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Orientador: Paulo Mazzafera / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T10:40:28Z (GMT). No. of bitstreams: 1 Geromel_Clara_D.pdf: 2807679 bytes, checksum: 87559525feb767ee465302f9b3380513 (MD5) Previous issue date: 2006 / Resumo: Sabendo-se que a produtividade da cultura de café está diretamente ligada a três fatores básicos de produção, climáticos, genéticos e fisiológicos, nesse estudo foram abordados alguns aspectos do metabolismo de carboidratos envolvidos no processo de enchimento dos frutos de café ao longo do seu desenvolvimento. A composição de carboidratos, principalmente de polissacarídeos do grão (endosperma) de café é conhecida, assim como a importância dos açúcares sobre a qualidade da bebida, porém a importação de sacarose a partir de folhas e seu desdobramento nos frutos ainda não são completamente esclarecidos. Os açúcares utilizados no metabolismo das sementes são de extrema importância, tendo como exemplo a regulação da relação fonte-dreno, além de controlar a expressão de genes que codificam algumas enzimas envolvidas no metabolismo de açúcares. O trabalho teve como principal objetivo estudar o metabolismo de sacarose nos frutos de café, ao longo do desenvolvimento. Análises histológicas e fornecimento de compostos marcados mostraram que não existem conexões vasculares entre os tecidos, pericarpo,perisperma e endosperma, mas vasos condutores que percorrem o funículo chegam até o perisperma, permitindo um descarregamento direto de fotoassimilados produzidos nas folhas e dele a transferência para o endosperma, além de receber fotoassimilados do pericarpo, pelo transporte de célula a célula. A fotossíntese no pericarpo diminui ao longo do desenvolvimento do fruto. Grãos de amido foram observados nos estados jovens do perisperma e à medida que surge o endosperma, esses grãos vão desaparecendo em regiões próximas ao tecido que está sendo formado. As enzimas e os teores de açúcares endógenos avaliados em tecidos separados ao longo da maturação do fruto apresentaram diferenças entre diferentes tratamentos da lavoura (¿pleno sol¿: condições normais de cultivo, sombreamento e carga reduzida). A sacarose sintase (SUS) apresentou valores de atividades bem maiores que das invertases ácidas (IAV). A sacarose fosfato sintase (SPS) acompanhou o acúmulo de sacarose no estádio inicial no pericarpo de frutos de café. Foi mostrada a existência de duas isoformas de SUS, codificadas por dos genes chamados CaSUS1 e CaSUS2 em C. arabica, que possivelmente desempenham diferentes funções metabólicas nos diferentes tecidos do fruto de café. Esses genes apresentam uma expressão diferencial, com CaSUS1 expressando-se nas fases jovens do desenvolvimento do perisperma e do endosperma; porém a expressão de CaSUS2 sobrepõe os picos de atividade SUS detectados e o acúmulo de sacarose nos estados finais de desenvolvimento do pericarpo e do endosperma. Isso sugere que a isoforma CaSUS1 está relacionada à degradação de sacarose, quando parece claro que a isoforma CaSUS2 está relacionada com a síntese desse açúcar. Foi mostrado que esses dois genes se expressam também in C. racemosa, pois as sondas CaSUS1 e CaSUS2 de C. arabica reconheceram transcritos (mRNA) no endosperma dessa espécie. Portanto, os genes CrSUS1 e CrSUS2 parecem codificar para isoformas de SUS que desempenham funções diferentes daquelas observadas em C. arábica; SUS1 parece estar relacionado com a síntese da sacarose. O sombreamento influenciou na duração do ciclo de desenvolvimento dos frutos, tornando-o mais longo que no pleno sol (respectivamente 260 e 231 dias após o florescimento) e alterando o acúmulo de sacarose nos frutos. Com base nesses resultados fica clara a complexidade do metabolismo de sacarose em frutos de café, visto que nem sempre as atividades enzimáticas seguem o mesmo padrão de acúmulo de açúcares e enzimas de degradação e ressíntese de sacarose atuam simultaneamente, assim como a transferência de açúcares entre os tecidos / Abstract: Since coffee culture productivity is straightly connected to three basic production factors: climate, genetics and physiology, herein some aspects of the carbohydrates metabolism involved in the process of coffee grains filling through its development were analyzed. It is known the carbohydrates composition, mainly the polysaccharides composition of the coffee grain (endosperm) as well as the importance of sugars in the beverage quality, nevertheless the sucrose import from the leaves and its sharing in the fruits are still to be completely clarified. The sugars utilized in the seeds metabolism are extremely important, such as in the regulation of the drain-source relation and in the expression control of genes that codify some enzymes involved in sugars metabolism. The main goal of this work was to study the sucrose metabolism in coffee fruits through their development. Histological analyses and marked compounds supply showed that there are no vascular connections among the tissues of the pericarp, perisperm and endosperm, but conduction vases that run through the funiculus get to the perisperm, enabling an unloading of photoassimilates produced in the leaves. From the perisperm, these assimilates are transferred to the endosperm; The pericarp photosynthesis diminishes through the fruit development. Starch grains were observed in juvenile stages of the perisperm and during the endosperm formation these grains start to disappear in regions close to the forming tissue. The enzymes and the endogenous sugar level evaluated in separated tissues through fruit maturation show some differences among distinct lavoura treatments (¿full sun¿: ordinary culture conditions, shadowing and reduced loading). The sucrose synthase (SUS) showed activity values much higher than the ones presented by the acid invertases (IAV). The sucrose phosphate synthase (SPS) followed the sucrose accumulation in the pericarp initial stage in coffee fruits. It was shown the existence of two isoforms of SUS codificated by genes called CaSUS1 e CaSUS2 in C. Arabica, that possibly play different metabolic roles in different tissues in coffee fruit. These genes show a differential expression, in which CaSUS1 is expressed in the juvenile stage of perisperm and endosperm development; however, CaSUS2 expression overlaps the detected activity peaks of SUS and the sucrose accumulation in the final stages of pericarp and endosperm development. This fact suggests that the CaSUS1 isoform is related to sucrose degradation while it seems clear that the CaSUS2 isoform is related to sucrose synthesis. Both genes were shown to be also expressed in C.racemosa since CaSUS1 and CaSUS2 C. arabica probes recognized transcripts (mRNA) in this species endosperm. Therefore, CrSUS1 and CrSUS2 genes seem to codify SUS isoforms that play different functions from the ones observed in C. arabica; SUS1 seems to be related to sucrose synthesis. The shadowing has influenced the fruits development cycle duration, turning it longer than in pleno sol (respectively, 260 and 231 days after flowering) and altering the sucrose accumulation in fruits. According to these results, it is clear how complex is the sucrose metabolism in coffee fruits, since it is not always that the same pattern of sugar accumulation is followed by the enzymatic activities and enzyme degradation and sucrose (re)synthesis act simultaneously as well as sugar transference among tissues / Doutorado / Biologia Vegetal / Doutor em Biologia Vegetal

Sucrose synthase (Susy) is a key enzyme of sucrose metabolism, catalysing the reversible conversion of sucrose and UDP to UDP-glucose and fructose. Therefore, its activity, localization and function have been studied in various plant species. It has been shown that Susy can play a role in supplying energy in companion cells for phloem loading (Fu and Park, 1995), provides substrates for starch synthesis (Zrenner et al., 1995), and supplies UDP-glucose for cell wall synthesis (Haigler et al., 2001). Analysis of the Arabidopsis genome identifies six Susy isoforms. The expression of these isoforms was investigated using promoter-reporter gene constructs (GUS) and real time RT-PCR. Although these isoforms are closely related at the protein level they have radically different spatial and temporal patterns of expression in the plant with no two isoforms showing the same distribution. More than one isoform is expressed in all organs examined. Some of them have high but specific expression in particular organs or developmental stages whilst others are constantly expressed throughout the whole plant and across various stages of development. The in planta function of the six Susy isoforms were explored through analysis of T-DNA insertion mutants and RNAi lines. Plants without the expression of individual isoforms show no differences in growth and development, and are not significantly different from wild type plants in soluble sugars, starch and cellulose contents under all growth conditions investigated. Analysis of T-DNA insertion mutant lacking Sus3 isoform that was exclusively expressed in stomata cells only had a minor influence on guard cell osmoregulation and/or bioenergetics. Although none of the sucrose synthases appear to be essential for normal growth under our standard growth conditions, they may be necessary for growth under stress conditions. Different isoforms of sucrose synthase respond differently to various abiotic stresses. It has been shown that oxygen deprivation up regulates Sus1 and Sus4 and increases total Susy activity. However, the analysis of the plants with reduced expression of both Sus1 and Sus4 revealed no obvious effects on plant performance under oxygen deprivation. Low temperature up regulates Sus1 expression but the loss of this isoform has no effect on the freezing tolerance of non acclimated and cold acclimated plants. These data provide a comprehensive overview of the expression of this gene family which supports some of the previously reported roles for Susy and indicates the involvement of specific isoforms in metabolism and/or signalling. / Saccharose spielt eine zentrale Rolle in höheren Pflanzen. Es zählt zu den wichtigsten Kohlenhydraten und wird als Nährstoff, Speicherstoff (z.B. in Zuckerrüben, Zuckerrohr, Mohrrüben) oder auch als potentielles Signalmolekül verwendet. Saccharose ist eines der primären Endprodukte der Photosynthese in den grünen Blättern der Pflanzen, kann aber auch in nicht-photosynthetisch aktiven Geweben (z.B. in keimenden Samen) synthetisiert und verstoffwechselt werden. Die Saccharosesynthase (Susy) stellt ein Schlüsselenzym im Saccharosestoffwechsel dar. Es katalysiert die reversible Umwandlung von Saccharose zu UDP-Glukose und Fruktose. Die Aktivität, die Lokalisierung und die Funktionen der Susy wurden bereits in verschiedenen Pflanzenarten untersucht. Dabei hatte sich herausgestellt, daß die Susy eine wichtige Rolle in der Bereitstellung von Energie für Transportprozesse spielt. Außerdem stellt Susy die Substrate für die Stärkesynthese in Speichergeweben, sowie fast alle Substrate für die Zellwandsynthese bereit. Eine Untersuchung des Genoms von Arabidopsis thaliana ergab, daß die Ackerschmalwand sechs Isoformen der Susy besitzt. Die Expression dieser Isoformen wurde mittels Echtzeit RT-PCR analysiert. Obwohl die verschiedenen Isoformen auf Proteinebene in ihrer Sequenz sehr ähnlich sind, zeigen sie Unterschiede in ihrem zeitlichen und räumlichen Auftreten innerhalb der Pflanze. Einige der Isoformen sind hoch exprimiert in speziellen Organen oder Entwicklungsstufen der Pflanze. Andere hingegen sind gleichmäßig in der ganzen Pflanze und über verschiedene Entwicklungsstufen hinaus exprimiert. In allen untersuchten Organen der Pflanze ist mehr als eine Isoform exprimiert. Um die spezifische Funktion der einzelnen Isoformen aufzuklären, wurden für alle sechs Saccharosesynthasen Mutanten-Linien isoliert und analysiert. Alle Pflanzen, bei denen die Expression einer bestimmten Isoform fehlte, zeigten im Vergleich zu Wildtyppflanzen keine signifikanten Unterschiede in Wachstum und Entwicklung. Des Weiteren waren die Gehalte an Stärke, Saccharose und Zellulose in Blättern und Wurzeln im Vergleich zu Wildtyppflanzen unverändert. Mutanten, denen die ausschließlich in Schließzellen lokalisierte Isoform Sus3 fehlte, zeigten nur geringe Veränderungen in der Osmoregulation und/oder der Bioenergetik der Schließzellen. Daraus kann gefolgert werden, dass in dem Ackerunkraut Arabidopsis keine der Saccharosesynthasen essentiell für normales Wachstum unter Standardbedingungen ist. Es ist jedoch möglich, dass Saccharosesynthasen unter Stressbedingungen benötigt werden. Es war bereits bekannt, dass einzelne Isoformen der Susy auf Stress reagieren und in ihrer Expression verändert sind. Es konnte gezeigt werden, daß Sauerstoffmangel zu einer Erhöhung der Expression der Isoformen Sus1 und Sus4 und zu einer Zunahme der Susy Gesamtaktivität führt. Die Analyse von Pflanzen mit reduzierter Expression von Sus1 und Sus4 zeigte jedoch, dass Sauerstoffmangel keinen offensichtlichen Einfluss auf das Wachstum dieser Pflanzen hat. Niedrige Temperaturen führen zu einer Erhöhung der Sus1 Expression, aber auch ein Verlust dieser Isoform hat keinen Einfluss auf die Gefriertoleranz von normalen oder an Kälte akklimatisierten Pflanzen. Diese Ergebnisse bieten einen umfassenden Einblick in die Expression der Genfamilie der Saccharosesynthase; sie untermauern die genannten Funktionen der Saccharosesynthase und weisen auf eine mögliche Beteiligung mehrerer Isoformen am Saccharosestoffwechsel und/oder der Signaltransduktion hin.

Sucrose is the major transported photoassimilate in plants and is degraded concurrently by two enzymes: invertases and sucrose synthase. Sucrose synthase catalyzes the reversible conversion of UDP and sucrose to form fructose and UDP-glucose, the latter being the activated substrate for many metabolic processes including cellulose biosynthesis. There is evidence that sucrose synthase is phosphorylated as a regulatory mechanism of carbon allocation at a conserved N-terminal serine residue. The phosphorylation or dephosphorylation at this specific site has also been found to shift the protein localization in a tissue and species specific manner. A literature study of the functional regulation of sucrose synthase in plants has highlighted several scientific questions: Is sucrose synthase cellular localization regulated by phosphorylation of an N-terminal conserved serine residue? What are the regulatory mechanisms underlying within and between species variation in sucrose synthase localization? Does sucrose synthase associate with the cellulose synthase enzyme complex? Can cellulose biosynthesis be increased by over-expression of the membrane-associated form of sucrose synthase? The aim of this M.Sc study was to determine the subcellular localization of Eucalyptus grandis sucrose synthase 1 (EgSUSY1) fusion proteins expressed in Arabidopsis thaliana plants. This was investigated through modifying the 11th serine residue of EgSUSY1 into either a non-polar alanine residue that cannot be phosphorylated (S11A), or into a negatively charged glutamic acid residue which may mimic phosphorylation at this site (S11E). The modified proteins were translationally fused to green fluorescent protein (GFP) and expressed in transgenic Arabidopsis thaliana. The proteins’ subcellular localization were analysed in planta using laser scanning confocal microscopy (LSCM). Findings in this study point to the peripheral localization of modified and unmodified GFPEgSUSY1 proteins with a prominent cytoplasmic component. No evidence was found for the localization of modified or unmodified GFP-EgSUSY1 proteins within the extracellular matrix. The current study did not establish nor negate plasma membrane association of any of the GFP-EgSUSY1 fusion proteins. It was concluded that alternative methodologies need to be explored to further address issues surrounding subcellular localization of sucrose synthase. These studies will not only aid in defining the role of this enzyme in carbon allocation, but also add to our expanding knowledge of cellulose biosynthesis and cell wall formation. Copyright 2011, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Sauer, J 2011, The subcellular localization of eucalyptus grandis sucrose synthase 1 (EgSUSY1) fusion proteins expressed in Arabidopsis thaliana, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-02102012-102209 / > C12/4/111/gm / Dissertation (MSc)--University of Pretoria, 2011. / Genetics / unrestricted

Chez les végétaux supérieurs, lembryogenèse est une phase clé du développement au cours de laquelle lembryon établit les principales structures de la future plante. La compréhension des processus moléculaires et physiologiques menant à la formation de la graine est donc dun intérêt agronomique majeur. Chez Phaseolus la caractérisation moléculaire de lembryogenèse permet de mieux comprendre les mécanismes du développement embryonnaire et de son dysfonctionnement observé chez les hybrides interspécifiques. Cette thèse sinscrit dans ce cadre et vise à identifier et caractériser des gènes clés impliqués dans le développement de l'embryon chez Phaseolus. Des hybridations interspécifiques ont été réalisées entre lespèce P.vulgaris L. (cultivar NI637) utilisée comme parent mâle et lespèce P. coccineus L. (cultivar NI16) utilisée comme parent femelle. Des analyses ont aussi été effectuées sur un mutant obtenu par mutagenèse chimique à l'EMS (Ethyl Méthyl Sulfonate) de graines de la variété BAT93 de P.vulgaris. Une étude histologique comparative a permis de suivre la dynamique de lembryogenèse du haricot commun à partir dembryons prélevés 3 à 12 jours après la pollinisation et provenant de plantes normales et déficients dans la production de graines. Les embryons de P. vulgaris se développent plus rapidement par rapport à ceux issus du mutant EMS. Ces derniers présentent des anomalies au niveau de lembryon et du suspenseur. La caractérisation fonctionnelle de deux gènes candidats MIPS (myo-inositol phosphate synthase) et Sus (sucrose synthase) a été réalisée par RT-PCR quantitative et hybridation in situ suite à une étude spatio-temporelle dexpression de ces deux gènes candidats au cours de développement embryonnaire chez Phaseolus. Lanalyse du profil dexpression de ces deux gènes montre quils sont exprimés différemment au niveau des tissus de lembryon et du suspenseur. Lanalyse in silico nous a permis de sélectionner 22 gènes candidats dont nous avons vérifié l'expression au cours de développement de la graine chez Phaseolus. Des variations au niveau de la méthylation de lADN ont été déterminées chez les hybrides interspécifiques comparativement à leurs parents. La technique de lHSS a permis disoler des fragments dADNs complémentaires différemment exprimés au cours de développement de la graine chez Phaseolus. Lanalyse des séquences de ces ADNs complémentaires montre quils codent pour plusieurs protéines intervenant dans le développement cellulaire et embryonnaire, en particulier le "storage protein activator" (SPA), le "pentatricopeptide repeat-containing protein" (PPR) et lacetyl-CoA carboxylase (ACCase). La caractérisation de ces différents gènes exprimés au cours du développement de la graine, fournit de nouveaux outils susceptibles de mettre en évidence des mécanismes de dysfonctionnement embryonnaire chez le genre Phaseolus.

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