Even though the structure of the plant cell wall is by and large quite well characterized, its synthesis and regulation remains largely obscure. However, it is accepted that the building blocks of the polysaccharidic part of the plant cell wall are nucleotide sugars. Thus to gain more insight into the cell wall biosynthesis, in the first part of this thesis, plant genes possibly involved in the nucleotide sugar interconversion pathway were identified using a bioinformatics approach and characterized in plants, mainly in Arabidopsis. For the computational identification profile hidden markov models were extracted from the Pfam and TIGR databases. Mainly with these, plant genes were identified facilitating the “hmmer” program. Several gene families were identified and three were further characterized, the UDP-rhamnose synthase (RHM), UDP-glucuronic acid epimerase (GAE) and the myo-inositol oxygenase (MIOX) families. For the three-membered RHM family relative ubiquitous expression was shown using variuos methods. For one of these genes, RHM2, T-DNA lines could be obtained. Moreover, the transcription of the whole family was downregulated facilitating an RNAi approach. In both cases a alteration of cell wall typic polysaccharides and developmental changes could be shown. In the case of the rhm2 mutant these were restricted to the seed or the seed mucilage, whereas the RNAi plants showed profound changes in the whole plant. In the case of the six-membered GAE family, the gene expressed to the highest level (GAE6) was cloned, expressed heterologously and its function was characterized. Thus, it could be shown that GAE6 encodes for an enzyme responsible for the conversion of UDP-glucuronic acid to UDP-galacturonic acid. However, a change in transcript level of variuos GAE family members achieved by T-DNA insertions (gae2, gae5, gae6), overexpression (GAE6) or an RNAi approach, targeting the whole family, did not reveal any robust changes in the cell wall. Contrary to the other two families the MIOX gene family had to be identified using a BLAST based approach due to the lack of enough suitable candidate genes for building a hidden markov model. An initial bioinformatic characterization was performed which will lead to further insights into this pathway. In total it was possible to identify the two gene families which are involved in the synthesis of the two pectin backbone sugars galacturonic acid and rhamnose. Moreover with the identification of the MIOX genes a genefamily, important for the supply of nucleotide sugar precursors was identified. In a second part of this thesis publicly available microarray datasets were analyzed with respect to co-responsive behavior of transcripts on a global basis using nearly 10,000 genes. The data has been made available to the community in form of a database providing additional statistical and visualization tools (http://csbdb.mpimp-golm.mpg.de). Using the framework of the database to identify nucleotide sugar converting genes indicated that co-response might be used for identification of novel genes involved in cell wall synthesis based on already known genes. / Obwohl der Aufbau der pflanzlichen Zellwand im Großen und Ganzen relativ gut charakterisiert ist, ist relativ wenig über ihre Synthese bekannt. Allgemein akzeptiert ist jedoch, dass die Nukleotidzucker die Vorstufe für den polysaccharidären Teil der Zellwand stellen. Im Rahmen der vorliegenden Arbeit wurden neue Kandidatengene für die Zellwandbiosynthese mittels bioinformatorischer Analysen ermittelt und deren Rolle in Pflanzen, hauptsächlich Arabidopsis thaliana untersucht. Zur Identifizierung von Arabidopsis thaliana Kandidatengenen des Nukleotidzucker-Stoffwechselweges wurden „hidden Markov Modelle“ für Gene desselben aus den Datenbanken Pfam und TIGR extrahiert. Unter anderem wurden diese dann unter Zuhilfenahme des Programms hmmer zur Identifikation von pflanzlichen Genen benutzt. Es wurden einige Genfamilien identifiziert und drei von diesen wurden weiter charakterisiert. Hierbei handelte sich um eine UDP-Rhamnose Synthase Familie (RHM), eine UDP-Glucuronsäurepimerase Familie (GAE) und eine myo-Inositol Oxygenase Familie (MIOX). Für die RHM Kandidatengenfamilie, mit drei Mitgliedern, wurde die relativ ubiquitäre Expression aller Gene mittels verschiedener Methoden gezeigt und für eines der Gene, RHM2, konnten T-DNA Linien bezogen werden. Außerdem wurde die Transkription der gesamten Familie mittels eines RNAi Konstruktes herunter geregelt. In beiden Fällen konnte eine Veränderung von zellwandtypischen Polysacchariden sowie schwere Entwicklungsstörungen gezeigt werden. Diese waren bei der rhm2 Funktionsverlustpflanze auf den Samenschleim bzw. den Samen reduziert, bei den RNAi Pflanzen hingegen war die gesamte Pflanze betroffen. Im Falle der zweiten Kandidatengenfamilie, GAE, wurde das höchst-exprimierte Gen (GAE6) kloniert, heterolog exprimiert und die Funktion charakterisiert. So konnte gezeigt werden, dass GAE6 für ein Enzym kodiert, welches UDP-Glukuronsäure in UDP-Galakturonsäure wandelt. Allerdings zeigten Pflanzen mit veränderter Transkriptmenge, erreicht durch T-DNA Insertionen (gae2, gae5, gae6), Überexpression (GAE6) oder RNAi / keine robuste Veränderung der Zellwand. Die letzte betrachtete Kandiatengenfamilie myo-Inositol Oxygenase wurde im Gegensatz zu den beiden anderen Familien, durch eine BLAST Suche gefunden, da zur Zeit der Durchführung noch zu wenig myo-inositol Oxygenasen bekannt waren, um daraus „hidden Markov Modelle“ abzuleiten. Dennoch konnten erste bioinformatorische Analysen zu dieser Genfamilie gemacht werden. Insgesamt gesehen wurden in diesem Teil der Arbeit die beiden Genfamilien identifiziert und charkterisiert, die bei der Synthese der beiden Pektinrückgradzucker Rhamnose und Galakturonsäure die tragende Rolle spielen. Weiterhin wurde mit der Identifizierung der MIOX Genfamilie, eine Genfamilie identifiziert, die wichtige Vorstufen in der Synthese der Nukleotidzucker liefert. In einem zweiten Teil der Arbeit wurden öffentlich zugängliche Mikroarray-Daten durch ihr Gleich -oder Ungleichverhalten charakterisiert. Dieses erfolgte auf globaler Ebene für zunächst fast 10.000 Gene. Die Daten wurden in Form einer allgemein zugänglichen Datenbank der Allgemeinheit zur Verfügung gestellt (http://csbdb.mpimp-golm.mpg.de). Eine Anwendung der Methode auf Gene des Nukleotidzuckerstoffwechsels, deutet darauf hin, dass so neue Kandiatengene, die bei der Zellwandsynthese eine Rolle spielen, von bereits bekannten Genen abgeleitet werden können.

Bibliography : leaves 197-209 / 210 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Botany, 1991

Thesis (Ph.D.) -- University of Western Sydney, 2002. / "A thesis submitted in fulfilment of the requirement for the degree of Doctor of Philosophy" Bibliography: leaves 161-189.

Orientadores: Márcia Regina Braga, Simone de Pádua Teixeira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T00:05:19Z (GMT). No. of bitstreams: 1 Caccere_Rodrigo_M.pdf: 5287310 bytes, checksum: 7433584cc6dacc1485f7ed86dd51116e (MD5) Previous issue date: 2010 / Resumo: Inga vera Pennington produz sementes com alta sensibilidade à dessecação, o que dificulta seu armazenamento. Diversos mecanismos estão relacionados tolerância à dessecação, dentre eles o acúmulo de reservas insolúveis e de moléculas protetoras, redução do metabolismo e dobramento da parede celular. Desse modo, o objetivo deste trabalho foi analisar o comportamento de eixos embrionários e cotilédones de I. vera com respeito a seus teores e composição de açúcares solúveis, de reserva e de parede celular e quanto à ultra-estrutura durante a maturação e após a secagem. Eixos embrionários e cotilédones de I. vera durante a maturação, acumulam altos níveis de amido e armazenam também outras substâncias como compostos fenólicos. O acúmulo de massa seca e o processo de vacuolização durante todo o desenvolvimentos dos embriões desta espécie indicam alta atividade metabólica até o momento da dispersão. Além disso, as paredes celulares de eixos embrionários e cotilédones acumulam galactanos que podem lhe conferir maior rigidez. Embriões de I. vera secos até 35% de teor de água apresentaram redução da capacidade germinativa. Esta desidratação parcial provocou um estímulo metabólico, evidenciado pela mobilização de reservas e deposição de material nas paredes celulares, além de intensa atividade do retículo endoplasmático rigoso, observada nos eixos embrionários. A secagem severa (17% de teor de água) provocou rompimento das membranas resultando no aparecimento de células completamente colapsadas. Dessa forma, pode-se concluir que embriões I. vera mantém o metabolismo ativo durante a desidratação até que os processos de injúria comecem a ocorrer. / Abstract: Inga vera Pennington produces recalcitrant seeds, characterized by desiccation sensibility and short post-harvest life span, no longer than one mouth. Mechanisms proposed to explain the ability of organisms to survive desiccation include accumulation of insoluble reserves and protective molecules, metabolic "switch off" and cell wall folding, among others. The aim of the present study was to analyze the behavior of I.vera embryos (axes and cotyledons) with respect to sugar content, cell wall composition and ultrastructure during different stages of development and after desiccation. Axes and cotyledons accumulate starch and phenolic compounds and also showed vacuolization all over development, suggesting high metabolic activity up to the end of the maturation period. Moreover, cell walls of axes and cotyledons cantain polysaccharides, like galactans, that can provide more rigidity to the cell wall. Mature I.vera seeds were dried 35% or 17% and seed variability was skilghtly reduced due to drying to 35% of water content, but no seeds survived to severe desiccation (17% water content). Starch mobilization, increase in the cell wall thickness in axes and cotyledons, and high degree of development of the rough endoplasmic reticulum in axes suggest that drying to 35% of water content enhanced metabolic activity. Severe desiccation resulted in membrane breaking leading to collapsed protoplasm. Therefore we can conclude that I.vera embryos keep high metabolic activity during desiccation until damage processes start. / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

A ocorrência de mamões Golden com casca verde e polpa mole, em determinadas épocas do ano, tem sido relatada por produtores da região Norte do Espírito Santo. Não sendo possível distinguir frutos com o distúrbio do amolecimento precoce (DAP) apenas pela análise da cor da casca e da firmeza da polpa no momento da colheita, o objetivo deste trabalho foi estudar aspectos da fisiologia e bioquímica do amadurecimento que pudessem auxiliar no entendimento da perda precoce da firmeza. As coletas ocorreram no período de maior frequência de frutos com o distúrbio (meados de verão a final de outono). Os frutos foram coletados no estádio 1 de maturação, armazenados a 10º±1oC e 85±10% UR, durante 10 dias e submetidos à metodologia do Índice de Amolecimento (IA). O sintoma do amolecimento precoce foi observado claramente no 4°dia após o armazenamento sendo verificada a perda de firmeza nos frutos com DAP entre o 2° e o 8°dia a 10°C, sendo o decréscimo na firmeza equivalente a 71% da firmeza inicial, comportamento atípico para frutos armazenados sob refrigeração. Frutos com e sem DAP não apresentaram diferença significativa no teor de sólidos solúveis, acidez titulável e ácido ascórbico. A atividade respiratória e a produção de etileno foram reduzidas com o armazenamento refrigerado, sem diferença entre os frutos com e sem DAP, o que sugere que o aparecimento do distúrbio não se deve ao aumento na produção de etileno no período póscolheita. Os resultados indicam forte associação entre o amolecimento precoce e mudanças na parede celular. O rendimento das frações solúvel em água (FSA) e em NaOH (FSH) nos frutos com DAP, foi superior ao observado nos frutos sem DAP, durante todo o período de armazenamento. O menor nível da fração solúvel em imidazol (FSI) no tempo 0 pode estar associado à maior atividade da pectinametilesterase nos frutos com DAP. O decréscimo na fração celulose (CEL) em frutos com DAP pode indicar modificação na parede celular, ao nível celulose-hemicelulose. A produção de etileno não acompanhou as modificações na parede celular, sugerindo que alterações na estrutura da parede podem ter ocorrido durante o crescimento do fruto. Frutos com DAP apresentaram níveis de auxina (AIA) 6 vezes menor do que frutos sem DAP, no dia 0, sem diferença durante o armazenamento. Possivelmente os níveis de AIA nos frutos com DAP estavam reduzidos quando o fruto ainda estava ligado à planta. O distúrbio possivelmente está relacionado à alteração na parede celular e níveis de AIA. Desta forma, uma alteração hormonal durante o desenvolvimento do fruto anterior à colheita -, associada à sensibilidade do fruto à essa sinalização hormonal, levaria à mudanças na parede celular, o que determinaria a ocorrência do distúrbio do amolecimento precoce. / The occurrence of green skin and soft pulp in Golden papaya fruit during certain seasons has been reported by orchards in northern Espírito Santo State, Brazil. It is not possible solely by skin color and pulp firmness analysis to distinguish early softening disorder fruits (DAP), at the time of harvest, from those without the disorder. The aim of this work was to study the physiological and biochemical ripening aspects that might help to understand the early softening disorder. Fruits were harvested on the most common seasons (mid-summer to mid-autumn) in maturity stage 1 and evaluated for 10 days. Fruits were stored at 10°C, and submitted to softening index (IA). The symptoms of early softening disorder were clearly observed on the 4th day after storage. However, the firmness decrease in DAP fruits happened between the 2nd and 8th day at 10°C, with a decrease of 71% of initial firmness, presenting an atypical behavior for fruits stored under refrigeration. There was no difference between fruits with DAP and without DAP for soluble solids, titratable acidity and ascorbic acid content. Besides that, there was no difference between fruits with DAP and without DAP for respiration rate and ethylene production under refrigerated storage, which suggests that the disorder occurrence is not related to an increase in ethylene in the postharvest period. The results indicate a strong association between early softening disorder and changes in the cell wall composition. The cell wall polymers fractionation show that the yield of water soluble polymers (FSA) and NaOH soluble polymers (FSH) was higher in fruits with DAP than on fruits without DAP during the entire storage period. The smallest level of the imidazole soluble fraction (FSI) in DAP fruit, on day 0, may be related to a higher activity of pectinmethylesterase. The decrease in cellulose-rich polysaccharides (CEL) in the DAP fruit suggest modifications in the cellulose-hemicellulose domain. Ethylene production did not follow cell wall modification, suggesting that changes in the cell wall structure could be happening during fruit growth. DAP fruit presented an AIA level 6 times smaller than fruit without DAP, in day 0, without storage differences. Possibly, AIA levels in DAP fruit were reduced while fruits were still connected to the plant. The disorder may be related to cell wall changes and to AIA level. Should a hormonal change occur during fruit development i. e. prior to harvesting -- and still depending on fruit sensibility to hormonal signaling, cell wall changes could have different intensities, which determine if the fruit will or not have the flesh early softening observed after harvest.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21808号 / 農博第2321号 / 新制||農||1066(附属図書館) / 学位論文||H31||N5180(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 渡邊 隆司, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

ARP2/3 is an evolutionarily conserved heteroheptameric protein complex. Its main activity lies in the nucleation of dendritic actin filaments that are involved in membrane remodeling. ARP2/3 takes part in plasma membrane remodeling and the formation of cytoplasmic protrusions that serve in the amoeboid motion of mammalian cells and some protists and plays role in exocytosis and endocytosis of animal and yeast cells. The main objective of this work was to find a connection between the ARP2/3 complex and the regulation of the plant endomembrane system. Using TIRF microscopy we visualized the localization of the ARP2/3 complex in the cortical layer of plant cells and compared it to the localization of several endomembrane markers from the Rab family and an exocytotic marker Exo84b. In the vicinity of the plasma membrane, the ARP2/3 complex subunits localized to dynamic dots very similar to the localization of Exo84b protein. Colocalization analysis showed that a small portion of Exo84b marker and ARP2/3 complex signals colocalize and this result was seconded by the biochemical approach of coimmunoprecipitation. Key words: ARP2/3, endomembrane system, cortical layer, RabA1g, RabC1, RaD2a, Exo84b

<p> The plant cytoskeleton is commonly considered a vital component of cell growth and development; however, it also plays a critical role in plant immunity. During plant immunity, the cytoskeleton orchestrates rapid and precise immune-associated processes. For instance, the cytoskeleton mobilizes and orients the movement of organelles, proteins, and chemical signaling. To counter plant immunity, bacterial pathogens deliver virulence proteins, known as T3Es (type III effectors), into plant cells through a needle-like apparatus called the type III secretion system (T3SS). A novel T3E, called RipU, interacts with the cytoskeleton. Data has shown that RipU co-localizes with cytoskeletal markers in tobacco leaves. Ectopic expression of RipU can suppress PTI responses like ROS bursts or seedling growth inhibition. Tomato plants inoculated with <em>Rs</em> K60 lacking RipU showed less wilting and root colonization, suggesting that RipU plays a role in pathogenesis and virulence. Furthermore, inducible expression of RipU in Arabidopsis dramatically alters plant development. These plants have wavy roots, branching root hairs, and underdeveloped true leaves. Our results suggest that by targeting the cytoskeleton, RipU contributes to <em>Rs</em> K60s pathogenicity and virulence. </p>

<p>Nitrogen and phosphorus are the two macronutrients which play important roles in the plant, both structurally and functionally, e.g., starting from being constituents of cellular integrity to being signal molecules in signal transduction. Since they are required by plants in higher concentrations, it becomes indispensable to replenish their pools in soils by the application of chemical fertilizers. However, this practice is not only costly, the sources of Phosphorus and Nitrogen are not renewable and the excessive application in the form of fertilizers is not environmentally sustainable. Therefore, it warrants a better understanding of the plant responses during the nutrient deficiency because such knowledge will help implement strategies for breeding crops with more efficient use of minerals.</p><p>Most prior efforts in studying the molecular and physiological responses to low minerals were focused on roots. However, recently it has been found that shoot-to-root long distance signaling plays an important role in the adaptation of roots to low nitrogen or phosphorus. Here, we measured different physiological and morphological parameters and used RNA-Seq to elucidate the physiological and molecular responses in the vascular tissues of <i>Plantago major</i>, a new model species established in our laboratory, to low nitrogen, low phosphate or combined nitrogen and phosphate starvation<i>. </i>In this study, <i>P major </i>showed reduced photosynthesis and Fv/Fm, increased catalase and ascorbate peroxidase activity, reduced phosphate and nitrate contents in respective treatments. In addition, assessment of root morphological parameters revealed that nutrient deficiencies could lead to higher root densities and increased root to shoot ratios.</p><p>For molecular analysis of transcriptome changes, 24 hours of nutrient starvation exhibited an alteration of 33, 221, and 329 genes for the deficiencies of phosphorus, nitrogen and combined nitrogen and phosphorus, respectively. Our study helped to dissect several novel pathways associated with the vascular system in response to the deficiencies of major macronutrients. </p>

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24670号 / 農博第2553号 / 新制||農||1099(附属図書館) / 学位論文||R5||N5451(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 梅澤 俊明, 教授 矢﨑 一史, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM