Orientador : Yong Kun Park / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Tecnologia de Alimentos / Made available in DSpace on 2018-07-14T13:03:32Z (GMT). No. of bitstreams: 1 Draetta_IacydosSantos_M.pdf: 1636727 bytes, checksum: 664e0c1d46b8b09ec3a94a8379877eb0 (MD5) Previous issue date: 1973 / Resumo: Invértase (ß-d-fructofuranosido-fructosídase E.C.3.2.1.26) foi obtida por autólise prolongada de leveduras, em presença de acetato de etila. Obtém-se, dessa maneira, duas formas de invértases ¿ solúvel e insolúvel ¿ as quais foram separadas por centrifugação a 1.200 g. A cinética da hidrolise enzímica de sacarose pela invertase tem sido estudada com ênfase, principalmente em altas concentrações de substratos. A velocidade da reação foi determinada pela produção de açúcares redutores diretamente em função do tempo. Os dados revelam que a velocidade de hidrolise de soluções de sacarose pela invertase diminui quando as concentrações de substrato aumentam. Os efeitos do pH, da temperatura e da ação de inibidores foram experimentalmente estudados, com os respectivos resultados incorporados, sempre que possível, em equações cinéticas. A relação entre a velocidade de inversão e a concentração de sacarose, para aplicação cooercial de preparações de açúcar invertido, também foi um dos propósitos deste trabalho. / Abstract: Invertase (ß-d-fructofuranoside-fructohydrolase, E.C.3.2.1.26) was obtained by extensive autolysis of baker's yeast in the presence of ethyl acetate. Two forms of the enzyme were obtained by centrifugation at 1200 g. The kinetics of the enzymatic hydrolysis of sucrose by invertase has been examined, with emphasis on high substrate concentration. Initial rate of reaction was determined by the production of reducing sugar, directly, as function of time. The data shows that the rate of hydrolysis by invertase of sucrose solutions decrease as the substrate concentrate increase. The effect of pH, temperature and inhibitors were experimentally studied and the results incorporated into kinetics equations. The relation between the rate of invertase and the sugar concentration for the commercial aplication of inverted sugar preparations, was one of the objectives of our paper. / Mestrado / Mestre em Ciência de Alimentos

Orientador: Yong Kun Park / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-07-20T17:38:00Z (GMT). No. of bitstreams: 1 Ikegaki_Masaharu_M.pdf: 2892590 bytes, checksum: 717b0a2cb516ced54801b9a6329b962e (MD5) Previous issue date: 1995 / Resumo: Foram isoladas 1341 linhagens de microrganismos a partir de amostras de solos, flores e frutos e testados quanto à produção de (3-frutofuranosidase que, além da atividade hidrolítica sobre a sacarose, catalisa a formação de lactosacarose através da transferência de resíduo de frutosil provenientes da sacarose para o aceptor lactose. Seis linhagens de microrganismos foram preliminarmente selecionadas visando a produção de lactosacarose. Entre estas uma linhagem identificada como Bacillus sp n° 417 apresentou alta produtividade de (3-frutofuranosidase com atividade de transferência para produção de lactosacarose a partir de lactose e sacarose. O pH e a temperatura ótima de atividade da (3-frutofuranosidase foram 5,6 e 45°C, respectivamente. Verificou-se que, após 8 horas de reação, a proporção de lactose e sacarose 1: 1 (P/p) e 20% de concentração de açúcares totais, a uma temperatura de 45°C e pH 5,6, foram as melhores condições para produção de lactosacarose. A enzima (3-frutofuranosidase de Bacillus sp n° 417 foi purificada em coluna de troca iônica e o peso molecular da enzima foi estimado em 89.000 Da através de cromatografia em Sephadex G-200 / Abstract: One thousand and three hundred forty one strains of microorganisms were isoleted from soils, flowers and fruits samples and examinated for production of P-fructofuranosidase which hydrolyze sucrose to glucose and fructose. Those strains of p-fructofuranosidase producing microorganisms were tested for production of lactosucrose from mixture of lactose and sucrose. Six strains of microorganisms were selected as lactosucrose producers. Among them, one strain, identified as Bacillus sp, showed high ß fructofuranosidase transfer activity to lactosucrose production. The optimum pH and temperature for lactosucrose production were 5,6 and 45°C, respectively. The best condition for the lactosucrose production were: 8 hours of reaction; a ratio lactose to sucrose of 1:1 (w/w) and a total sugar concentration of20% (1:1, w/w) The molecular weight of 89.000 Da for P-fructofuranosidase was estimated by Sephadex G-200 chromatography / Mestrado / Mestre em Ciência de Alimentos

La leishmaniose est une maladie tropicale négligée pour laquelle l’arsenal thérapeutique actuel est limité. Ce travail de thèse s’est intéressé à rechercher des nouvelles cibles thérapeutiques en ciblant la paroi des leishmanies. Le lipophosphoglycane (LPG), constituant majoritaire de la paroi, présente un motif glucidique particulier, le galactofuranose, qui semble une cible thérapeutique intéressante car il est absent des membranes de mammifères. Les galactofuranosyl-transférases sont impliquées dans le métabolisme de ce furanose, et ce travail a débuté par l’étude de ces enzymes et par la caractérisation d’une mutase, également nécessaire au métabolisme du galactofuranose. Une fois les cibles caractérisées dans les 2 stades du parasite, des analogues du galactofuranose ont été testés quant à leur capacité antiparasitaire sur les formes promastigotes et amastigotes de Leishmania donovani. Un composé s’est révélé intéressant et a été plus étudié, le n-octyl-galactofuranose (Galf). Différentes approches ont été utilisées pour caractériser son mode d’action sur les promastigotes et les amastigotes : résonance paramagnétique électronique, microscopie électronique à transmission, cytométrie en flux ou résonance magnétique nucléaire. L’observation d’une activité inductrice du métabolisme oxydatif des macrophages nous a conduits à nous intéresser aux capacités immunomodulatrices de ces analogues galacto-furanosidiques. Ainsi la dernière partie de ce travail est consacrée à l’étude de la polarisation des macrophages par les galactofuranosides, sur un modèle in vitro de macrophages humains. Nous avons pu montrer que le Galf exerçait une activation des macrophages en faveur d’une polarisation de type M1, ce qui pourrait expliquer l’effet limitateur de croissance des amastigotes. / Leishmaniasis is a neglected tropical disease for which the current therapeutic arsenal is limited. This work aimed at finding new therapeutic drugs by targeting the Leishmania cell wall. Lipophosphoglycan (LPG) is the major glycoconjugate in promastigotes cell wall, consisting of a hexasaccharide core including a galactofuranose motif. Galactofuranose is absent in mammalian membranes, thus could be a therapeutic target. First, this work studied the galactofuranosyl-transferases involved in the metabolism of this furanose, as well as a mutase, also necessary for the metabolism of galactofuranose. Once targets were identified in the two parasitic stages, galactofuranose derivatives were tested for antileishmanial activity on promastigotes and amastigotes forms of Leishmania donovani. A compound showed interesting results and has been studied further, the n-octyl-galactofuranose (Galf). Different techniques have been used to characterize its mode of action on promastigotes and amastigotes: electron paramagnetic resonance, transmission electron microscopy, nuclear magnetic resonance or flow cytometry. Infected macrophages treated with Galf were able to produce oxygen derivatives species, leading us to look at the immunomodulatory capacity of Galf derivatives. Thus, the last part of this work focused on the study of macrophage polarization by galactofuranosides on an in vitro model of human macrophages. We were able to show that Galf stimulates macrophages towards M1 polarization, which could explain the decreased growth of amastigotes inside macrophage cells.

Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: The goals of this project were to (i) determine maximum extractable neutral invertase (NI) activity in the sugarcane culm, (ii) sequence a cDNA encoding for the sugarcane NI (SNI), (iii) determine SNI copy number in the genome, (iv) describe SNI transcript and protein expression patterns throughout the plant, and (v) attempt to determine the contribution of hydrolysis to sucrose accumulation. SNI and sugars were extracted from the developing culm tissues of sugarcane, commercial variety N19. Tissues were divided according to developmental stage (internodes 3, 6 and 9) and anatomical differentiation (enriching for elongating, vascular or storage tissues). The lowest sucrose content was found in the core of the bottom of each of the internodes. The ratio between hexoses and sucrose was highest in the young internodes. In these internodes hexose content was higher in the bottom than the top. There was a significant correlation between sucrose content and NI. Fluxes involved in sucrose synthesis and hydrolysis were investigated. The hexoses glucose and fructose were supplied as a carbon source for tissue discs of young and maturing internodal tissues of sugarcane, varieties N19 and US6656-15. Sucrose content was 10-fold higher in maturing internodes of N19 than US6656-15. Calculated sucrose hydrolysis rates via invertase were higher in maturing internodes of US6656-15 than N19. Taking metabolic compartmentation into account, hydrolysis of sucrose via invertase made a significant contribution to the net turnover of sucrose. Along with this, it would appear that the ability to partition sucrose between the vacuole and cytosol causes a significant difference in sucrose content between varieties. A full-length cDNA for SNI was sequenced. This expressed gene showed significant homology to known NI sequences on both nucleic and amino acid levels. The SNI sequence did not contain the putative invertase catalytic amino acid sequence, suggesting it developed separately from the other classes of invertases. Approximately 1.8 kb of the SNI cDNA was incorporated into a vector suitable for direct bombardment into sugarcane tissue. Southern blot analysis showed the enzyme has a low copy number. SNI transcript expression was observed in all tissues of the sugarcane plant: roots, internodes, leaf roll and leaves. In culm tissues where sucrose content was low and hexose contents were high, SNI transcript and protein levels were high. This suggests that SNI is involved in growth metabolism. / AFRIKAANSE OPSOMMING: Die doel van die projek was om (i) maksimum ekstaheerbare neutrale invertase (NI) aktiwiteit in die suikerriet stingel te bepaal, (ii) die volgorde van 'n eDNA wat vir suikerriet NI (SNI) kodeer te bepaal, (iii) die SNI kopie-getal in die genoom te bepaal, (iv) SNI m- RNA en proteïenuitdrukkingspatrone deur die plant te beskryf, en (v) te poog om die bydrae van hidrolise op sukrose akkumulering te bepaal. SNI en suikers is geëkstraheer uit 'n kommersiële varieteit, N19. Weefsels was volgens ontwikkelingstadiums (internodes 3, 6 en 9) en anatomiese verskille (verryking vir groeiende, vaat- en bergings-weefsels) verdeel. Die laagste sukrose inhoud is in die middel van die onderste helfte van elke internode gevind. Die verhouding van heksoses tot sukrose was die hoogste in die jong internodes. Die inhoud heksoses was hoër in die onderste deel van die internode as die boonste deel. 'n Betekenisvolle korrelasie tussen sukrose inhoud en SNI is gevind. Flukse betrokke by sukrose sintese en hidrolise is ondersoek. Glukose en fruktose is as koolstofbron aan stingelweefsel van twee variëteite (US6656-15 and N19) toegedien. Sukrose-inhoud het tienvoudig tussen volwasse weefsels van die twee variëteite verskil. Hidrolise via invertase was hoër in ouer weefsels van US6656-15 as N19, en het In noemenswaardige bydrae tot sukroseomset gemaak. Die verdeling van sukrose tussen die vakuool en die sitosol kan moontlik 'n groot rol speel in die vermoë van die sel om sukrose te akkumuleer. Die volgorde van 'n volledige SNI eDNA is bepaal. The uitgedrukte geen het, op beide In nukleïen- en aminosuur vlak, betekenisvolle ooreenkoms getoon met ander bekende plant NI volgordes. Die SNI volgorde bevat nie die kenmerkende invertase katalitiese setel nie, wat daarop kan dui dat dit onafhanklik van ander klasse invertases ontwikkel het. Min of meer 1.8 kb van die SNI eDNA is in 'n vektor geskik vir bioliestiese transformering van suikerrietweefsel, geïnkorporeer. Southern klad analise het gewys dat die ensiem 'n lae kopiegetal op geen vlak het. SNI mRNA uitdrukking is waargeneem in elke weefseltipe van die suikerriet plant: wortels, internodes, blaarrol en blare. In stingelweefsels met lae sukrose- en hoë heksose-inhoud, was die vlakke van beide SNI-mRNA en -proteïen hoog. Dit dui daarop dat SNI moontlik betrokke is by groei-metabolisme.

Thesis (Ph. D.)--Columbia University, 1926. / Vita. Bibliography: p. [47].

Thesis (MSc)--University of Stellenbosch, 2006. / Bibliography.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2012. / Made available in DSpace on 2013-07-16T04:37:22Z (GMT). No. of bitstreams: 0 / As leveduras Saccharomyces são amplamente utilizadas na produção de álcool combustível graças à sua capacidade de adaptação ao ambiente industrial, e à habilidade de fermentar açúcares eficientemente. A sacarose, açúcar majoritário nos mostos utilizados no Brasil, é um dissacarídeo hidrolisado pela enzima invertase, que é codificada pelos genes SUC e secretada pelas leveduras. Dessa forma, obtêm-se, no meio, glicose e frutose, monossacarídeos que são transportados para o interior da célula e fermentados até etanol e CO2. No entanto, recentemente foi descrita uma outra via para a eficiente fermentação de sacarose envolvendo o transporte ativo do açúcar para o interior da célula, mediado pela permease AGT1, e sua hidrolise pela invertase intracelular. No presente trabalho uma linhagem industrial da levedura S. cerevisiae, que tende a dominar as dornas de fermentação, foi modificada utilizando técnicas de engenharia genômica visando a fermentação da sacarose através da sua captação direta. A estratégia envolveu sobre-expressar a invertase intracelular (iSUC2) e utilizar o gene da permease AGT1 (flanqueado por sequências que permitem sua sobre-expressão) para deletar a outra cópia do gene SUC2 presente no genoma diploide da levedura, impedindo portanto sua hidrólise extracelular. Embora as modificações genéticas tenham sido realizadas com sucesso, os resultados indicam que na linhagem industrial modificada (iSUC2 + suc2::AGT1) existem outros genes SUC que expressam a invertase extracelular.<br> / Abstract : Saccharomyces yeasts are widely used in the production of fuel ethanol due to its capacity to adapt to the industrial environment, and the ability to ferment sugars efficiently. Sucrose, the major sugar in musts used in Brazil, is a disaccharide hydrolyzed by the enzyme invertase secreted by yeasts and encoded by the SUC genes. Thus, glucose and fructose are obtained and transported into the cell in order to be fermented into ethanol and CO2. However, another pathway for efficient sucrose fermentation was recently described involving the active transport of the sugar into the cell mediated by the AGT1 permease, and its hydrolysis by the intracellular invertase. In this work an industrial S. cerevisiae yeast strain, which tends to dominate the fermentation vats, was modified using genomic engineering techniques aiming the fermentation of sucrose by its direct uptake. The strategy involved over-expression of the intracellular invertase (iSUC2) and the use of the AGT1 permease gene (flanked by sequences that allow its over-expression) to delete the other copy of the SUC2 gene present in the diploid genome of yeasts, preventing its extracellular hydrolysis. Although the genetic modifications were performed successfully, the results indicate that in the modified industrial strain (iSUC2 + suc2::AGT1) there are other SUC genes that express the extracellular invertase.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biotecnologia e Biociências, Florianópolis, 2015. / Made available in DSpace on 2016-10-19T12:47:14Z (GMT). No. of bitstreams: 1 338219.pdf: 1806932 bytes, checksum: cb4e68c860e46d1c5b4be98ef954ceed (MD5) Previous issue date: 2015 / Na levedura S. cerevisiae os carboidratos sacarose, maltose e maltotriose são fermentados por vias metabólicas diferentes: a sacarose é hidrolisada pela invertase extracelular (codificada pelos genes SUC), enquanto maltose e maltotriose são ativamente transportadas para dentro da célula e hidrolisadas pelas a-glicosidases presentes no citoplasma (ambas proteínas codificadas pelos genes MAL). Os genes SUC também permitem a síntese de uma forma intracelular da invertase, uma enzima com nenhuma função óbvia em leveduras. No entanto, a sacarose pode também ser metabolizada por células de levedura através dos transportadores e a-glicosidases codificados pelos genes MAL. Nossos resultados mostram que a maltotriose pode ser também eficientemente fermentada pelas células de S. cerevisiae através de seu transporte ativo mediado pela permease AGT1, um transportador MAL necessário para utilização de maltotriose, e sua hidrólise intracelular mediada pela invertase intracelular. A cepa brasileira industrial utilizada na produção de etanol combustível CAT-1 não pode fermentar maltotriose eficientemente devido ao promotor do AGT1 ser defeituoso. Para aumentar a fermentação de maltotriose por esta levedura, colocamos um promotor constitutivo (PGPD) à frente do gene AGT1 presente na cepa CAT-1, gerando a linhagem GMY05. No entanto, esta linhagem não foi capaz de fermentar eficientemente a maltotriose. Por outro lado, quando esta linhagem foi modificada para sobre-expressar a forma intracelular da invertase, por substituição da sequência sinal do gene SUC2 com o promotor constitutivo PADH1, a cepa iSUC2 obtida (GMY08) fermentou maltotriose eficientemente. Utilizando condições em que os genes MAL não são expressos, foi possível mostrar que a forma intracelular da invertase é capaz de hidrolisar a maltotriose (mas não maltose ou p-nitrofenil-a-glicosídico). Assim, os nossos resultados indicam uma sobreposição inesperada no metabolismo de sacarose e maltotriose por células de levedura, indicando que a invertase intracelular pode hidrolisar da maltotriose, e oferece novas abordagens a ser aplicadas para otimizar várias fermentações industriais que utilizam hidrolisados de amido, incluindo a panificação, produção de bebidas destiladas e cerveja, ou inclusive bioetanol.<br> / Abstract : It is well known that in the yeast S. cerevisiae the sugars sucrose, maltose and maltotriose are metabolized by different pathways: sucrose is hydrolyzed by the extracellular invertase (encoded by SUC genes), while maltose and maltotriose are actively transported into the cell and hydrolyzed by intracellular a-glucosidases (both proteins encoded by MAL genes). Furthermore, the SUC genes also allow the synthesis of an intracellular form of invertase, an enzyme with no obvious function in yeasts. We have already shown that sucrose can be metabolized by yeast cells through MAL-encoded transporters and a glucosidases. Now, our results will show that maltotriose can be efficiently fermented by S. cerevisiae cells through its active transport mediated by the AGT1 permease, a MAL transporter required for maltotriose utilization, and its intracellular hydrolysis mediated by the cytoplasmic invertase. The Brazilian industrial fuel-ethanol strain CAT-1 cannot ferment maltotriose efficiently due to a defective promoter of the AGT1 gene. To increase maltotriose fermentation by this strain, we placed a strong promoter (PGPD) in the AGT1 gene of strain CAT-1, generating strain GMY05. While the AGT1 gene was indeed over-expressed in this strain (measured by real-time PCR), maltotriose was still not fermented efficiently. However, when we over-expressed the intracellular form of invertase, by replacing the signal sequence of the SUC2 gene with the strong PPGK promoter, the resulting iSUC2 strain GMY08 fermented maltotriose efficiently. Using conditions were the MAL-encoded a glucosidases could not be expressed, we showed that the intracellular form of invertase could hydrolyze maltotriose (but not maltose or p-nitrophenyl-a-glucoside). Thus, our results indicate an unexpected overlap in sucrose-maltotriose metabolism by yeast cells, showing that the intracellular invertase allows efficient maltotriose hydrolysis, and offers new approaches that can be applied to optimize several industrial fermentation processes that use starch hydrolysates, including production of bread, distilled beverages and beer, or even bioethanol.

No description available.

Wachstum und Entwicklung pflanzlicher Gewebe bedingen eine fortwährende Veränderung von Source-Sink Beziehungen. Gewebe mit einem Nettoexport (Source) oder - import (Sink) von Kohlenhydraten müssen ihren aktuellen Bedarf an Assimilaten entsprechend dem Entwicklungsstadium anpassen. Darüber hinaus haben Pflanzen als ortsgebundene Lebewesen Regulationsmechanismen entwickelt, die eine flexible Antwort der Assimilatverteilung auf spezielle Anforderungen des Habitats, wie biotische oder abiotische Stressfaktoren und wechselnde Lichtbedingungen, ermöglichen. Die Assimilatverteilung ist vielfältig reguliert und erfordert spezifische Enzymfunktionen, wie Zuckertransporter und saccharosespaltende Enzyme. Extrazelluläre Invertasen nehmen eine essentielle Funktion in der apoplastischen Phloementladung und in der Regulation von Source-Sink Übergängen ein. Dies spiegelt sich in dem Auftreten verschiedener Invertase- Isoenzyme mit speziellen Expressions- und Regulationsmustern wider, welche eine Koordination des Kohlenhydratmetabolismus in unterschiedlichen Geweben, zu unterschiedlichen Entwicklungsstufen und unter sich ändernden Umweltbedingungen ermöglichen. Ein detailliertes Wissen über die Funktion extrazellulärer Invertasen könnte eingesetzt werden, um Wachstum, Entwicklung oder Pathogenresisitenz von Nutzpflanzen gezielt zu verändern. In der vorliegenden Studie wurden die Regulationsmuster und die Funktion dreier extrazellulärer Invertasen aus Tomate, Lin5, Lin6 und Lin7 untersucht. Durch umfangreiche Promotorstudien konnte eine gewebe- und entwicklungsspezifische Expression dieser Isoenzyme und entsprechende Regulationsmuster offengelegt werden. Lin5 zeigt eine entwicklungsabhängige Expression in Früchten. Lin6 wird in frühen Entwicklungsstadien, beginnend mit der Samenkeimung, exprimiert; in ausgewachsenen Pflanzen ist eine Lin6 Expression nur in Pollen oder nach Verwundungsinduktion nachweisbar. Lin7 wird ausschließlich in Tapetum-Gewebe und Pollen exprimiert. Die hormonelle Regulation der Isogene wurde im Detail untersucht, hierbei konnten bekannte Phänotypen, welche durch Gibberellinsäure und Jasmonate bedingt werden, mit Invertasefunktionen in Korrelation gebracht werden. Darüber hinaus konnte in einem funktionalen Ansatz gezeigt werden, dass Lin7 eine wichtige Rolle in der Pollenkeimung zukommt. Die vorliegende Arbeit stellt die umfassendste Untersuchung extrazellulärer Invertasen während der Blütenentwicklung dar, an der drei Isoenzyme aus Tomate beteiligt sind. Dadurch, dass den einzelnen Invertasen Lin5, Lin6 und Lin7 individuelle Funktionen zugewiesen werden konnten, eröffnen sich neue Erkenntnisse über die Kohlenhydratversorgung während der Blüten- und Fruchtentwicklung. Für die untersuchten gewebespezifischen Promotoren eröffnen sich zudem Anwendungsmöglichkeiten in der Biotechnologie, was insbesondere für den pollenspezifischen Lin7 Promotor zutrifft. Es konnte gezeigt werden, dass der Lin6 Promotor das Ziel von hormon-, zucker- und verwundungsvermittelten Signalwegen ist. Darüber hinaus konnte nachgewiesen werden, dass Elemente des circadianen Oszillators von A. thaliana mit dem Lin6 Promotor funktionell interagieren und die Lin6 Expression einem diurnalen Rhythmus unterliegt. Dieses komplexe Regulationsmuster spiegelt sich in vielen cis-aktiven Elementen wider, die im Lin6 Promotor vorgefunden wurden. Durch dieses Merkmal wird die These gestützt, dass verschiedene Stimuli über die extrazelluläre Invertase integriert werden und so eine koordinierte Zellantwort auf sich ändernde interne und externe Bedingungen ermöglicht wird. Nachdem Zuckermoleküle ihrerseits die Expression von Lin6 induzieren, wird dadurch eine Amplifikation von Signalen über eine positive Rückkopplungsschleife ermöglicht. Die Vielzahl an cis-aktiven Elementen und deren Anordnung im Lin6 Promotor stellen ein ideales Modellsystem dar, um Fragen in Bezug auf Signalinteraktion und -integration zu untersuchen. In einer umfangreichen Studie wurde der Lin6 Promotor erfolgreich als induzierbares Expressionssystem eingesetzt. Hierbei wurde ein Invertaseinhibitor unter der Kontrolle des cytokinininduzierbaren Lin6 Promotors in transgenen Tabakpflanzen exprimiert. Mit diesem Ansatz ist es gelungen einen kausalen Zusammenhang zwischen dem Hormon Cytokinin und extrazellulären Invertasen in der Seneszenzverzögerung herzustellen. Diese Studie zeigt, dass induzierbare Expressionssysteme essentiell sind, um spezifische Fragestellungen auf molekularer Ebene klären zu können. Bei der Klonierung obig genannter Promotorsequenzen haben sich zudem zwei interessante strukturelle Besonderheiten ergeben. Zum einen sind die Gene von Lin5 und Lin7 in einem Tandem auf dem Genom angeordnet, zum anderen konnte eine Transposoninsertion im Intron I des Lin5 Gens gezeigt werden. Mit einem Primerpaar, das aus der Transposaseregion dieses Transposons abgeleitet wurde, konnten entsprechende Sequenzen von mehreren Solanaceae Spezies gewonnen werden. / Because of growth and development, plant tissues are characterised by a permanent change in source-sink relations. Tissues with a net carbohydrate export (source) or import (sink) have to adopt their actual demand for assimilates according to the developmental status. Furthermore, plants, as sessile life forms, have developed regulatory mechanisms that enable a flexible response of assimilate partitioning to specific requirements of the habitat, like biotic and abiotic stress factors and changing light conditions. The distribution of assimilates involves specific enzyme functions including sugar transporters and sucrose cleaving enzymes and is regulated by a variety of stimuli. Extracellular invertases cover an essential function in apoplastic phloem unloading and play an important role in regulating source-sink relations. This property is reflected by the occurrence of different invertase isoenzymes with specific expression and regulation patterns that enable a co-ordination of the carbohydrate metabolism in diverse tissues, at different developmental stages, and under varying environmental conditions. Improved knowledge of extracellular invertase function might allow altering growth, development or pathogen resistance of crop plants in a specific way. The present study is aimed at elucidating the regulation patterns and functions of three members of the extracellular invertase gene family of tomato, Lin5, Lin6, and Lin7. Detailed promoter analysis revealed a tissue- and developmental-specific expression of isoenzymes and corresponding regulation patterns. Lin5 shows a developmental regulated expression in fruits. Lin6 is expressed in early developmental stages starting in germinating seeds; in grown up plants Lin6 is solely expressed in pollen and upon wound-stimulation. Lin7 is exclusively expressed in tapetum and pollen tissue. The hormonal regulation of all three isogenes was analysed in detail, whereby known GA- and JA-mediated flower phenotypes could be correlated with invertase functions. In addition, an important role of Lin7 invertase in pollen germination was demonstrated in a functional approach. This is the most profound analysis of extracellular invertases in the delicate process of floral organ development that includes three tomato isoenzymes. In particular, dissection of the individual roles of Lin5, Lin6, and Lin7 reveals novel insights in carbohydrate supply during flower and fruit development. The analysed tissue-specific promoters are profitable tools in plant biotechnology, which in particular applies to the pollen-specific Lin7 promoter. It has been demonstrated that the Lin6 promoter serves as target for hormonal-, sugar-, and wound-mediated signalling pathways. Moreover, a functional interaction of circadian oscillator elements of A. thaliana with the Lin6 promoter and a diurnal rhythm of Lin6 expression have been substantiated. This complex regulation pattern is reflected by the identification of many well-defined cis-acting elements within the Lin6 promoter. This feature supports an integration of various stimuli mediated via extracellular invertase expression resulting in a co-ordinated cellular response to changing internal and external conditions. As sugars on their part induce Lin6 expression, this could result in signal amplification via a positive feedback loop. Furthermore, the extensive appearance and constellation of cisacting elements within the Lin6 promoter provides the basis to answer questions in signal cross-talk and signal integration in plant gene expression. In addition, the Lin6 promoter was successfully used as an inducible expression system. In transgenic tobacco lines an invertase inhibitor was expressed under control of the cytokinin-inducible Lin6 promoter. Thereby, a causal relationship between cytokinin and extracellular invertase for the delay of senescence was demonstrated. This study emphasises the importance of inducible expression systems to address specific questions on a molecular basis. The above-mentioned promoter sequences were obtained via sequential genome walks. Hereby two interesting structural features appeared. First, Lin5 and Lin7 genes are arranged in a direct tandem repeat on the genome. Second, a CACTA-like transposon insertion in intron I of the Lin5 gene was revealed. A primer pair deduced from the transposase region of this transposon allowed the amplification of similar sequences of various Solanaceae species.