involved in plant defense against pathogens. By the search for orthologous genes to AtPROPEP1 in different species of Solanaceae was possible to identify an ortholog in Solanum lycopersicum - SlPROPEP. The ethephon treatment increased resistance of plants to P. dissotocum compared to untreated plants. This treatment also induced the expression of SlPROPEP, PR-1, PR-5 and DEF2 defensin. As ethephon treatment, the silencing of CTR1 gene, which acts as a negative regulator of defense signaling pathway by ET, increased plant resistance to P. dissotocum. Whereas the plants containing ERF1 and SlPROPEP genes silenced were more susceptible. SlPROPEP silencing repressed the relative expression of the genes PR-1, PR-5, ERF1, LOX-D e DEF2 involved in plant defense to pathogens. These results, together with increased resistance in the plants treated with ethephon, suggest that ET signaling pathway acts in tomato to resistance P. dissotocum and this resistance is likely mediated by SlPROPEP.