Transformação genética em espécies florestais.

Volume growth of pau-ferro trees (Astronium balansae) of a native forest and a twelve-years old plantation was studied using stem analysis technique. Analysis of cross-sectional discs allowed only to quantify the growth of the plantation trees because it was not possible to identify the tree rings in the native forest  cross-sections. Growth data was modeled with a stepwise regression procedure which resulted in  equations of high precision and good fit to describe the mean and current annual increment of a tree from the dominant and another from the dominated stratum. It was not possible to detect the point of maximum increment because of the little age of the trees.A transformação genética, que compreende a introdução de genes exógenos de forma controlada no genoma de uma célula vegetal e posterior regeneração da planta transgênica, tem contribuído com os programas de melhoramento genético de plantas pela obtenção de genótipos com novas características de interesse. O melhoramento de espécies florestais é limitado por características intrínsecas a tais espécies, como a altura dos indivíduos e o ciclo longo de vida. A transformação genética constitui, portanto, uma alternativa para a obtenção de espécies florestais com características desejáveis em um menor espaço de tempo. Plantas transgênicas com resistência a determinadas pragas, com melhor qualidade de madeira, maior produção de biomassa, tolerância a herbicidas, entre outras características de interesse, já foram obtidas para diferentes espécies florestais de importância econômica como álamo, eucalipto e pinheiros em geral. Este trabalho mostra a importância da transformação genética, associada a outras técnicas biotecnológicas no melhoramento de espécies florestais, as técnicas de transformação mais utilizadas e as características que já foram introduzidas nessas espécies pela transformação

Funktionelle Analyse der Rolle des Enzyms Succinyl Coenzym A-Ligase im photosynthetischen Metabolismus der Tomate

Title page,Acknowledgements, Abstract, Zusammenfassung, Table of Contents, List of Figures, List of Tables, List of Abbreviations 1\. General introduction 1 2\. Materials and Methods 24 2.1. Chemicals 25 2.2. Bacterial and yeast strains 25 2.3. Expression vectors 25 2.4. Transformation and cultivation of bacteria 26 2.5. Complementation, transformation and cultivation of yeast 26 2.6. Yeast mitochondria isolation 27 2.7. DNA manipulation 27 2.8. Plant material 27 2.9. Screening of putative full-length cDNA from the tomato EST collection 28 2.10. Subcloning of selected cDNA from tomato into pENTR vector 29 2.11. Subcellular localisation experiments 30 2.12. Phylogenetic analysis tomato SCoAL α1, α2 and β subunits 32 2.13. Expression analysis tomato SCoAL α1, α2 and β subunits 32 2.14. Analysis of enzyme activities 33 2.14.1. Succinyl CoA ligase enzyme assay 33 2.14.2 Glutamate decarboxylase enzyme assay 34 2.14.3 Glutamate dehydrogenase enzyme assay 34 2.15. Oxygen consumption measurements in yeast 35 2.16. Determination of metabolite levels in tomato leaves 35 2.17. Measurements of photosynthetic parameters 36 2.18. Measurement of respiratory parameters 36 2.19. Microarray 36 2.20. Statistical analysis 37 3\. Molecular cloning of tomato TCA cycle full-length cDNAs and characterisation of succinyl CoA ligase α and β subunits 38 4\. Phenotypic and metabolic effects in tomato plants with reduced activity of succinyl CoA ligase 60 5\. General Discussion 92 6\. Bibliography 100Despite the central importance of the TCA cycle in plant metabolism not all of the genes encoding its constituent enzymes have been functionally identified. In this work I report the isolation of tomato cDNAs coding for α1- and α2 and one coding for the β-subunit of succinyl CoA ligase, for E2- and E3-subunits of 2-oxoglutarate dehydrogenase complex, for iron sulphur-subunit of succinate dehydrogenase, for E1α and β-subunits of pyruvate dehydrogenase complex, and for chloroplastic-, cytosolic-, mitochondrial- and glyoxysomal-subunits of malate dehydrogenase. Emphasis was given to the cDNAs coding for α1- and α2- and for the β-subunit of succinyl CoA ligase. These three cDNAs were used to complement the respective Saccharomyces cerevisiae mutants deficient in the α\- and β-subunit, demonstrating that they encode functionally active polypeptides. The genes encoding for the subunits were expressed in all plant organs, but most strongly in flowers and leaves, with the two α-subunit genes being expressed to equivalent levels in all plant organs. In all instances GFP fusion expression studies confirmed an expected mitochondrial location of the proteins encoded. Following the development of a novel assay to measure succinyl CoA ligase activity, in the direction of succinate formation, the evaluation of the maximal catalytic activities of the enzyme in a range of plant organs revealed that these paralleled those of mRNA levels. I also utilized this assay to perform a preliminary characterisation of the regulatory properties of the enzyme suggesting allosteric control of this enzyme may regulate flux through the TCA cycle in a manner consistent with its position therein. Transgenic tomato (Solanum lycopersicum) plants expressing the complete coding region of α1- and β-subunit of succinyl CoA ligase separately in antisense orientation and using the RNAi approach were produced. Transformants were screened for reduced succinyl CoA ligase activity and selected lines were used for molecular, biochemical and physiological characterisation. Transgenic tomato plants harbouring the β-subunit of succinyl CoA ligase showed an increase in plant high, and a decrease in fruit production and leaf dry matter, but no change in photosynthetic parameters (for example CO2 assimilation rate) and about 30% decrease in the respiration rate. Accumulation of glutamate and γ-aminobutyric acid amino acids in leaves, and a higher activity of glutamate dehydrogenase and glutamate decarboxylase in the transformants suggest an alternative route, the GABA shunt which bypasses the succinyl CoA ligase deficiency and supplies the mitochondria with succinate to support the respiratory processes. Further analysis of other steady-state metabolite levels suggests a link between succinyl CoA ligase activity with other metabolic pathways such as the shikimate and isoprenoid pathways. It was observed that end products of the shikimate pathway, such as aromatic amino acids, were increased, as well as tocopherols, chlorophylls and carotenoids, which are end products of the isoprenoid pathways. Analysis of transgenic tomato plants displaying reduced expression of α1-subunit of succinyl CoA ligase performed in parallel with the tomato plants described above surprisingly did not show similar phenotype as the first plants, most likely because the selected lines showed not strongly enough decrease in activity.Trotz der bedeutenden Rolle des Citratzyklus im Stoffwechsel der Pflanze, wurden bisher nur wenige Enzyme innerhalb dieses Stoffwechselweges näher charakterisiert. Das Ziel der vorliegenden Arbeit war es daher eben diese Lücke zu schliessen und die Aktivitäten einiger der relevanten Citratzyklus- Enzyme genauer zu analysieren. Zu diesem Zweck wurden zunächst die cDNAs, die fur folgende Enzyme kodieren, aus Tomate isoliert: die α1-, α2 und β-Untereinheiten des Enzyms Succinyl Coenzym A-Ligase, die E2- und E3-Untereinheiten des 2-Oxoglutarat Dehydrogenase Komplexes, die Eisen- Schwefel-Untereinheit der Succinat Dehydrogenase, die E1α-und β-Untereinheiten des Pyruvat Dehydrogenase Komplexes, und die chloroplastidare-, cytosolische-, mitochondriale- and glyoxysomale-Untereinheiten der Malat Dehydrogenase. Der Schwerpunkt der Arbeit lag dann auf der Untersuchung der Funktionen der α1-, α2 und β-Untereinheiten der Succinyl Coenzym A-Ligase. Diese drei cDNAs wurden zunächst zur Komplementation von Hefe-Mutanten (Saccharomyces cerevisiae) mit einem entsprechenden Defekt in der jeweiligen Untereinheit verwendet. Es konnte somit gezeigt werden, dass die isolierten cDNAs funktionsfähige Polypeptide kodieren. Hybridisierungexperimente haben im Weiteren ergeben, dass die Untereinheiten der Succinyl Coenzym A-Ligase in allen pflanzlichen Organen exprimiert werden. Besonders starke Expression fand man in Blüten und Blättern, wohingegen die beiden α-Untereinheiten in allen Organen zu gleichen Teilen exprimiert wurden. Durch die Analyse der Expression der GFP- fusionierten Untereinheiten konnte weiterhin die erwartete mitochondriale Lokalisierung der Proteine bestätigt werden. Zur Aktivitätbestimmung des Enzyms Succinyl Coenzym A-Ligase wurde ein neues Protokoll in Richtung der Bildung von Succinat entwickelt. Die Auswertung der gemessenen Enzymeaktivitäten in verschiedenen pflanzlichen Organe ergab, dass diese mit den Transkriptgehalten der Gene korrelierten. Eine umfassende Charakterisierung der regulatorischen Eigenschaften des Enzyms konnte ebenfalls mithilfe des neu entwickelten Enzymassays durchgeführt werden. Dabei ergaben sich Hinweise auf eine allosterische Kontrolle des Enzyms das den Fluss durch den Zyklus übereinstimmend mit seiner Position kontrolliert. Anschliessend wurden verschiedene, genetisch modifizierte Tomatenpflanzen (Solanum lycopersicum) hergestellt, die die vollständige Kodierungsregion der α1- und β-Untereinheiten von Succinyl Coenzym A-Ligase einzeln in antisense Orientierung und als RNAi exprimieren. Einzelne Transformanden dieser Pflanzen wurden nach ihrer verminderten Enzymaktivität ausgewählt und dann fur molekulare, biochemische und physiologische Analysen verwendet. Hierbei zeigten die fur das Gen der β-Untereinheit der Succinyl Coenzym A-Ligase reprimierten Tomatenpflanzen ein gesteigertes Pflanzenwachstum, eine verminderte Fruchtproduktion, ein verringertes Trockengewicht der Blätter und eine ungefähr 30%ige Verminderung der zellulären Atmungsrate. Sie zeigten jedoch keine Änderung der photosynthetischen Parameter (zum Beispiel CO2 Assimilationsrate). Die Anreicherung an Glutamat und γ-Aminobuttersäure (GABA) in Blättern, und die Erhöhung der Enzymaktivitäten der Glutamat Dehydrogenase und der Glutamat Decarboxylase in diesen Pflanzen, weisen somit darauf hin, dass der "GABA-shunt", als ein alternativer Weg, den Mangel an Succinyl Coenzym A-Ligase überbrückt und die Mitochondrien mit Succinat beliefert, um somit die Atmungsprozesse zu sichern. Weiterhin durchgeführte Metaboliten- Analysen in Blättern, weisen auf eine Verbindung zwischen der Succinyl Coenzym A-Ligase Enzymaktivität und anderen Stoffwechselwege, wie zum Beispiel dem Shikimat- und dem Isoprenoidweg, hin. So konnte beobachtet werden, dass Endprodukte des Shikimatwegs, wie aromatische Aminosäure, als auch Tocopherol-, Chlorophyll- und Karotenoidemengen des Isoprenoidstoffwechselweges, erhöht waren. Die Analysen der Tomatenpflanzen mit verminderter Expression der Succinyl Coenzym A-Ligase α1-Untereinheit, die parallel durchgeführt wurden, wiesen überraschenderweise, nicht dieselben Eigenschaften wie die zuvor beschriebenen Transformanden der β-Untereinheit auf. Es besteht hierbei die Möglichkeit, dass die Verminderung der Enzymaktivität der ausgewählten Pflanzen nicht stark genug ausgeprägt war

Transformação genética em espécies florestais

Breeding of forest species is limited by intrinsic characteristics such as individuals height and long life cycle. Plant genetic transformation, the integration of known foreign genes into the plant genome, represents a less time consuming alternative for the recovery of forest species with desirable traits. This technology has contributed to plant breeding programs by facilitating the recovery of genotypes containing novel exciting traits of agricultural importance. Many of them including resistance to insect pests, improvement of wood quality and biomass production, and tolerance to herbicides have been introduced in forest species such as poplar, eucalyptus and pine trees using this technology. This review highlights current transformation methods, and illustrates the importance of finally defining the most important traits that have already been introduced into these valuable species

Transformação genética em espécies florestais.

Volume growth of pau-ferro trees (Astronium balansae) of a native forest and a twelve-years old plantation was studied using stem analysis technique. Analysis of cross-sectional discs allowed only to quantify the growth of the plantation trees because it was not possible to identify the tree rings in the native forest  cross-sections. Growth data was modeled with a stepwise regression procedure which resulted in  equations of high precision and good fit to describe the mean and current annual increment of a tree from the dominant and another from the dominated stratum. It was not possible to detect the point of maximum increment because of the little age of the trees.A transformação genética, que compreende a introdução de genes exógenos de forma controlada no genoma de uma célula vegetal e posterior regeneração da planta transgênica, tem contribuído com os programas de melhoramento genético de plantas pela obtenção de genótipos com novas características de interesse. O melhoramento de espécies florestais é limitado por características intrínsecas a tais espécies, como a altura dos indivíduos e o ciclo longo de vida. A transformação genética constitui, portanto, uma alternativa para a obtenção de espécies florestais com características desejáveis em um menor espaço de tempo. Plantas transgênicas com resistência a determinadas pragas, com melhor qualidade de madeira, maior produção de biomassa, tolerância a herbicidas, entre outras características de interesse, já foram obtidas para diferentes espécies florestais de importância econômica como álamo, eucalipto e pinheiros em geral. Este trabalho mostra a importância da transformação genética, associada a outras técnicas biotecnológicas no melhoramento de espécies florestais, as técnicas de transformação mais utilizadas e as características que já foram introduzidas nessas espécies pela transformação

TRANSFORMAÇÃO GENÉTICA EM ESPÉCIES FLORESTAIS

A transformação genética, que compreende a introdução de genes exógenos de forma controlada no genoma de uma célula vegetal e posterior regeneração da planta transgênica, tem contribuído com os programas de melhoramento genético de plantas pela obtenção de genótipos com novas características de interesse. O melhoramento de espécies florestais é limitado por características intrínsecas a tais espécies, como a altura dos indivíduos e o ciclo longo de vida. A transformação genética constitui, portanto, uma alternativa para a obtenção de espécies florestais com características desejáveis em um menor espaço de tempo. Plantas transgênicas com resistência a determinadas pragas, com melhor qualidade de madeira, maior produção de biomassa, tolerância a herbicidas, entre outras características de interesse, já foram obtidas para diferentes espécies florestais de importância econômica como álamo, eucalipto e pinheiros em geral. Este trabalho mostra a importância da transformação genética, associada a outras técnicas biotecnológicas no melhoramento de espécies florestais, as técnicas de transformação mais utilizadas e as características que já foram introduzidas nessas espécies pela transformação

Transformação genética em espécies florestais.

<p>A transformação genética, que compreende a introdução de genes exógenos de forma controlada no genoma de uma célula vegetal e posterior regeneração da planta transgênica, tem contribuído com os programas de melhoramento genético de plantas pela obtenção de genótipos com novas características de interesse. O melhoramento de espécies florestais é limitado por características intrínsecas a tais espécies, como a altura dos indivíduos e o ciclo longo de vida. A transformação genética constitui, portanto, uma alternativa para a obtenção de espécies florestais com características desejáveis em um menor espaço de tempo. Plantas transgênicas com resistência a determinadas pragas, com melhor qualidade de madeira, maior produção de biomassa, tolerância a herbicidas, entre outras características de interesse, já foram obtidas para diferentes espécies florestais de importância econômica como álamo, eucalipto e pinheiros em geral. Este trabalho mostra a importância da transformação genética, associada a outras técnicas biotecnológicas no melhoramento de espécies florestais, as técnicas de transformação mais utilizadas e as características que já foram introduzidas nessas espécies pela transformação.</p

Reduced Expression of Succinyl-Coenzyme A Ligase Can Be Compensated for by Up-Regulation of the γ-Aminobutyrate Shunt in Illuminated Tomato Leaves1[W]

Increasing experimental evidence suggests that the tricarboxylic acid cycle in plants is of greater importance in illuminated photosynthetic tissues than previously thought. In this study, transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the β-subunit of succinyl-coenzyme A ligase in either the antisense orientation or using the RNA interference approach, however, revealed little alteration in either photosynthesis or plant growth despite exhibiting dramatic reductions in activity. Moreover, the rate of respiration was only moderately affected in the transformants, suggesting that this enzyme does not catalyze a crucial step in mitochondrial respiration. However, metabolite and transcript profiling of these lines alongside enzyme and label redistribution experiments revealed that, whereas considerable activity of this enzyme appears to be dispensable, the reason for such a mild phenotype in extremely inhibited lines was an up-regulation of an alternative pathway for succinate production—that offered by the γ-aminobutyric acid shunt. When taken together, these data highlight the importance both of succinate production for mitochondrial metabolism and the interplay between various routes of its production. The results are discussed in the context of current models of plant respiration in mitochondrial and cellular metabolism of the illuminated leaf