Das Ziel der vorliegenden Arbeit war es den Zusammenhang zwischen dem endogenen und internen ABA-Gehalt des Wurzelgewebes und dem radialen ABA- und Wasserfluss zu untersuchen und zu überprüfen ob diese Faktoren durch unterschiedliche Nährstoffbedingungen beeinflusst werden. Der radiale Transportweg von ABA wurde ebenfalls untersucht. • In dieser Arbeit konnte das erste Mal gezeigt werden, dass ein direkter Zusammenhang zwischen dem endogenen und internen ABA-Gehalt des Wurzelgewebes und dem radialen Wasser- und ABA-Transport besteht. Unter vergleichbaren Bedingungen können aus einem gegebenen ABA-Gehalt Rückschlüsse auf die radialen Wasser- und ABA-Flüsse gezogen werden. • Während Kalium- und Calciummangel und die Kultur in CaSO4 den radialen Wasserfluss von Maiskeimlingen stimulierten, war Jv unter Nitratmangel reduziert. Phosphat- und Sulfatmangel wirkten sich nicht auf den Wasserhaushalt von Maiskeimlingen aus, trotz einem deutlich reduzierten P- bzw. S-Gehalt konnten keine klaren Defizienzsymptome festgestellt werden. • Der endogene ABA-Gehalt im Wurzelgewebe von Maiskeimlingen war nur unter Kalium- und Nitratmangel erhöht. • Der radiale ABA-Transport wurde unter Kalium-, Nitrat-, Calciummangel und in CaSO4-Kultur gesteigert. Der erhöhte ABA-Fluss in Kaliumdefizienten Keimlingen resultiert aus einer gesteigerten ABA-Biosynthese und dem erhöhten Wassertransport. Unter Nitratmangelbedingungen lässt sich der gesteigerte ABA-Fluss anhand des erhöhten ABA-Gehaltes im Wurzelgewebe erklären. Die erhöhte ABA-Konzentration im Xylemsaft von Keimlingen aus Calciummangel- und CaSO4-Kultur ist das Ergebnis des gesteigerten Wassertransportes. Phosphat- und Sulfatmangel hatten keine Auswirkungen auf den ABA-Fluss. • Salzstress (50 mM) reduzierte den radialen Wasserfluss deutlich. Der erhöhte endogene ABA-Gehalt im Wurzelgewebe hatte keinen Einfluss auf Jv und JABA. Die Auswirkungen von Salzstress waren voll reversibel. • 100 nM externe ABA wirkte sich unter allen untersuchten Nährstoffbedingungen gleichermaßen stimulierend auf Jv und JABA aus. In NaCl-gestressten Keimlingen zeigte externe ABA keinen Effekt. • Eine Möglichkeit zur Immunolokalisation von ABA in Wurzelquerschnitten von Maiskeimlingen wurde entwickelt und optimiert. • Die Visualisierung des radialen ABA-Transportes anhand der Immunolokalisation mit monoclonalen Antikörpern zeigte, dass Endo- und Exodermis eine apoplastische Barriere für den ABA-Transport darstellen. Die Ergebnisse lassen den Rückschluss zu, dass die Exodermis die wirksamere Barriere für den ABA-Transport ist. • Wurzeln von Maiskeimlingen bildeten unter Nitratmangelbedingungen eine Exodermis aus und verstärkten die Suberinisierung der Endodermis. Unter Kaliummangel konnten keine verstärkten Barriereeigenschaften beobachtet werden. In der vorliegenden Arbeit konnte zum ersten Mal aufgezeigt werden, dass eine signifikant hohe Korrelation zwischen dem endogenen ABA-Gehalt des Wurzelgewebes und dem ABA- bzw. Wassertransport besteht. Die ebenfalls positiv signifikant hohe Korrelation zwischen dem radialen Wasser- und ABA-Transport zeigt einen apoplastischen ABA-Transport an. Mit zunehmendem Wasserfluss steigt auch die ABA-Konzentration im Xylem. Ein apoplastischer radialer bypass der ABA konnte auch mit Hilfe der Immunolokalisation nachgewiesen werden. / The objective of this study has been to investigate the relations between the endogenous and internal ABA content in root tissues and the radial ABA- and water-flows and how these individual factors can be affected by different conditions of nutrient deficiency. The radial transport paths also have been studied. • The experiments of this study, for the first time, show a direct correlation between endogenous and internal ABA content in root tissue and radial water- and ABA-transport. From differences of the endogenous ABA content, conclusions can be drawn about changes of the radial water- and ABA-flows under comparable transpiring conditions. • Whereas potassium and calcium deficiencies and CaSO4-culture are stimulating the radial water flow of maize seedlings, nitrate-deficiency will reduce Jv. Phosphorus and sulphur deficiencies do not have an effect on the water balance of maize seedlings because, despite clearly reduced internal P- and S-content no serious deficiency symptoms developed. • The endogenous ABA-content of maize root tissues is enhanced by potassium and nitrate deficiencies only. • Radial ABA-transport is enhanced by potassium, nitrate, calcium deficiencies and in CaSO4-culture. The increased ABA-flow in potassium deficient seedlings is a result of the enhanced ABA-biosynthesis and the increased water-transport. Under conditions of nitrate deficiency the enhanced ABA-content in root tissue results in an increased ABA-flow. In maize seedlings cultivated under calcium deficiency or in CaSO4 the enhanced ABA-concentration of xylem sap is a result of the stimulated water-flow. No effect can be seen under phosphate and sulphate deficiencies. • Salt stress (50 mM) reduces the radial water flow drastically. Although endogenous ABA is accumulated under salt stress Jv remains unaffected. The salt effect is fully reversible. • Under all nutrient deficient and hypoxic conditions, 100 nM external ABA stimulates water and ABA-flows in a comparable way. In NaCl-stressed seedlings external ABA proved to be ineffective. • A technique of immunolocalisation of ABA in cross sections of maize roots has been developed and optimised. • Visualisation of the radial ABA-transport by immunolocalisation with monoclonal antibodies demonstrated the barrier properties of endodermis and exodermis for radial ABA-transport. From the results of immunolocalisation it is concluded that the exodermis only is a significant barrier for radial ABA transport. • Roots of maize seedling build up an exodermis and enhance the suberinisation of the endodermis under nitrogen deficiency, whereas under potassium deficiency no increased barrier properties could be observed. The presented work, for the first time, shows the tight and significant correlation between the endogenous and internal ABA-content of root tissue and the radial ABA- respectively water-transport. Likewise, there is a positive highly significant correlation between the radial water- and ABA-transport, indicating an apoplastic bypass of ABA. With increasing water flow, the ABA-concentration in xylem-sap is increasing as well. A radial apoplastic ABA-flow could also be demonstrated by immunolocalisation.

This work was performed in the framework of two application-oriented research projects that focus on the generation and evaluation of fluorescent Saccharomyces (S.) cerevisiae-based sensor and reporter cells for white biotechnology as well as the extension of the conventional single-cell/single-construct principle of ordinary yeast biosensor approaches. Numerous products are currently generated by biotechnological processes which require continuous and precise process control and monitoring. These demands are only partially met by physical or physiochemical sensors since they measure parameters off-line or use surrogate parameters that consequently provide only indirect information about the actual process performance. Biosensors, in particular whole cell-based biosensors, have the unique potential to near-line and long-term monitor parameters such as nutrient availability during fermentation processes. Moreover, they allow for the assessment of an analyte’s biological relevance. Prototype yeast sensor and reporter strains derived from common laboratory strains were transformed with multicopy expression plasmids that mediate constitutive or inducible expression of a fluorescence reporter gene. Performance of these cells was examined by various qualitative and quantitative detection methods – representative of putative transducer technologies. Analyses were performed on the population level by microplate reader-based fluorometry and Western blot as well as on the single-cell level by fluorescence microscopy and flow cytometry. ‘Signature’ promoters that are activated or repressed during particular nutrient-limited growth conditions were selected in order to generate yeast nutrient sensor strains for monitoring the biological availability of nitrogen, phosphorus or sulphur. For each category, at least one promoter mediating at least threefold changed green fluorescence levels between sensor cells in non-limited and nutrient-limited conditions was identified. Sensor strains were evaluated in detail regarding sensitivity, analyte selectivity and the ability to restore basic fluorescence after shift from nutrient-limited to non-limited conditions (regeneration). The applicability for bioprocess monitoring purposes was tested by growth of yeast nutrient sensor cells in microalgae media and supernatants. Despite successful proof of principle, numerous challenges still need to be solved to realise prospective implementation in this field of white biotechnology. The major drawback of plasmid-borne detection constructs is a high fluorescence variance between individual cells. By generation of a nitrogen sensor strain with a genome-integrated detection construct, uniform expression on the single-cell level and simultaneous maintenance of basic properties (ability of fluorescence induction/regeneration and lack of cross-reactivity) was achieved. However, due to the singular detection construct per cell, significantly weaker overall fluorescence was observed. The traditional single-cell/single-construct approach was expanded upon in two ways. Firstly, a practical dual-colour sensor strain was created by simultaneous, constitutive expression of a red fluorescence reporter gene in green fluorescent nitrogen sensor cells. Secondly, an innovative cellular communication and signal amplification system inspired by the natural S. cerevisiae pheromone system and mating response was established successfully. It features the yeast pheromone alpha-factor as a trigger and alpha-factor-responsive reporter cells which express a fluorescence reporter gene from the pheromone-inducible FIG1 promoter as an output signal. The system was functional both with synthetic and cell-secreted alpha-factor, provided that recombinant cells were deleted for the alpha-factor protease Bar1p. Integration of amplifier cells which secrete alpha-factor in response to stimulation with the pheromone itself could increase the system\'s sensitivity further. Signal amplification was demonstrated for phosphorus sensor cells as a proof of concept. Therefore, the alpha-factor-based cellular communication and signal amplification system might be useful in applications that suffer from poor signal yield. Due to its modular design, the system could be applied in basically any cell-based biosensor or sensor-actor system. Immobilisation of the generated sensor and reporter cells in transparent natural polymers can be beneficial considering biosensor fabrication. Functionality of sensor and reporter cells in calcium-alginate beads or nano-printed arrays was successfully demonstrated. For the latter setup, fluorescence scanning and software-assisted fluorescence quantification was applied as a new detection method. In an experiment using an agarose-based two-compartment setup proposed by Jahn, 2011, properties of the alpha-factor-based cellular communication and signal amplification system after immobilisation were tested. These studies provide an initial experimental basis for an appropriate geometry of miniaturised immobilisation matrices with fluorescent yeast sensor and reporter cells in prospective biosensor designs.

Ziel dieser Arbeit war es, das Verständnis über Reaktionen von Tomaten auf limitiertes Nährstoffangebot zu verbessern. In der Kontrollbehandlung (100% Biomassewachstum) wurden Pflanzen ohne Nährstofflimitierung kultiviert. Bei den Mangelvarianten wurde entweder K, Mg oder N kontinuierlich in Raten angeboten, die das Wachstum auf etwa 80% (leichter Mangel) oder 60% (starker Mangel) der Kontrolle reduzierten. Dieser Versuchsansatz ermöglichte es, pflanzliche Reaktionen auf eine gut definierte Intensität von Mangel an K, Mg und N zu vergleichen. Nährstofflimitierung veränderte die Biomasse- und Nährstoffallokation auf die verschiedenen Pflanzenorgane, wobei diese Veränderungen sich je nach Nährstoff unterschieden, aber nicht von der Intensität des Mangels abhängig waren. Die Wirkungen von Nährstofflimitierung auf morphologische Wurzel- und Sprosseigenschaften waren ebenfalls Nährstoff-spezifisch und nicht abhängig von der Intensität des Mangels. Die Wirkungen auf die Fruchtqualität, z.B. den Gehalt an Zuckern, Lycopin und Vitamin C waren Nährstoff-spezifisch und bei einigen Parametern auch von der Intensität des Mangels abhängig. Die Pflanzenreaktionen auf Mg-Mangel wurden in größerem Detail untersucht. In den ersten 6 Tagen nach Beginn der Limitierung nahmen die Mg-Konzentrationen in allen Organen stark ab, ohne Wirkung auf das Wachstum und die Photosynthese. Mangel erhöhte die Blattzuckergehalte, aber die Zuckerakkumulation stand in keiner Beziehung zur Photosyntheserate. Diese nahm erst ab, wenn die Blatt-Konzentrationen auf Werte unterhalb von 0,1 bis 0,2 mg Mg g-1 Frischmasse abgesunken waren. In Mg-Mangelpflanzen akkumulierten Zucker in den Sourceblättern sogar wenn das Source/Sink-Verhältnis in den Pflanzen durch Beschattung der basalen Blätter stark verringert wurde. Das stimmt mit der Annahme überein, dass Mg-Mangel aufgrund einer Inhibierung der Phloembeladung den Zuckerexport aus den Blättern verringert. / This thesis aims to increase our understanding about plant responses to K, Mg and N limitation and their relationship with fruit quality of tomato. In the control treatment (100% biomass growth), plants were grown at high nutrient concentration. In the nutrient-limitation treatments, either K or Mg or N was supplied continuously at rates, which reduced biomass growth to about 80% (mild deficiency) or 60% (severe deficiency) of the control. This experimental approach allowed comparing long-term plant responses to deficiency of K, Mg or N at well-defined intensities of nutrient limitation. Nutrient limitation was associated with changes of biomass and nutrient allocation among various plant organs, whereby these changes were nutrient specific (e.g., biomass allocation to leaves was increased in Mg deficient plants, and decreased in N deficient plants), but not much dependent on the intensity of nutrient limitation. Effects of nutrient limitation on root and shoot morphological traits were also nutrient-specific and independent on the intensity of deficiency. Effects of nutrient limitation on fruit quality, e.g, sugar, lycopene and ascorbic acid content, were also nutrient-specific, and for specific parameter dependent on the intensity of deficiency. Responses to Mg deficiency were studied in more detail. In the first 6 days after start of Mg limitation, Mg concentrations in all plant organs drastically decreased without any effects on growth and leaf photosynthetic rates. Mg-deficiency increased leaf sugar concentrations, but sugar accumulation was not directly related to leaf photosynthesis which only decreased after leaf Mg concentrations were below 0.10 to 0.20 mg Mg g-1 leaf fresh mass. In Mg-deficient plants, sugars accumulated in source leaves even when the source/sink ratio was strongly decreased by shading of basal leaves. This is in accordance with the suggestion that sugar export is reduced due to inhibition of phloem loading.

This work was performed in the framework of two application-oriented research projects that focus on the generation and evaluation of fluorescent Saccharomyces (S.) cerevisiae-based sensor and reporter cells for white biotechnology as well as the extension of the conventional single-cell/single-construct principle of ordinary yeast biosensor approaches. Numerous products are currently generated by biotechnological processes which require continuous and precise process control and monitoring. These demands are only partially met by physical or physiochemical sensors since they measure parameters off-line or use surrogate parameters that consequently provide only indirect information about the actual process performance. Biosensors, in particular whole cell-based biosensors, have the unique potential to near-line and long-term monitor parameters such as nutrient availability during fermentation processes. Moreover, they allow for the assessment of an analyte’s biological relevance. Prototype yeast sensor and reporter strains derived from common laboratory strains were transformed with multicopy expression plasmids that mediate constitutive or inducible expression of a fluorescence reporter gene. Performance of these cells was examined by various qualitative and quantitative detection methods – representative of putative transducer technologies. Analyses were performed on the population level by microplate reader-based fluorometry and Western blot as well as on the single-cell level by fluorescence microscopy and flow cytometry. ‘Signature’ promoters that are activated or repressed during particular nutrient-limited growth conditions were selected in order to generate yeast nutrient sensor strains for monitoring the biological availability of nitrogen, phosphorus or sulphur. For each category, at least one promoter mediating at least threefold changed green fluorescence levels between sensor cells in non-limited and nutrient-limited conditions was identified. Sensor strains were evaluated in detail regarding sensitivity, analyte selectivity and the ability to restore basic fluorescence after shift from nutrient-limited to non-limited conditions (regeneration). The applicability for bioprocess monitoring purposes was tested by growth of yeast nutrient sensor cells in microalgae media and supernatants. Despite successful proof of principle, numerous challenges still need to be solved to realise prospective implementation in this field of white biotechnology. The major drawback of plasmid-borne detection constructs is a high fluorescence variance between individual cells. By generation of a nitrogen sensor strain with a genome-integrated detection construct, uniform expression on the single-cell level and simultaneous maintenance of basic properties (ability of fluorescence induction/regeneration and lack of cross-reactivity) was achieved. However, due to the singular detection construct per cell, significantly weaker overall fluorescence was observed. The traditional single-cell/single-construct approach was expanded upon in two ways. Firstly, a practical dual-colour sensor strain was created by simultaneous, constitutive expression of a red fluorescence reporter gene in green fluorescent nitrogen sensor cells. Secondly, an innovative cellular communication and signal amplification system inspired by the natural S. cerevisiae pheromone system and mating response was established successfully. It features the yeast pheromone alpha-factor as a trigger and alpha-factor-responsive reporter cells which express a fluorescence reporter gene from the pheromone-inducible FIG1 promoter as an output signal. The system was functional both with synthetic and cell-secreted alpha-factor, provided that recombinant cells were deleted for the alpha-factor protease Bar1p. Integration of amplifier cells which secrete alpha-factor in response to stimulation with the pheromone itself could increase the system\'s sensitivity further. Signal amplification was demonstrated for phosphorus sensor cells as a proof of concept. Therefore, the alpha-factor-based cellular communication and signal amplification system might be useful in applications that suffer from poor signal yield. Due to its modular design, the system could be applied in basically any cell-based biosensor or sensor-actor system. Immobilisation of the generated sensor and reporter cells in transparent natural polymers can be beneficial considering biosensor fabrication. Functionality of sensor and reporter cells in calcium-alginate beads or nano-printed arrays was successfully demonstrated. For the latter setup, fluorescence scanning and software-assisted fluorescence quantification was applied as a new detection method. In an experiment using an agarose-based two-compartment setup proposed by Jahn, 2011, properties of the alpha-factor-based cellular communication and signal amplification system after immobilisation were tested. These studies provide an initial experimental basis for an appropriate geometry of miniaturised immobilisation matrices with fluorescent yeast sensor and reporter cells in prospective biosensor designs.

Der Mangel an Nährstoffen im Boden, hauptsächlich an Phosphor (P) und Stickstoff (N), verbunden mit einem hohen Salzgehalt und der generellen Verarmung landwirtschaftlicher Böden , sind ein ernstes Problem für die landwirtschaftliche Produktion weltweit. Daher besteht ein dringender Bedarf an Forschung und Entwicklung geeigneter landwirtschaftlicher Praktiken, um ungünstige Bodenbedingungen zu verringern und wenn möglich die Fruchtbarkeit von Kulturland wiederherzustellen. Die Verwendung von Rhizobakterien, die das Pflanzenwachstum (PGPR) fördern, kann sich bei der Entwicklung von Strategien zur Erleichterung des Pflanzenwachstums unter normalen Wachstumsbedingungen sowie unter abiotischen Stress als nützlich erweisen. Diese Bakterien bieten ihren pflanzlichen Wirten Vorteile, indem sie die Aufnahme von Bodenmineralien fördern und Pflanzen vor schädlichen Umwelteinflüssen schützen. Die vorliegende Arbeit bewertet die Rolle von in Kamerun natürlich vorkommenden PGPR an Mais und untersucht deren Potenzial als Bioimpfstoffe zur Steigerung des Pflanzenwachstums in Kamerun. Wir prüfen die Hypothese, dass einheimische Bakteriengemeinschaften aus Kamerun einen hohen Anteil an Bakterien aufweisen, deren Eigenschaften Kulturpflanzen helfen, mit ungünstigen Bedingungen umzugehen. In der vorliegenden Arbeit wurden dazu Bakteriengemeinschaften der Rhizosphäre von in Kamerun angebautem Mais isoliert und untersucht. Zum ersten Mal erfolgte eine umfassende phylogenetische Zuordnung aller kultivierbaren Bakterien, auf Grundlage ihrer potenziellen Fähigkeiten zur Förderung des Pflanzenwachstums. / Nutrient deficiencies in soil, mainly in phosphorus (P) and nitrogen (N), coupled to salinity and the impoverishment of agricultural soils, are a severe problem for agricultural production worldwide. Therefore, there is an urgent need for research and development of more suitable agricultural practices in order to reduce unfavorable conditions, and if possible, to restore the fertility of cultivated lands. The use of rhizobacteria, which promote plant growth (PGPR), can prove useful in developing strategies to facilitate plant growth under normal as well as under abiotic stress conditions. These bacteria offer benefits to plant hosts by promoting the uptake of soil minerals and protecting plants from environmental stresses. The thesis evaluates the role of native PGPR associated with maize as potential bio-inoculants for plants growth in Cameroon. We hypothesized that native bacterial communities from Cameroon include a high potential of bacteria helping the plant cope with unfavorable conditions. Here, we provide for the first time a comprehensive phylogenetic affiliation of cultivable bacterial communities associated with maize rhizosphere grown in Cameroon in relationship to their potential plant growth-promoting abilities.

No description available.