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In den letzten 20 Jahren hat sich der Maiszünsler (Ostrinia nubilalis HÜBNER), aus der Schmetterlingsfamilie der Pyralidae oder Zünsler, zum bedeutendsten tierischen Schädling des Maises (Zea mays) entwickelt. Eine Möglichkeit den Befall des Maiszünslers abzuwenden, bietet der Anbau von Bacillus thuringiensis-Mais (Bt-Mais). Mit Hilfe der Gentechnik wurden Gene des Bakteriums Bacillus thuringiensis übertragen, die einen für Fraßinsekten giftigen Wirkstoff bilden, wodurch die Pflanzen während der kompletten Vegetation vor den Larven des Maiszünslers geschützt sind. Ziel des vorliegenden Projektes war es, in einer 3-jährigen Studie die Auswirkungen des großflächigen Anbaus von Bt-Mais auf die ökologische Situation und den Handlungsrahmen des integrierten Pflanzenschutzes komplex zu untersuchen. Dazu wurden in Betrieben im Oderbruch, das als permanentes Befallsgebiet des Maiszünslers gilt, in den Jahren 2002 bis 2004 jährlich zwei Felder mit jeweils einer Bt-Sorte und einer konventionellen Sorte angelegt. Zusätzlich wurden biologische und chemische Maiszünsler-Bekämpfungsvarianten geprüft. Durch verschiedene Methoden wie Bonituren, Ganzpflanzenernten, Bodenfallenfänge und Beobachtungen des Wahlverhaltens von (Flug-)insekten konnten Aussagen zum Vorkommen von Insekten und Spinnentieren getroffen werden, wobei hierfür Daten aus Untersuchungen der Jahre 2000 und 2001 im Oderbruch ergänzend herangezogen werden konnten. Durch Ertragsmessungen, Energie- und Qualitätsermittlungen, sowie Fusarium- und Mykotoxinanalysen konnte der Anbau von Bt-Mais als neue Alternative zur Bekämpfung des Maiszünslers bewertet werden. Bezüglich des Auftretens von Insekten und Spinnentieren wurden im Mittel der fünfjährigen Datenerhebung beim Vergleich der Bt-Sorte zur konventionellen Sorte, mit Ausnahme der fast 100 %igen Bekämpfung des Maiszünslers, keine signifikanten Unterschiede festgestellt. Hierfür wurde ein besonderes Augenmerk auf Thripse, Wanzen, Blattläuse und deren Fraßfeinde, sowie mittels Bodenfallenfängen auf Laufkäfer und Spinnen gerichtet. Die erwarteten ökonomischen Vorteile wie etwa Ertragsplus oder bessere Nährstoff- und Energiegehalte durch geringeren Schaden beim Anbau von Bt-Mais als Silomais blieben in den Untersuchungsjahren aus. Allerdings zeigten Fusarium- und Mykotoxinanalysen eine geringere Belastung des Bt-Maises, was möglicherweise auf den geringeren Schaden zurückzuführen ist, da beschädigte Pflanzen für Fusarium und Mykotoxine anfälliger sind. Desweiteren konnten erste methodische Ansätze für ein auf EU-Ebene gefordertes, den Anbau von Bt-Mais begleitendes Monitoring, erarbeitet werden. So konnten Vorschläge für geeignete Methoden, deren Umfang sowie des Zeitpunktes der Durchführungen gemacht werden.
Regional variation in nitrogen (N) deposition increases plant productivity and decreases species diversity, but landscape- or local -scale influences on N deposition are less well-known. Using ion-exchange resin, we measured variation of N deposition and soil N availability within Elk Island National Park in the ecotone between grassland and boreal forest in western Canada. The park receives regionally high amounts of atmospheric N deposition (22 kg ha(-1) yr(- 1)). N deposition was on average higher ton clay-rich luvisols than on brunisols, and areas burned 1-15 years previously received more atmospheric N than unburned sites. We suggest that the effects of previous fires and soil type on deposition rate act through differences in canopy structure. The magnitude of these effects varied with the presence of ungulate grazers (bison, moose, elk) and vegetation type (forest, shrubland, grassland). Available soil N (ammonium and nitrate) was higher in burned than unburned sites in the absence of grazing, suggesting an effect of deposition. On grazed sites, differences between fire treatments were small, presumably because the removal of biomass by grazers reduced the effect of fire. Aspen invades native grassland in this region, and our results suggest that fire without grazing might reinforce the expansion of forest into grassland facilitated by N deposition
Investigation of the ectoparasitic fauna (Siphonaptera: Ischnopsyllidae; Diptera: Nycteribiidae) on bats in Brandenburg, Germany. During the investigation of bats in hibernation places four species of fleas (Ischnopsyllus hexactenus (Kolenati, 1856) on Myotis myotis and Myotis nattereri, Nycteridopsylla pentactena (Kolenati, 1856)on Myotis daubentoni, Ischnopsyllus elongatus (Curtis, 1832) and Nycteridopsylla eusarca Dampf, 1908 on Nyctalus noctula) and one bat fly (Nycteribia kolenatii Theodor and Moscona, 1954 on Myotis daubentoni, Myotis natereri and Plecotus auritus were found. For the first time specimens of Pipistrellus pipistrellus and Pipistrellus pygmaeus were investigated separately in summer quarters to reveal differences in the parasitic flea fauna. Ischnopsyllus octactenus (Kolenati, 1856) was present on both bat species. The host-parasite relationship of autochthonous flea species in Brandenburg is discussed.
Die Etablierung der Transkription von kompletten Genen auf planaren Oberflächen soll eine Verbindung zwischen der Mikroarraytechnologie und der Transkriptomforschung herstellen. Darüber hinaus kann mit diesem Verfahren ein Brückenschlag zwischen der Synthese der Gene und ihrer kodierenden Proteine auf einer Oberfläche erfolgen. Alle transkribierten RNAs wurden mittels RT-PCR in cDNA umgeschrieben und in einer genspezifischen PCR amplifiziert. Die PCR-Produkte wurden hierfür entweder per Hand oder maschinell auf die Oberfläche transferiert. Über eine Oberflächen-PCR war es möglich, die Gensequenz des Reportergens EGFP direkt auf der Oberfläche zu synthetisieren und anschließend zu transkribieren. Somit war eine Transkription mit weniger als 1 ng an Matrize möglich. Der Vorteil einer Oberflächen-Transkription gegenüber der in Lösung liegt in der mehrfachen Verwendung der immobilisierten Matrize, wie sie in dieser Arbeit dreimal erfolgreich absolviert wurde. Die Oberflächen-Translation des EGFP-Gens konnte ebenfalls zweimal an einer immobilisierten Matrize gezeigt werden, wobei Zweifel über eine echte Festphasen-Translation nicht ausgeräumt werden konnten. Zusammenfassend kann festgestellt werden, dass die Transkription und Translation von immobilisierten Gensequenzen auf planaren Oberflächen möglich ist, wofür die linearen Matrizen direkt auf der Oberfläche synthetisiert werden können.
TPK1 ( formerly KCO1) is the founding member of the family of two-pore domain K 1 channels in Arabidopsis ( Arabidopsis thaliana), which originally was described following expression in Sf9 insect cells as a Ca2(+)- and voltage- dependent outwardly rectifying plasma membrane K 1 channel. In plants, this channel has been shown by green fluorescent protein fusion to localize to the vacuolar membrane, which led to speculations that the TPK1 gene product would be a component of the nonselective, Ca2+ and voltage- dependent slow-vacuolar (SV) cation channel found in many plants species. Using yeast ( Saccharomyces cerevisiae) as an expression system for TPK1, we show functional expression of the channel in the vacuolar membrane. In isolated vacuoles of yeast yvc1 disruption mutants, the TPK1 gene product shows ion channel activity with some characteristics very similar to the SV-type channel. The open channel conductance of TPK1 in symmetrically 100mM KCl is slightly asymmetric with roughly 40 pS at positive membrane voltages and 75 pS at negative voltages. Similar to the SV-type channel, TPK1 is activated by cytosolic Ca2+, requiring micromolar concentration for activation. However, in contrast to the SV- type channel, TPK1 exhibits strong selectivity for K+ over Na+, and its activity turned out to be independent of the membrane voltage over the range of +/- 80mV. Our data clearly demonstrate that TPK1 is a voltage- independent, Ca2+- activated, K+- selective ion channel in the vacuolar membrane that does not mediate SV- type ionic currents
We tested whether neighborhood density affects the clonal life history of the stoloniferous plant Ranunculus reptans through selection and genetic drift. After three generations of sexual reproduction of 16 low- and 16 high- density lines, we studied traits related to growth form and reproduction in a common competition free environment. A 7.7% lower branching frequency and slightly longer internodes indicated an evolutionary shift towards a less compact growth form under high neighborhood density, but because stolons grew also more vertically, horizontal spread per ramet was slightly decreased. Neighborhood density had no directional effects on the evolution of allocation to sexual and vegetative reproduction in R. reptans. Variation among replicated high-density lines was significantly lower than among replicated low-density lines in both growth form and reproductive characteristics, indicating less pronounced genetic drift under high neighborhood density. This study demonstrates that a clonal plant can respond to selection imposed by neighborhood density. Moreover, it shows that the effect of random genetic drift increases with decreasing neighborhood density. In a declining species, such as R. reptans in central Europe, this may lower the potential for adaptive evolutionary change and increase extinction risk
Electrochemical investigations of the blood oxygen carrier protein include both mediated and direct electron transfer. The reaction of haemoglobin (Hb) with typical mediators, e.g., ferricyanide, can be quantified by measuring the produced ferrocyanide which is equivalent to the Hb concentration. Immobilization of the mediator within the electrode body allows reagentless electrochemical measuring of Hb. On the other hand, entrapment of the protein within layers of polyclectrolytes, lipids, nanoparticles of clay or gold leads to a fast heterogeneous electron exchange of the partially denatured Hb. (c) 2005 Elsevier B.V. All rights reserved
The thermal unfolding of the wild-type lambda Cro repressor and of two designed variants, Cro K56-[DGEVK] and Cro K56-[DGEVK] Q16L, was studied by Fourier transform infrared spectroscopy and dynamic light scattering. The engineered Cro K56-[DGEVK] monomer has five additional amino acids inserted after position 56 of the wild-type sequence, while the K56-[DGEVK] Q16L variant differs only in one position (Gln-16 to Leu substitution) from the Cro K56-[DGEVK] sequence. The temperature dependence of selected protein backbone infrared `marker' bands revealed that Cro K56- [DGEVK] is slightly more stable than the wild-type protein, while the replacement of Gln-16 by Leu increases the thermal transition temperature by similar to 20 degrees C. Moreover, thermal unfolding of the two Cro variants was found to proceed through equilibrium unfolding intermediates and to involve the formation of oligomers. The first thermal transition of Cro K56-[DGEVK] involves the melting of major parts of its native secondary structure and is accompanied by the formation of dinners and non-native beta-sheet structures. These structures unfold during a second transition at higher temperatures, accompanied by the dissociation of the dimers. In contrast to the Cro K56-[DGEVK] protein, the intermediate state of the Cro K56-[DGEVK] Q16L variant is less well defined, and involves the formation of oligomers of different size. (c) 2005 Elsevier B.V. All rights reserved
The multiplication and antibody production of murine hybridoma cells cultured on five different polymer membranes were tested and compared with conventional tissue culture polystyrene (TCPS). Membranes were prepared from polyacrylonitrile (PAN) and acrylonitrile copolymerized with N-vinylpyrrolidone (NVP20, NVP30), Na-methallylsulfonate (NaMAS) and N-(3-amino-propyl-methacrylamide-hydrochloride) (APMA). Cell number and antibody concentration were quantified as criteria for viability and productivity. Adhesion of hybridoma cells was characterized by vital and scanning electron microscopy. The results suggest that a strong adhesion of cells, observed on APMA and TCPS, increased cell growth but reduced monoclonal antibody production. In contrast membranes with lowered adhesivity such as NVP20 provided favourable conditions for monoclonal antibody production. In addition it was shown that this membrane also possessed a minor fouling as indicated by the low decrease of water flux across the membrane after protein adsorption. It was concluded that NVP20 could be a suitable material for the development of hollow fibre membranes for bioreactors.
The present study aimed at assessing genetic purity of black wildebeest (Connochoetes gnou) at Abe Bailey Nature Reserve, Gauteng Province, South Africa, using a multitocus microsatellite approach. Five loci were studied in black and blue (C. taurinus) wildebeest, the latter being a closely related species and known to produce hybrids with the morphologically very similar black wildebeest. In fact, the entire national black wildebeest population of South Africa potentially contains a significant proportion of introgressed blue wildebeest genes. In our case, eight out of 39 alleles were unique to black and 22 to blue wildebeest, with nine alleles shared between pure populations of the two species in Line with their taxonomic proximity. A possible Limited past introgression of blue wildebeest genes into the Abe Bailey population, corresponding to documents on population history, was only supported by the presence of a single allele otherwise exclusively found in samples of four pure blue but not in samples of two pure black wildebeest control populations. However, an assignment test and coefficients of population divergence did not support an extended introgression of C. taurinus alleles into the C. gnou population under study. Average heterozygosity at Abe Bailey proved to be intermediate between black and blue wildebeest, the tatter species generally harbouring more genetic variation than the former owing to larger population sizes and the absence of population bottlenecks in historical times. The implications of our data are discussed with reference to the persistence of introgressed genes and the conservation of pure black wildebeest gene pools
Among plant remains from Mesolithic layers dating from 9249 to 7779 B.C. at the excavation site of Friesack IV in north-eastern Germany, nutlets of Urtica kioviensis were identified. Morphological studies have shown that they clearly differed from all other European Urtica species investigated. In contrast, pollen morphological investigations revealed only slight differences between the central European Urtica species, which could hardly have been noticed during routine or normal pollen analyses. The records of U. kioviensis nutlets are the first subfossil finds reported and prove the indigenous status of this taxon in north-eastern Germany. The records are discussed in the context of the overall species spectrum of the Mesolithic layers and consequences for the interpretation of pollen analytical studies concerning human impact are pointed out
After heterologous expression in Escherichia coli, the Azotobacter vinelandii rhodanese RhdA is purified in a persulfurated form (RhdA-SSH). We identified L-cysteine as the most effective sulfur source in producing RhdA-SSH. An E. coli soluble extract was required for in vitro persulfuration of RhdA, and the addition of pyridoxal-5'-phosphate increased RhdA-SSH production, indicating a likely involvement of a cysteine desulfurase. We were able to show the formation of a covalent complex between IscS and RhdA. By combining a time-course fluorescence assay and mass spectrometry analysis, we demonstrated the transfer of sulfur from E. coli IscS to RhdA. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved
Testing for ecological and genetic Allee effects in the invasive shrub Senna didymobotrya (Fabaceae)
(2005)
For an introduced plant species to become invasive, it must be able to reproduce even in initially small populations We tested for Aliee effects (reduced reproductive performance of individuals in small populations) in the nonclonal, NW-pollinated shrub Senna didymobotrya in its invasive range in South Africa. The species is self-compatible. but we found that in its invasive range in South Africa it requires pollinators to set seed. Nearly all stigmas (90%) received pollen. but natural fruit set has very low (3-20%), Pollen receipt and fruit set were not significantly correlated with population size. We thus found no evidence for an ecological Alice effect arising from pollen limitation in small populations. Offspring seedling performance, measured in terms of stern volume and leaf area, was also not significantly correlated with the number of plants in the source population. indicating that genetic Alice effects, such as inbreeding depression, are either absent or of such a small magnitude that they would be unlikely to limit further spread of S. didymobotrya in South Africa
Molybdenum cofactor deficiency (MIM#252150) is a severe autosomal- recessive disorder with a devastating outcome. The cofactor is the product of a complex biosynthetic pathway involving four different genes (MOCS1, MOCS2, MOCS3 and GEPH). This disorder is caused almost exclusively by mutations in the MOCS1 or MOCS2 genes. Mutations affecting this biosynthetic pathway result in a lethal phenotype manifested by progressive neurological damage via the inactivation of the molybdenum cofactor-dependent enzyme, sulphite oxidase. Here we describe a total of ten novel disease-causing mutations in the MOCS1 and MOCS2 genes. Nine out of these ten mutations were classified as pathogenic in nature, since they create a stop codon, affect constitutive splice site positions, or change strictly conserved motifs. The tenth mutation abolishes the stop codon of the MOCS2B gene, thus elongating the corresponding protein. The mutation was expressed in vitro and was found to abolish the binding affinities of the large subunit of molybdopterin synthase (MOCS2B) for both precursor Z and the small subunit of molybdopterin synthase (MOCS2A)
Synthesis, characterisation and theoretical calculations of 2,6-diaminopurine etheno derivatives
(2005)
Four derivatives of 2,6-diaminopurine (1) were synthesised and characterised. When 1 was reacted with chloroacetaldehyde, 5-aminoimidazo[2,1- i] purine (2), 9-aminoimidazo[2,1-b]purine (3), 9-aminoimidazo[1,2- a]purine (4) and diimidazo[2,1-b: 2', 1'-i]purine (5) were formed. The purified products (3 - 5) were fully characterised by MS, complete NMR assignments as well as fluorescence and UV spectroscopy. The purified, isolated yields of these products ( 3 - 5) varied from 2.5 to 30%. The relative stability of different tautomers was investigated by theoretical calculations. Fluorescence characteristics are also discussed and compared to the starting material 1 and a reference molecule 2-aminopurine
Empirical evidence suggests that the direction and intensity of plant-plant interactions may depend on the favourability of the environment. Previous studies have mainly focused on steep gradients of environmental stress or disturbance, while the interplay of competition and environment has not been tested for subtle environmental differences. Here, we present results from a study on plant communities of temporary wetlands in East-German farmland. Due to yearly ploughing in autumn, the vegetation is composed of annual species. Flooding does not affect adult plants and the elevation on the gradient expresses differences in the length of the growing season rather than in disturbance intensity or severe environmental stress. We tested whether such subtle differences in environmental stress may affect the importance of interspecific competition by the dominant species. Two treatments were applied at two elevations: removal of the dominant species (Matricaria maritima ssp. inodora) and reciprocal transplants of the seed-bank of the two elevations. At both elevations, removal of Matricaria inodora led to an increase in total species richness and number of wetland species, but the effects were substantially stronger at high elevations. Removal and the elevation on the flooding gradient significantly influenced the plant community composition. In particular, the weed communities became more similar to the wetland communities after the removal. Transplanted weed species did not emerge at low elevations. While two of four target species had significantly higher densities after the removal at high elevations, none of them was influenced by removal at low elevations. This indicates that, consistent with previous studies from other habitat types, competition by the dominant species was more intense under conditions of low environmental stress. The overall results suggest that both flooding as well as interspecific competition are important in structuring the plant communities along the freshwater gradient studied
In this study we determined the complete sequence of the mitochondrial DNA (mtDNA) control region of the Eurasian otter (Lutra lutra). We then compared these new sequences with orthologues of nine carnivores belonging to six families (Mustelidae, Mephitidae, Canidae, Hyaenidae, Ursidae, and Felidae). The comparative analyses identified all the conserved regions previously found in mammals. The Eurasian otter and seven other species have a single location with tandem repeats in the right domain, while the spotted hyena (Hyaenidae) and the tiger (Felidae) have repeated sequences in both the right and left domains. To assess the degree of genetic heterogeneity of the Eurasian otter in Italy we sequenced two fragments of the gene and analyzed length polymorphisms of repeated sequences and heteroplasmy in 32 specimens. The study includes 23 museum specimens collected in northern, central, and southern Italy; most of these specimens are from extinct populations, while the southern Italian samples belong to the sole extant Italian population of the Eurasian otter. The study also includes all the captive-reared animals living in the colony "Centro Lontra, Caramanico Terme" (Pescara, central Italy). The colony is maintained for reintroduction of the species. We found a low level of genetic polymorphism; a single haplotype is dominant, but our data indicate the presence in central and southern Italy of two slightly divergent haplotypes. One haplotype belongs to an extinct population, the other is present in the single extant Italian population. Analyses of length polymorphisms and heteroplasmy indicate that the autochthonous Italian samples are characterized by a distinct array of repeated sequences from captive-reared animals
Background: Protein sequence motifs are by definition short fragments of conserved amino acids, often associated with a specific function. Accordingly protein sequence profiles derived from multiple sequence alignments provide an alternative description of functional motifs characterizing families of related sequences. Such profiles conveniently reflect functional necessities by pointing out proximity at conserved sequence positions as well as depicting distances at variable positions. Discovering significant conservation characteristics within the variable positions of profiles mirrors group-specific and, in particular, evolutionary features of the underlying sequences. Results: We describe the tool PROfile analysis based on Mutual Information (PROMI) that enables comparative analysis of user-classified protein sequences. PROMI is implemented as a web service using Perl and R as well as other publicly available packages and tools on the server-side. On the client-side platform-independence is achieved by generally applied internet delivery standards. As one possible application analysis of the zinc finger C2H2-type protein domain is introduced to illustrate the functionality of the tool. Conclusion: The web service PROMI should assist researchers to detect evolutionary correlations in protein profiles of defined biological sequences. It is available at http:// promi.mpimpgolm. mpg.de where additional documentation can be found
From its first use in the field of biochemistry, instrumental analysis offered a variety of invaluable tools for the comprehensive description of biological systems. Multi-selective methods that aim to cover as many endogenous compounds as possible in biological samples use different analytical platforms and include methods like gene expression profile and metabolite profile analysis. The enormous amount of data generated in application of profiling methods needs to be evaluated in a manner appropriate to the question under investigation. The new field of system biology rises to the challenge to develop strategies for collecting, processing, interpreting, and archiving this vast amount of data; to make those data available in form of databases, tools, models, and networks to the scientific community. On the background of this development a multi-selective method for the determination of phytohormones was developed and optimised, complementing the profile analyses which are already in use (Chapter I). The general feasibility of a simultaneous analysis of plant metabolites and phytohormones in one sample set-up was tested by studies on the analytical robustness of the metabolite profiling protocol. The recovery of plant metabolites proved to be satisfactory robust against variations in the extraction protocol by using common extraction procedures for phytohormones; a joint extraction of metabolites and hormones from plant tissue seems practicable (Chapter II). Quantification of compounds within the context of profiling methods requires particular scrutiny (Chapter II). In Chapter III, the potential of stable-isotope in vivo labelling as normalisation strategy for profiling data acquired with mass spectrometry is discussed. First promising results were obtained for a reproducible quantification by stable-isotope in vivo labelling, which was applied in metabolomic studies. In-parallel application of metabolite and phytohormone analysis to seedlings of the model plant Arabidopsis thaliana exposed to sulfate limitation was used to investigate the relationship between the endogenous concentration of signal elements and the ‘metabolic phenotype’ of a plant. An automated evaluation strategy was developed to process data of compounds with diverse physiological nature, such as signal elements, genes and metabolites – all which act in vivo in a conditional, time-resolved manner (Chapter IV). Final data analysis focussed on conditionality of signal-metabolome interactions.
A highly sensitive piezoelectric biosensor has been developed for detection of cholinesterase inhibitors. The inhibitor benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO) was immobilized on a monolayer of 11- mercaptomonoundecanoic acid (MUA) self-assembled on the gold surface of the sensor. The binding of high-molecular-weight cholinesterase to the immobilized cocaine derivative was monitored with a mass sensitive piezoelectric quartz crystal (quartz crystal nanobalance; QCN). In the presence of an inhibiting substance in the sample, the binding of cholinesterase to the immobilized inhibitor was reduced. The decrease of the rate of mass change was proportional to the concentration of free inhibitor in the sample. This way the affinity sensor followed anti-cholinesterase toxicity and the enzyme activity of ChE was not addressed. A assay for detection of organophosphates (OP) was optimized. Regeneration of the sensor surface was achieved with 1 mol L-1 formic acid, which enabled 40 measurements with one sensor. All assays were carried out in a flow-through arrangement. The total measurement time (binding + regeneration) was 25 min and the detection limit for different OP (paraoxon, diisopropylfluorophosphate, chlorpyriphos, and chlorfenvinphos) was down to 10(-10) mol L-1 (0.02 mu g L-1). This sensor was used for determination of organophosphate (diisopropylfluorophosphate) levels in river water samples
Different aspects of the structure formation for a class of molecules containing the diphenyl-1,3,4-oxadiazole fragment are discussed. Starting from the bulk state with the ideal crystal lattice and the derivation of some common packing motifs the formation of liquid-crystalline states are described. This leads to the consideration of structures found in Langmuir-Blodgett films and those obtained by organic molecular beam deposition. These structures may again be compared to those for the bulk crystalline state. Common features as well as characteristic differences due to peculiarities of the individual molecular structures are discussed. (c) 2005 Elsevier B.V. All rights reserved
The synthesis of materials via self-assembly typically involves the spontaneous and reversible organization,of small building blocks for the purpose of creating conglomerate assembly structures over larger length scales. This introductory article, describes self processes on several length scales, from subnanometer, up to millimeter scales, and briefly summarizes some of the incredible diversity of materials that exhibit self-assembly. Articles in this issue cover self-assembly using zeolitic structures, organic molecular crystals; block copolymers, surfactants, mesoscale templates, and soluble. crystallization additives
Im Norddeutschen Tiefland wurde die Ausbreitung von Gefäßpflanzen durch Rehe, Dam- und Rothirsche sowie Wildschweine untersucht. Diese Tiere transportieren zahlreiche Pflanzenarten in teilweise erheblichen Mengen über größere Distanzen, sowohl durch den Kot nach Darmpassage (Endozoochorie) als auch durch Anheftung an Fell und Schalen (Epizoochorie). Besondere Bedeutung kommt dabei Wildschweinen zu, die potenziell fast alle Pflanzenarten ausbreiten können. Bevorzugt werden im Wald wie im Offenland vorkommende Pflanzen und Arten des Offenlands ausgebreitet, während Arten mit enger Waldbindung nur in geringem Maße transportiert werden. Zoochorie durch Schalenwild bietet Erklärungsansätze sowohl für Ausbreitungsphänomene wie auch für das weitgehend fehlende Ausbreitungspotenzial vieler Pflanzenarten. Der Einfluss des Schalenwilds auf die Artenzusammensetzung und Gefäßpflanzen-Diversität in der mitteleuropäischen Kulturlandschaft sollte in seine naturschutzfachliche Neubewertung miteinbezogen werden. Die Einschränkung von Aktionsradien der Tiere durch die Zerschneidung von Lebensräumen sowie die Wildfütterung können für Ausbreitungsprozesse bisher kaum beachtete Konsequenzen haben.
This Red List of grasshoppers and crickets for Berlin is the first that treats the whole area of Berlin, since the two preceding lists focused only on the western parts of the formerly divided city. Until now 54 species of grasshoppers and crickets have been recorded in the area. 46 species have been found to be well established in the wild, while eight species were only non breeding accidental immigrants or species whose successful reproduction has only been observed in greenhouses or similar structures. Of the 46 species being well established in the past and/or present 22 must be included in one of the threat categories, indicating that the populations of these species are already endangered. Additionally, seven species belong to the category "Near Threatened". Only 17 of the grasshopper and cricket species of Berlin are not threatened.
Insect cells convert vitamin A into a number of retinoids that are evolutionarily conserved with those of mammalian cells. However, insect cells also produce additional natural retinoids. Namely, two retinoic acid peptides, N- trans-retinoylserine (1) and N-trans-retinoylalanine (2), have been isolated from a cell line of the common cabbage looper, Trichoplusia ni. These are the first examples of naturally occurring retinoic acid linked to amino acids through an amide bond; the amino acid moieties are depicted in the more common L-configuration, although the absolute configuration was not determined due to the minuscule sample amount
Post-translational redox-regulation is a well-known mechanism to regulate enzymes of the Calvin cycle, oxidative pentose phosphate cycle, NADPH export and ATP synthesis in response to light. The aim of the present thesis was to investigate whether a similar mechanism is also regulating carbon storage in leaves. Previous studies have shown that the key-regulatory enzyme of starch synthesis, ADPglucose pyrophosphorylase (AGPase) is inactivated by formation of an intermolecular disulfide bridge between the two catalytic subunits (AGPB) of the heterotetrameric holoenzyme in potato tubers, but the relevance of this mechanism to regulate starch synthesis in leaves was not investigated. The work presented in this thesis shows that AGPase is subject to post-translational redox-regulation in leaves of pea, potato and Arabidopsis in response to day night changes. Light was shown to trigger posttranslational redox-regulation of AGPase. AGPB was rapidly converted from a dimer to a monomer when isolated pea chloroplasts were illuminated and from a monomer to a dimer when preilluminated leaves were darkened. Conversion of AGPB from dimer to monomer was accompanied by an increase in activity due to changes in the kinetik properties of the enzyme. Studies with pea chloroplast extracts showed that AGPase redox-activation is mediated by thioredoxins f and m from spinach in-vitro. In a further set of experiments it was shown that sugars provide a second input leading to AGPase redox activation and increased starch synthesis and that they can act as a signal which is independent from light. External feeding of sugars such as sucrose or trehalose to Arabidopsis leaves in the dark led to conversion of AGPB from dimer to monomer and to an increase in the rate of starch synthesis, while there were no significant changes in the level of 3PGA, an allosteric activator of the enyzme, and in the NADPH/NADP+ ratio. Experiments with transgenic Arabidopsis plants with altered levels of trehalose 6-phosphate (T6P), the precursor of trehalose synthesis, provided genetic evidence that T6P rather than trehalose is leading to AGPase redox-activation. Compared to Wt, leaves expressing E.coli trehalose-phosphate synthase (TPS) in the cytosol showed increased activation of AGPase and higher starch level during the day, while trehalose-phosphate phosphatase (TPP) overexpressing leaves showed the opposite. These changes occurred independently of changes in sugar and sugar-phosphate levels and NADPH/NADP+ ratio. External supply of sucrose to Wt and TPS-overexpressing leaves led to monomerisation of AGPB, while this response was attenuated in TPP expressing leaves, indicating that T6P is involved in the sucrose-dependent redox-activation of AGPase. To provide biochemical evidence that T6P promotes redox-activation of AGPase independently of cytosolic elements, T6P was fed to intact isolated chloroplasts for 15 min. incubation with concentrations down to 100 µM of T6P, but not with sucrose 6-phosphate, sucrose, trehalose or Pi as controls, significantly and specifically increased AGPB monomerisation and AGPase activity within 15 minutes, implying T6P as a signal reporting the cytosolic sugar status to the chloroplast. The response to T6P did not involve changes in the NADPH/NADP+ ratio consistent with T6P modulating redox-transfer to AGPase independently of changes in plastidial redox-state. Acetyl-CoA carboxylase (ACCase) is known as key-regulatory enzyme of fatty acid and lipid synthesis in plants. At the start of the present thesis there was mainly in vitro evidence in the literature showing redox-regulation of ACCase by DTT, and thioredoxins f and m. In the present thesis the in-vivo relevance of this mechanism to regulate lipid synthesis in leaves was investigated. ACCase activity measurement in leaf tissue collected at the end of the day and night in Arabidopsis leaves revealed a 3-fold higher activation state of the enzyme in the light than in the dark. Redox-activation was accompanied by change in kinetic properties of ACCase, leading to an increase affinity to its substrate acetyl-CoA . In further experiments, DTT as well as sucrose were fed to leaves, and both treatments led to a stimulation in the rate of lipid synthesis accompanied by redox-activation of ACCase and decrease in acetyl-CoA content. In a final approach, comparison of metabolic and transcript profiling after DTT feeding and after sucrose feeding to leaves provided evidence that redox-modification is an important regulatory mechanism in central metabolic pathways such as TCA cycle and amino acid synthesis, which acts independently of transcript levels.
Das Superoxidradikal kann mit fast allen Bestandteilen von Zellen reagieren und diese schädigen. Die medizinische Forschung stellte eine Beteiligung des Radikals an Krebs, Herzinfarkten und neuraler Degeneration fest. Ein empfindlicher Superoxidnachweis ist daher zum besseren Verständnis von Krankheitsverläufen wichtig. Dabei stellen die geringen typischen Konzentrationen und seine kurze Lebensdauer große Anforderungen. Ziel dieser Arbeit war es zum einen, zwei neuartige Proteinarchitekturen auf Metallelektroden zu entwickeln und deren elektrochemisches Ansprechverhalten zu charakterisieren. Zum anderen waren diese Elektroden zur empfindlichen quantitativen Superoxiddetektion einzusetzen. Im ersten Teil der Arbeit wurde eine Protein-Multischichtelektrode aus Cytochrom c und dem Polyelektrolyten Poly(anilinsulfonsäure) nach dem Layer-by-layer-Verfahren aufgebaut. Für zwei bis 15 Schichten an Protein wurde eine deutliche Zunahme an elektrodenaktivem Cytochrom c mit jedem zusätzlichen Aufbringungsschritt nachgewiesen. Die Zunahme verlief linear und ergab bei 15 Schichten eine Zunahme der redoxaktiven Proteinmenge um deutlich mehr als eine Größenordnung. Während das formale Potential im Multischichtsystem sich im Vergleich zur Monoschichtelektrode nicht veränderte, wurde für die Kinetik eine Abhängigkeit der Geschwindigkeit des Elektronentransfers von der Zahl der Proteinschichten beobachtet. Mit zunehmender Scangeschwindigkeit trat ein reversibler Kontaktverlust zu den äußeren Schichten auf. Die lineare Zunahme an elektroaktivem Protein mit steigender Zahl an Depositionsschritten unterscheidet sich deutlich von in der Literatur beschriebenen Protein/Polyelektrolyt-Multischichtelektroden, bei denen ab etwa 6-8 Schichten keine Zunahme an elektroaktivem Protein mehr festgestelltwurde. Auch ist bei diesen die Zunahme an kontaktierbaren Proteinmolekülen auf das Zwei- bis Fünffache limitiert. Diese Unterschiede des neu vorgestellten Systems zu bisherigen Multischichtassemblaten erklärt sich aus einem in dieser Arbeit für derartige Systeme erstmals beschriebenen Elektronentransfermechanismus. Der Transport von Elektronen zwischen der Elektrodenoberfläche und den Proteinmolekülen in den Schichten verläuft über einen Protein-Protein-Elektronenaustausch. Dieser Mechanismus beruht auf dem schnellen Selbstaustausch von Cytochrom c-Molekülen und einer verbleibenden Rotationsflexibilität des Proteins im Multischichtsystem. Die Reduzierung des Proteins durch das Superoxidradikal und eine anschließende Reoxidation durch die Elektrode konnten nachgewiesen werden. In einem amperometrischen Messansatz wurde das durch Superoxidradikale hervorgerufene elektrochemische Signal in Abhängigkeit von der Zahl an Proteinschichten gemessen. Ein maximales Ansprechverhalten auf das Radikal wurde mit 6-Schichtelektroden erzielt. Die Empfindlichkeit der 6-Schichtelektroden wurde im Vergleich zum Literaturwert der Monoschichtelektrode um Faktor 14, also mehr als eine Größenordnung, verbessert. Somit konnte eine Elektrode mit 6 Schichten aus Cytochrom c und Poly(anilinsulfonsäure) als neuartiger Superoxidsensor mit einer 14-fachen Verbesserung der Empfindlichkeit im Vergleich zum bislang benutzten System entwickelt werden. Der zweite Teil dieser Arbeit beschreibt die Auswahl, Gewinnung und Charakterisierung von Mutanten des Proteins Cu,Zn-Superoxiddismutase zur elektrochemischen Quantifizierung von Superoxidradikalen. Monomere Mutanten des humanen dimeren Enzyms wurden entworfen, die durch Austausch von Aminosäuren ein oder zwei zusätzliche Cysteinreste besaßen, mit welchem sie direkt auf der Goldelektrodenoberfläche chemisorbieren sollten. 6 derartige Mutanten konnten in ausreichender Menge und Reinheit in aktiver Form gewonnen werden. Die Bindung der Superoxiddismutase-Mutanten an Goldoberflächen konnte durch Oberflächen-plasmonresonanz und Impedanzspektroskopie nachgewiesen werden. Alle Mutanten wiesen einen quasi-reversiblen Elektronentransfer zwischen SOD und Elektrode auf. Durch Untersuchung von kupferfreien SOD-Mutanten sowie des Wildtyps konnte nachgewiesen werden, das die Mutanten über die eingefügten Cysteinreste auf der Elektrode chemisorptiv gebunden wurden und der Elektronentransfer zwischen der Elektrode und dem Kupfer im aktiven Zentrum der SOD erfolgte. Die Superoxiddismutase katalysiert die Zersetzung von Superoxidmolekülen durch Oxidation und durch Reduktion der Radikale. Somit sind beide Teilreaktionen von analytischem Interesse. Zyklovoltammetrisch konnte sowohl die Oxidation als auch die Reduktion des Radikals durch die immobilisierten Superoxiddismutase-Mutanten nachgewiesen werden. In amperometrischen Messanordnungen konnten beide Teilreaktionen zur analytischen Quantifizierung von Superoxidradikalen genutzt werden. Im positiven Potentialfenster wurde die Empfindlichkeit um einen Faktor von etwa 10 gegenüber der Cytochrom c–Monoschichtelektrode verbessert.
The salivary glands in the cockroach Periplaneta americana secrete protein-containing saliva when stimulated by serotonin (5-HT) and protein-free saliva upon dopamine stimulation. In order to obtain information concerning the signalling pathways involved in 5-HT-induced protein secretion, we have determined the protein content of saliva secreted after experimental manipulations that potentially elevate intracellular Ca2+ and cyclic nucleotide concentrations in isolated glands. We have found that 5-HT stimulates the rate of protein secretion in a dose-dependent manner (threshold: 3 x 10(-8) M; EC50 1.5 x 10(-6) M). The maximal rate of 5-HT-induced protein secretion was 2.2 +/- 0.2 mu g/min. Increasing intracellular Ca2+ or cAMP by bath application of ionomycin (5 mu M), db cAMP (10 mM), forskolin (100 mu M) or IBMX (100 mu M), respectively, stimulated protein secretion at significantly lower rates, whereas db cGMP (1 mM) did not activate protein secretion. The high rates and the kinetics of 5-HT-induced protein secretion could only be mimicked by either applying forskolin together with IBMX (with or without ionomycin) or by applying IBMX together with ionomycin. Our measurements suggest that 5-HT-induced protein secretion is mediated by an elevation of [cAMP](i) and that Ca2+ may function as a co-agonist and augment the rate of protein secretion. (c) 2005 Elsevier Ltd. All rights reserved
We examined the population genetic structure of the diamondback terrapins (Malaclemys terrapin), within and among estuaries. Based on mark-recapture studies, these estuarine turtles have high site fidelity that is likely to make them vulnerable to local extinctions. We tested if observed site fidelity of adults would be reflected in intraestuarine population genetic structure of six highly polymorphic microsatellite loci (five tetranucleotide and one dinucleotide). No evidence was found for population structuring within the Charleston estuary nor among three different estuaries in South Carolina. We then examined four other terrapin populations from North Carolina to New York, as well as from the Florida Keys and from Texas. With increasing geographical distance, genetic differentiation increased from South Carolina through New York, but overall values were low. The dinucleotide locus contributed significantly more to the genetic differentiation of some population comparisons than any of the other loci. Interestingly, terrapins from South Carolina to New York were much more genetically similar to those from Texas (rho = 0.154) than to those from Florida (rho = 0.357). We attribute this pattern to extensive translocations of terrapins during the early 20th century to replenish diminished populations and to provide turtle farms with stocks. Terrapins collected in Texas were especially sought for shipment to the northeastern US because of their larger size. Our study indicates no population structure within or among adjacent estuaries. Thus, the mark-recapture information from adult and subadult feeding locations is a poor predictor of population genetic structure. Additionally, it appears that past human activities may have drastically altered the genetics of current populations. Finally, our data suggest that translocation of eggs or head starting of terrapins within estuaries or among adjacent estuaries is acceptable from a genetic standpoint
Members of the Shaker-like plant K+ channel family share a common structure, but are highly diverse in their function: they behave as either hyperpolarization-activated inward-rectifying (K-in) channels, or leak-like (K-weak) channels, or depolarization-activated outward-rectifying (K-out) channels. Here we created 256 chimeras between the K-in channel KAT1 and the K-out channel SKOR. The chimeras were screened in a potassium-uptake deficient yeast strain to identify those, which mediate potassium inward currents, i.e., which are functionally equivalent to KAT1. This strategy allowed Lis to identify three chimeras which differ from KAT1 in three parts of the polypeptide: the cytosolic N- terminus, the cytosolic C-terminus, and the putative voltage-sensor S4. Additionally, mutations in the K-out Channel SKOR were generated in order to localize molecular entities underlying its depolarization activation. The triple mutant SKOR-D312N-M313L-1314G, carrying amino-acid changes in the S6 segment, was identified as a channel which did not display any rectification in the tested voltage-range. (C) 2005 Elsevier Inc. All rights reserved
We report here the development of piezoelectric affinity sensors for cocaine and cholinesterase inhibitors based on the formation of affinity complexes between an immobilized cocaine derivative and an anti-cocaine antibody or cholinesterase. For both binding reactions benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO) was immobilized on the surface of the sensor. For immobilization. pre-conjugated BZE-DADOO with 11-mercaptomonoundecanoic acid (MUA) via 2- (5-norbornen-2,3-dicarboximide)-1,1,3,3-tetramethyluronium-tetrafluoro borate (TNTU) allowed the formation of a chemisorbed monolayer on the piezosensor surface. The detection of cocaine was based oil a competitive assay. The change of frequency measured after 300 s of the binding reaction was used as the signal. The maximum binding of the antibody resulted in a frequency decrease of 35 Hz (with an imprecision 3%, n = 3) while the presence of 100 pmol I-1 cocaine decreased the binding by 11%. The limit of detection was consequently below 100 pmol I-1 for cocaine. The total time of one analysis was 15 min. This BZE-DADOO-modified sensor was adapted for the detection of organophosphates. BZE-DADOO - a competitive inhibitor - served as binding element for cholinesterase in a competitive assay. (C) 2004 Elsevier B.V. All rights reserved
Organic materials have received considerable attention because of their large dipole moments and optical nonlinearities. The optically induced switching of material properties is important for studying the optoelectronic effects including second harmonic generation. Organic materials for photonic applications contain chromophore dipole which consist of acceptor and donor groups bridged by a delocalized pi-electron system. Both theoretical and experimental data show a reversible highly dipolar photoinduced intra molecular charge transfer in betaine type molecules accompanied by change of the sign and the value of the dipole moment. The arrangement of polar molecules in films is studied both by atom force microscopy and surface potential measurements. To understand the photo response of these materials, their spectroscopic and electrical properties are studied. The morphology and photoinduced surface potential switching of the self-assembled monolayers and polymer films are investigated. (c) 2005 Elsevier B.V. All rights reserved
Agent-based complex systems are dynamic networks of many interacting agents; examples include ecosystems, financial markets, and cities. The search for general principles underlying the internal organization of such systems often uses bottom-up simulation models such as cellular automata and agent-based models. No general framework for designing, testing, and analyzing bottom-up models has yet been established, but recent advances in ecological modeling have come together in a general strategy we call pattern-oriented modeling. This strategy provides a unifying framework for decoding the internal organization of agent-based complex systems and may lead toward unifying algorithmic theories of the relation between adaptive behavior and system complexity
Avian extrapair mating systems provide an interesting model to assess the role of genetic benefits in the evolution of female multiple mating behavior, as potentially confounding nongenetic benefits of extrapair mate choice are seen to be of minor importance. Genetic benefit models of extrapair mating behavior predict that females engage in extrapair copulations with males of higher genetic quality compared to their social mates, thereby improving offspring reproductive value. The most straightforward test of such good genes models of extrapair mating implies pail-wise comparisons of maternal half-siblings raised in the same environment, which permits direct assessment of Paternal genetic effects oil offspring traits. But genetic benefits of mate choice may be difficult to detect. Furthermore, the extent of genetic benefits (in terms of increased offspring viability or fecundity) may depend oil the environmental context Such that the proposed differences between extrapair offspring (EPO) and within-pair offspring (WPO) only appear under comparatively poor environmental conditions. We tested the hypothesis that genetic benefits of female extrapair mate choice are context dependent by analyzing offspring fitness-related traits in the coal tit (Parus ater) in relation to seasonal variation in environmental conditions. Paternal genetic effects on offspring fitness were context dependent. as shown by a significant interaction effect of differential paternal genetic contribution and offspring hatching date. EPO showed a higher local recruitment probability than their maternal half-siblings if born comparatively late in the season (i.e.. when overall performance had significantly declined), while WPO performed better early in the season. The same general pattern of context dependence was evident when using the number of grandchildren born to a cuckolding female via her female WPO or EPO progeny as the respective fitness measure. However, we were unable to demonstrate that cuckolding females obtained a general genetic fitness benefit from extrapair fertilizations in terms of offspring viability or fecundity. Thus, another type of benefit Could be responsible for maintaining female extrapair mating preferences in the study population. Our results suggest that more than a single selective pressure may have shaped the evolution of female extrapair mating behavior in socially monogamous passerines.
The glycosphingolipid globoside (globotetraosylceramide, Gb4Cer) has been proposed to be the cellular receptor of human parvovirus B19. Quantitative measurements of the binding of parvovirus B19 to Gb4Cer were performed to explore the molecular basis of the virus tropism. Solid-phase assays with fluorescence-labeled liposomes or (125)iodine-labeled empty capsids were used to characterize the specificity of binding. In addition, surface plasmon resonance on lipid layers, as well as isothermal titration microcalorimetry, was utilized for real-time analysis of the virus-receptor interaction. These studies did not confirm binding of Gb4Cer to recombinant B19 VP2 capsids, suggesting that Gb4Cer does not function on its own as the cellular receptor of human parvovirus B19, but might be involved in a more complex recognition event. The biochemical results were further confirmed by cryo-electron microscopy image reconstructions at 10 A resolution, in which the structures of empty capsids were compared with empty capsids incubated with Gb4Cer. (C) 2004 Elsevier Inc. All rights reserved
Expressed sequence tags (ESTs) represent a huge resource for the discovery of previously unknown genetic information and functional genome assignment. In this study we screened a collection of 178 292 ESTs from Arabidopsis thaliana by testing them against previously annotated genes of the Arabidopsis genome. We identified several hundreds of new transcripts that match the Arabidopsis genome at so far unassigned loci. The transcriptional activity of these loci was independently confirmed by comparison with the Salk Whole Genome Array Data. To a large extent, the newly identified transcriptionally active genomic regions do not encode 'classic' proteins, but instead generate non-coding RNAs and/or small peptide-coding RNAs of presently unknown biological function. More than 560 transcripts identified in this study are not represented by the Affymetrix GeneChip arrays currently widely used for expression profiling in A. thaliana. Our data strongly support the hypothesis that numerous previously unknown genes exist in the Arabidopsis genome
1. The biovolume-specific carbon content, relative egg volume (a measure of per-offspring reproductive investment), growth and grazing rates, and the gross growth efficiency (GGE) of the rotifer Cephalodella hoodi, isolated from an extremely acidic habitat (pH 2.65), were determined and compared with literature values for rotifers living in circum-neutral habitats in order to reveal potential special features or adaptations related to the extreme habitat of C. hoodi. 2. Of the two dominant phytoflagellates (Ochromonas sp. and Chlamydomonas acidophila) that occur in the natural habitat of C. hoodi, only C. acidophila promoted positive growth and reproduction and, thus, the following results were obtained with C. acidophila as a food alga. 3. The body volume-specific carbon content of C. hoodi is in the range of that found in rotifers from circum-neutral lakes, suggesting that no costly carbon investment, brought about by the thickening of the lorica, for example, was required to withstand low pH. 4. The egg volume of C. hoodi exhibited no phenotypic plasticity dependent on the food concentration and, thus, C. hoodi allocated a constant, absolute amount of energy to each individual offspring. No adaptation to low food densities was found. 5. A dome-shaped type II functional response curve was found to describe the ingestion of Chlamydomonas as a source of food. 6. Compared with other rotifers, C. hoodi had a high threshold and half-saturating food concentration (=low affinity) but also a high maximum growth rate and a relatively high GGE, suggesting no severe adverse effect of low pH
Nitrogen is an essential macronutrient for plants and nitrogen fertilizers are indispensable for modern agriculture. Unfortunately, we know too little about how plants regulate their use of soil nitrogen, to maximize fertilizers-N use by crops and pastures. This project took a dual approach, involving forward and reverse genetics, to identify N-regulators in plants, which may prove useful in the future to improve nitrogen-use efficiency in agriculture. To identify nitrogen-regulated transcription factor genes in Arabidopsis that may control N-use efficiency we developed a unique resource for qRT-PCR measurements on all Arabidpsis transcription factor genes. Using closely spaced, gene-specific primer pairs and SYBR® Green to monitor amplification of double-stranded DNA, transcript levels of 83% of all target genes could be measured in roots or shoots of young Arabidopsis wild-type plants. Only 4% of reactions produced non-specific PCR products, and 13% of TF transcripts were undetectable in these organs. Measurements of transcript abundance were quantitative over six orders of magnitude, with a detection limit equivalent to one transcript molecule in 1000 cells. Transcript levels for different TF genes ranged between 0.001-100 copies per cell. Real-time RT-PCR revealed 26 root-specific and 39 shoot-specific TF genes, most of which have not been identified as organ-specific previously. An enlarged and improved version of the TF qRT-PCR platform contains now primer pairs for 2256 Arabidopsis TF genes, representing 53 gene families and sub-families arrayed on six 384-well plates. Set-up of real-time PCR reactions is now fully robotized. One researcher is able to measure expression of all 2256 TF genes in a single biological sample in a just one working day. The Arabidopsis qRT-PCT platform was successfully used to identify 37 TF genes which transcriptionaly responded at the transcriptional level to N-deprivation or to nitrate per se. Most of these genes have not been characterized previously. Further selection of TF genes based on the responses of selected candidates to other macronutrients and abiotic stresses allowed to distinguish between TFs regulated (i) specifically by nitrogen (29 genes) (ii) regulated by general macronutrient or by salt and osmotic stress (6 genes), and (iii) responding to all major macronutrients and to abiotic stresses. Most of the N-regulated TF genes were also regulated by carbon. Further characterization of sixteen selected TF genes, revealed: (i) lack of transcriptional response to organic nitrogen, (ii) two major types of kinetics of induction by nitrate, (iii) specific responses for the majority of the genes to nitrate but not downstream products of nitrate assimilation. All sixteen TF genes were cloned into binary vectors for constitutive and ethanol inducible over expression, and the first generation of transgenic plants were obtained for almost all of them. Some of the plants constitutively over expressing TF genes under control of the 35S promoter revealed visible phenotypes in T1 generation. Homozygous T-DNA knock out lines were also obtained for many of the candidate TF genes. So far, one knock out line revealed a visible phenotype: retardation of flowering time. A forward genetic approach using an Arabidopsis ATNRT2.1 promoter : Luciferase reporter line, resulted in identification of eleven EMS mutant reporter lines affected in induction of ATNRT2.1 expression by nitrate. These lines could by divided in the following classes according to expression of other genes involved in primary nitrogen and carbon metabolism: (i) lines affected exclusively in nitrate transport, (ii) those affected in nitrate transport, acquisition, but also in glycolysis and oxidative pentose pathway, (iii) mutants affected moderately in nitrate transport, oxidative pentose pathway and glycolysis but not in primary nitrate assimilation. Thus, several different N-regulatory genes may have been mutated in this set of mutants. Map-based cloning has begun to identify the genes affected in these mutants.
We describe the first microsatellite loci for the gynogenetic Amazon molly, Poecilia formosa, an all-female species arisen through hybridization of the bisexual species Poecilia mexicana and Poecilia latipinna. The loci showed one to six alleles and an expected heterozygosity between zero and 0.75. As expected with parthenogenetic inheritance, most loci were either constantly homozygous (five loci) or constantly heterozygous (eight loci). For six loci, both heterozygotes and homozygotes occurred. This and the fact that some loci only showed alleles of one of the ancestral species could indicate genome homogenization through mitotic gene conversion. Our new loci conformed to the hybrid origin of Amazon molly and are also applicable to both ancestral bisexual species
The human MOCS3 protein contains an N-terminal domain similar to the Escherichia coli MoeB protein and a C- terminal segment displaying similarities to the sulfurtransferase rhodanese. MOCS3 is proposed to catalyze both the adenylation and the subsequent generation of a thiocarboxylate group at the C-terminus of the smaller subunit of molybdopterin (MPT) synthase during Moco biosynthesis in humans. Recent studies have shown that the MOCS3 rhodanese-like domain (MOCS3-RLD) catalyzes the transfer of sulfur from thiosulfate to cyanide and is also able to provide the sulfur for the thiocarboxylation of MOCS2A in a defined in vitro system for the generation of MPT from precursor Z. MOCS3-RLD contains four cysteine residues of which only C412 in the six amino acid active loop is conserved in homologous proteins from other organisms. ESI-MS/MS studies gave direct evidence for the formation of a persulfide group that is exclusively formed on C412. Simultaneous mutagenesis of the remaining three cysteine residues showed that none of them is involved in the sulfur transfer reaction in vitro. A disulfide bridge was identified to be formed between C316 and C324, and possible roles of the three noncatalytic cysteine residues are discussed. By ESI-MS/MS a partially gluconoylated N- terminus of the His(6)-tagged MOCS3-RLD was identified (mass increment of 178 Da) which resulted in a heterogeneity of the protein but did not influence sulfurtransferase activity
Die Honigbiene Apis mellifera gilt seit langem als Modell-Organismus zur Untersuchung von Lern- und Gedächtnisvorgängen sowie zum Studium des Sozialverhaltens und der Arbeitsteilung. Bei der Steuerung und Regulation dieser Verhaltensweisen spielt das Indolalkylamin Serotonin eine wesentliche Rolle. Serotonin entfaltet seine Wirkung durch die Bindung an G-Protein-gekoppelte Rezeptoren (GPCRs). In der vorliegenden Arbeit wird der erste Serotonin-Rezeptor aus der Honigbiene molekular charakterisiert. Durch die Anwendung zwei verschiedener Klonierungsstrategien konnten drei cDNA-Sequenzen isoliert werden, die für potentielle Serotonin-Rezeptoren kodieren. Die Sequenzen weisen die größte Ähnlichkeit zu dem 5-HT7- und 5-HT2-Rezeptor von Drosophila melanogaster bzw. dem 5-HT1-Rezeptor von Panulirus interruptus auf. Die isolierten Serotonin-Rezeptoren der Honigbiene wurden dementsprechend Am(Apis mellifera)5-HT1, Am5-HT2 und Am5-HT7 benannt. Das Hydropathieprofil des Am5-HT1-, Am5-HT2- und Am5-HT7-Rezeptors deutet auf das Vorhandensein des charakteristischen heptahelikalen Aufbaus G-Protein-gekoppelter Rezeptoren hin. Die abgeleiteten Aminosäuresequenzen zeigen typische Merkmale biogener Amin-Rezeptoren. Aminosäuren, die eine Bedeutung bei der Bildung der Liganden-Bindungstasche, der Rezeptor-Aktivierung und der Kopplung eines G-Proteins an den Rezeptor haben, sind in allen drei Rezeptoren konserviert. Interessanterweise ist jedoch das in den meisten biogenen Amin-Rezeptoren vorhandene DRY-Motiv in dem Am5-HT2- und Am5-HT7-Rezeptor nicht konserviert. Das Vorhandensein einer PDZ-Domäne in dem Am5-HT1- und Am5-HT7-Rezeptor lässt vermuten, dass diese Rezeptoren als Adapterproteine fungieren, die Signalmoleküle zu einem Signaltransduktionskomplex vereinigen. RT-PCR-Experimente zeigen die Expression der Rezeptoren in verschiedenen Geweben der Honigbiene. Auffallend ist die hohe Expression im Zentralgehirn. Des Weiteren konnte die Expression der Serotonin-Rezeptoren in den optischen Loben, Antennalloben sowie in der Peripherie, d.h. in der Flugmuskulatur und den Malpighischen Gefäßen nachgewiesen werden. Durch in situ Hybridisierungen wurde die Expression in Gefrierschnitten von Gehirnen adulter Sammlerinnen im Detail untersucht. Transkripte der Rezeptoren sind in den Somata von intrinsischen Pilzkörperzellen, Neuronen der optischen Loben und Neuronen der Antennalloben vorhanden. In einem heterologen Expressionssystem wurde der intrazelluläre Signalweg des Am5-HT7-Rezeptors untersucht. Die Aktivierung des stabil exprimierten Rezeptors durch Serotonin führt zur Bildung von cAMP. Der 5-HT7-Rezeptor spezifische Agonist 5-CT zeigt eine mit Serotonin vergleichbare Fähigkeit, die intrazelluläre cAMP-Konzentration zu erhöhen. Am5-HT7 gehört daher funktionell zu der Gruppe der 5-HT7-Rezeptoren. Der EC50-Wert von 1,06~nM (5-HT), ist im Vergleich zu anderen 5-HT7-Rezeptoren äußert niedrig. Des Weiteren wurde gezeigt, dass das basale cAMP-Niveau in den transfizierten Zellen im Vergleich zu nicht transfizierten Zellen deutlich erhöht ist. Das heißt, dass der Rezeptor auch in der Abwesenheit eines Liganden aktiv ist. Diese konstitutive Aktivität ist auch von anderen biogenen Amin-Rezeptoren bekannt. Methiothepin wurde als wirksamer inverser Agonist des Am5-HT7-Rezeptors identifiziert, da es in der Lage ist, der konstitutiven Aktivität entgegenzuwirken. Die Ergebnisse der vorliegenden Arbeit deuten darauf hin, dass die Serotonin-Rezeptoren in verschiedenen Regionen des ZNS der Honigbiene an der Informationsverarbeitung beteiligt sind. Es kann eine Beeinflussung von Lern- und Gedächtnisprozessen sowie des olfaktorischen und visuellen Systems durch diese Rezeptoren vermutet werden. Mit der Klonierung und funktionellen Charakterisierung des ersten Serotonin-Rezeptors der Honigbiene ist eine Grundlage für die Untersuchung der molekularen Mechanismen der serotonergen Signaltransduktion geschaffen worden.
Patterns of sequence divergence in about 1 kb of mitochondrial DNA coding for two genes (16s rRNA and cytochrome oxidase I, COI) were analysed in 13 populations of the fairy shrimp Tanymastix stagnalis from Norway, Germany, France, Italy (northern and central Italy plus insular populations from Sardinia and the Tuscan Archipelago) and Spain, and in one presumed population of Tanymastix stellae from Corsica. The latter species was originally known only from a single locality in Sardinia, which has been destroyed by urbanization; the Corsican population was referred to T stellae by some French authors on the basis of the collection of several cysts from mud. mtDNA data revealed a very low level of genetic divergence between the presumed population of T stellae and the other T stagnalis populations included in the study. Our genetic findings do not support the presence of T stellae in Corsica and are in line with previous SEM studies revealing that all species belonging to the genus Tanymastix produce cysts with identical morphology. The results indicate complex phylogeographic relationships and pronounced genetic differentiation among T stagnalis populations. The islands of Corsica and Sardinia on the one hand and the island of Capraia (Tuscan Archipelago) on the other were probably colonized independently at different times. Genetic relationships among continental populations do not follow a clear geographical trend, indicating that geographical distance is not the main force shaping the pattern of genetic structuring of the species. Stochastic factors such as multiple and independent founder events probably contributed to the striking pattern of genetic differentiation along with subsequent local adaptation. These results agree with previously published molecular work on several groups of aquatic organisms and further support the high potential for dispersal-low gene flow paradox shown by a large array of animals living in lentic habitats
Molecular characterization of the ebony gene from the American cockroach, Periplaneta americana
(2005)
Biogenic amines are an important class of primary messengers in the central (CNS) and peripheral nervous systems and in peripheral organs. These substances regulate and modulate many physiological and behavioral processes. Various inactivation mechanisms for these substances exist to terminate biogenic amine-mediated signal transduction. In vertebrates, the enzymes monoamine oxidase and/or catechol-O-methyl-transferase are involved in these processes. In insects, however, in which both enzymes are low in abundance or absent, biogenic amines are inactivated mainly by N- acetylation or O-sulphation. In Droso-philo, beta-alanyl conjugation mediated by the Ebony protein has recently been shown to be a novel and alternative pathway for biogenic amine inactivation. Here, we report the cloning of ebony cDNA (Peaebony) from a brain-specific cDNA library of the cockroach Periplaneta americana. The open reading frame encodes a protein of 860 amino acid residues (PeaEbony). The PeaEbony polypeptide shares homology to Ebony sequences from Anopheles gambiae, Apis mellifera, and Drosophila melonogaster. In addition, PeaEbony exhibits sequence similarity to a family of microbial non-ribosomal peptide synthetases. The mRNA encoding PeaEbony is highly expressed in the cockroach brain and to a lesser extent in the salivary glands. PeaEbony is, therefore, probably involved in the inactivation of various biogenic amines through beta-alanyl conjugation in the cockroach CNS. Since the salivary glands in Periplaneta are innervated by dopaminergic and serotonergic neurons, PeaEbony probably also biochemically modifies dopamine and serotonin in these acinar glands. Arch. Insect Biochem. (c) 2005 Wiley-Liss, Inc
The predicted climate change causes deep concerns on the effects of increasing temperatures and changing precipitation patterns on species viability and, in turn, on biodiversity. Models of Population Viability Analysis (PVA) provide a powerful tool to assess the risk of species extinction. However, most PVA models do not take into account the potential effects of behavioural adaptations. Organisms might adapt to new environmental situations and thereby mitigate negative effects of climate change. To demonstrate such mitigation effects, we use an existing PVA model describing a population of the tawny eagle (Aquila rapax) in the southern Kalahari. This model does not include behavioural adaptations. We develop a new model by assuming that the birds enlarge their average territory size to compensate for lower amounts of precipitation. Here, we found the predicted increase in risk of extinction due to climate change to be much lower than in the original model. However, this "buffering" of climate change by behavioural adaptation is not very effective in coping with increasing interannual variances. We refer to further examples of ecological "buffering mechanisms" from the literature and argue that possible buffering mechanisms should be given due consideration when the effects of climate change on biodiversity are to be predicted. (c) 2004 Elsevier B.V. All rights reserved
Bei konventionellen Mikroarray-Experimenten zur Genexpressionsanalyse wird fluoreszenz- oder radioaktiv-markierte cDNA oder RNA mit immobilisierten Proben hybridisiert. Für ein gut detektierbares und auswertbares Ergebnis werden jedoch pro Array mindestens 15 - 20 µg Hybridisierungstarget benötigt. Dazu müssen entweder 15 - 20 µg RNA direkt durch Reverse Transkription in markierte cDNA umgeschrieben werden oder bei Vorhandensein von weniger Startmaterial die RNA amplifiziert werden (Standard- Affymetrix-Protokolle, Klur et al. 2004). Oft sind damit zeit- und kostenintensive Probenpräparationen verbunden und das Ergebnis ist nicht immer reproduzierbar. Obwohl es inzwischen einige Protokolle gibt, die dieses Problem zu lösen versuchen (Zhang et al. 2001, Iscove et al. 2002, McClintick et al. 2003, Stirewalt et al. 2004), eine optimale, leicht handbare und reproduzierbare Methode gibt es weiterhin nicht, weshalb in dieser Arbeit ein weiterer Lösungsansatz gesucht wurde. In der vorgestellten Arbeit werden zwei einfache Methoden beschrieben, mit denen Gene aus geringen RNA-Mengen nachgewiesen werden können: erstens die On Chip- RT-PCR mit cDNA als Matrize und zweitens diese Methode als One-Step-Reaktion mit RNA als Matrize. Beide Methoden beruhen auf dem Prinzip der PCR an immobilisierten Primern auf einer Chipoberfläche. Diese Möglichkeit der exponentiellen Amplifikation ist reproduzierbar und sensitiv. In Experimenten zur Etablierung des On-Chip-PCR-Systems wurden für die Immobilisierung der Primer verschiedene Kopplungsmethoden verwendet. Die affine Kopplung über Biotin- Streptavidin erwies sich als geeignet. Die On-Chip-Reaktion an kovalent gebundenen Primern wurde für amino-modifizierte Primer auf Epoxy-Oberflächen sowie für EDC-Kopplung auf silanisierten Oberflächen gezeigt. Für die letztgenannte Methode wurde die On-Chip-PCR optimiert, dass Spottingkonzentrationen der Primer von 5 - 10µM schon ausreichend sind. Der Einsatz von fluoreszenz-markierten Primern während der PCR ermöglicht eine unmittelbare Auswertung nach der Synthese ohne zusätzliche Detektionsschritte. In dieser Arbeit konnte außerdem mit der vorgestellten Methode der simultane Nachweis zweier Gene gezeigt werden. Die Methode kann noch als Multiplex-Analyse ausgebaut werden, um so mehrere Gene in gleichzeitig einem Ansatz nachweisen zu können. Die Ergebnisse der Versuche mit Matrizen aus unterschiedlichen Zelltypen deuten darauf hin, dass die On-Chip-RT-PCR eine weitere optimale Methode für den Nachweis von gering exprimierten Genen bietet.
Habitat fragmentation is known to cause genetic differentiation between small populations of rare species and decrease genetic variation within such populations. However, common species with recently fragmented populations have rarely been studied in this context. We investigated genetic variation and its relationship to population size and geographical isolation of populations of the common plant species, Lychnis flos-cuculi L., in fragmented fen grasslands. We analysed 467 plants from 28 L. flos-cuculi populations of different sizes (60 000-54 000 flowering individuals) in northeastern Switzerland using seven polymorphic microsatellite loci. Genetic differentiation between populations is small (F-ST = 0.022; AMOVA; P < 0.001), suggesting that gene flow among populations is still high or that habitat fragmentation is too recent to result in pronounced differentiation. Observed heterozygosity (H-O = 0.44) significantly deviates from Hardy-Weinberg equilibrium, and within-population inbreeding coefficient F-IS is high (0.30-0.59), indicating a mixed mating breeding system with substantial inbreeding in L. flos-cuculi. Gene diversity is the only measure of genetic variation which decreased with decreasing population size (R = 0.42; P < 0.05). While our results do not indicate pronounced effects of habitat fragmentation on genetic variation in the still common L. flos-cuculi, the lower gene diversity of smaller populations suggests that the species is not entirely unaffected
Long-Term Impact of grazing on vegetation under stochastic climate : a cross-scale simulation study
(2005)
Animals depend on a large set of essential compounds in their food. A reduced supply of specific lipid compounds has been shown to be critical for growth, reproduction and survival of aquatic molluscs and might also be critical for terrestrial gastropods. The effects were investigated of different fatty acid and cholesterol supplies on the growth, reproduction, and survival of the land snail Arianta arbustorum. Surprisingly, differences in diet did not affect shell growth, time to reach adulthood, and reproductive traits such as the number of eggs, dry mass and volume of spermatophores. However, snails reared on low cholesterol diets and low polyunsaturated fatty acid (PUFA) diets decreased their consumption rates and had a higher mortality. Snails fed a PUFA-rich diet showed higher mating activity than snails fed a diet deficient in PUFAs. A depletion of internal PUFA reserves may affect the regulation of mating behaviour because PUFAs play an important role as precursors for signal-transduction involved in the regulation of mating and reproduction. In contrast, when the diets were deficient in cholesterol, mating activity was not affected
1. The unicellular green alga Chlamydomonas acidophila accumulates in a thin phytoplankton layer in the hypolimnion (deep chlorophyll maximum, DCM) of an extremely acidic lake (Lake 111, pH 2.6, Lusatia, Germany), in which the underwater light spectrum is distorted and red-shifted. 2. Chlamydomonas acidophila exhibited a significantly higher absorption efficiency and a higher cellular chlorophyll b content when incubated in the red shifted underwater light of Lake 111 than in a typical, blue-green dominated, light spectrum. 3. Chlamydomonas acidophila has excellent low light acclimation properties (increased chlorophyll b content, increased oxygen yield and a low light saturation point for photosynthesis) that support survival of the species in the low light climate of the DCM. 4. In situ acclimation to the DCM under low light and temperature decreased maximum photosynthetic rate in autotrophic C. acidophila cultures, whereas the presence of glucose under these conditions enhanced photosynthetic efficiency and capacity. 5. The adaptive abilities of C. acidophila to light and temperature shown in this study, in combination with the absence of potent competitors because of low lake pH, most probably enable the unusual dominance of the green alga in the DCM of Lake 111
The absence of essential biochemical nutrients, such as polyunsaturated fatty acids or sterols, has been considered as a mechanism determining trophic interactions between the herbivore Daphnia and its phytoplankton food source. Here, we experimentally quantify the sensitivity of two Daphnia species to decreasing amounts of dietary sterols by measuring variations in life history traits. The two species Daphnia magna and D. galeata were fed different mixtures of the sterol-containing green alga Scenedesmus obliquus and the sterol-free cyanobacterium Synechococcus elongatus; a higher proportion of Synechococcus in the food is equivalent to a decrease in dietary sterols. To address the significance of sterol limitation, the Daphnia species were also fed Synechococcus supplemented with cholesterol. In both species, somatic and population growth rates, maternal dry mass, the number of viable offspring, and the probability of survival were significantly reduced with the lower availability of sterols. A high correlation between the sterol content of the mixed diet and the somatic and population growth rates was found, and growth on cholesterol- supplemented Synechococcus fitted well into this correlation. Somatic growth of first-clutch neonates grown on 100% Synechococcus exhibited a pattern similar to that of somatic growth of their mothers grown on the different food regimes, which demonstrated the significance of maternal effects for sterol-limited population growth. Daphnia galeata had a twofold higher incipient limiting sterol level than D. magna, which indicated interspecific differences in sterol requirements between the two Daphnia species. The results suggest a strong impact of dietary sterols on life history traits and therefore, population dynamics of the keystone species Daphnia
During the day, plants accumulate starch in their leaves as an energy source for the coming night. Based on recent findings, the prevailing view of how the transitory starch is remobilized needs considerable revision. Analyses of transgenic and mutant plants demonstrate that plastidic glucan phosphorylase is not required for normal starch breakdown and cast doubt on the presumed essential role of alpha-amylase but do show that beta-amylase is important. Repression of the activity of a plastidic beta-amylase, the export of its product (maltose) or further metabolism of maltose by a newly identified transglucosidase impairs starch degradation. Breakdown of particulate starch also depends on the activity of glucan-water dikinase, which phosphorylates glucosyl residues within the polymer
Die Tailspike Proteine (TSP) der Bakteriophagen P22, Sf6 und HK620 dienen der Erkennung von Kohlenhydratstrukturen auf ihren gram-negativen Wirtsbakterien und zeigen, von den ersten 110 Aminosäuren des N-Terminus abgesehen, keine Sequenzübereinstimmung. Mit Röntgenkristallstrukturanalyse konnte gezeigt werden, dass HK620TSP und Sf6TSP ebenfalls zu einer parallelen, rechtsgängigen beta-Helix falten, wie dies schon für P22TSP bekannt war. Die Kohlenhydratbindestelle ist bei Sf6TSP im Vergleich zu P22TSP zwischen die Untereinheiten verschoben.
The important fodder grass Poa alpina L. occurs at several ploidy levels with common aneuploidy. We isolated and characterized five polymorphic microsatellite markers for the study of molecular genetic variation of this species. As first examples of the value of the developed markers for population genetic analyses, we show that plants with more chromosomes have more microsatellite bands and that isolation by distance plays a small role in shaping microsatellite diversity of P. alpina in the Swiss Alps
Interkonversion zellulärer Signaltransduktionsprozesse durch Phosphorylierung und Redoxregulation
(2005)
Inorganic carbon limitation and mixotrophic growth in Chlamydomonas from an acidic mining lake
(2005)
Plankton communities in acidic mining lakes (pH 2.5-3.3) are species-poor because they face extreme environmental conditions, e.g. 150 mg l(-1) Fe2++Fe3+. We investigated the growth characteristics of the dominant pigmented species, the flagellate Chlamydomonas acidophila, in semi-continuous culture experiments under in situ conditions. The following hypotheses were tested: (1) Low inorganic carbon (IC) concentrations in the epilimnion (e.g. 0.3 mg l(-1)) arising from the low pH limit phototrophic growth (H-1); (2) the additional use of dissolved organic carbon (mixotrophy) leads to higher growth rates under IC-limitation (H-2), and (3) phagotrophy is not relevant (H-3). H- 1 was supported as the culture experiments, in situ PAR and IC concentrations indicated that IC potentially limited phototrophic growth in the mixed surface layers. H-2 was also supported: mixotrophic growth always exceeded pure phototrophic growth even when photosynthesis was saturated. Dark growth in filtered lake water illuminated prior to inoculation provided evidence that Chlamydomonas was able to use the natural DOC. The alga did not grow on bacteria, thus confirming H-3. Chlamydomonas exhibited a remarkable resistance to starvation in the dark. The compensation light intensity (ca. 20 mu mol photons m(-2) s(-1)) and the maximum phototrophic growth (1.50 d(-1)) fell within the range of algae from non-acidic waters. Overall, Chlamydomonas, a typical r-strategist in circum-neutral systems, showed characteristics of a K-strategist in the stable, acidic lake environment in achieving moderate growth rates and minimizing metabolic losses. (c) 2005 Elsevier GmbH. All rights reserved
Chlamydomonas acidophila Negoro had a higher maximum growth rate upon aeration with 5% CO2 (v/v) than in nonaerated conditions at an external pH above 2. In medium with a pH of 1.0 or 2.0, a decrease in the maximum growth rate was observed upon CO2 aeration in comparison with nonaerated conditions. At both very low and very high external pH conditions, an induction of external carbonic anhydrase was detected; this being more pronounced in CO2-aerated cells than in nonaerated cells. It is therefore suggested that the induction of carbonic anhydrase is part of a stress response in Chlamydomonas acidophila. Comparison of some physiological characteristics of Chlamydomonas acidophila acclimated at pH 2.65 and at pH 6.0, revealed that CO2 aeration increased gross maximum photosynthesis at both pHs, whereas respiration, light acclimation, and photoinhibition were not effected. At pH 2.65, Chlamydomonas acidophila was found to have a carbon-concentrating mechanism under nonaerated conditions, whereas it did not under CO2-aerated conditions at pH 6. The affinity for CO2 use in O-2 production was not dependent on CO2 aeration, but it was much lower at pH 6 than it was at pH 2.65. CO2 kinetic characteristics indicate that the photosynthesis of Chlamydomonas acidophila in its natural environment is not limited by inorganic carbon
The gene family of subtilisin-like serine proteases (subtilases) in Arabidopsis thaliana comprises 56 members, divided into six distinct subfamilies. Whereas the members of five subfamilies are similar to pyrolysins, two genes share stronger similarity to animal kexins. Mutant screens confirmed 144 T-DNA insertion lines with knockouts for 55 out of the 56 subtilases. Apart from SDD1, none of the confirmed homozygous mutants revealed any obvious visible phenotypic alteration during growth under standard conditions. Apart from this specific case, forward genetics gave us no hints about the function of the individual 54 non-characterized subtilase genes. Therefore, the main objective of our work was to overcome the shortcomings of the forward genetic approach and to infer alternative experimental approaches by using an integrative biolinformatics and biological approach. Computational analyses based on transcriptional co-expression and co-response pattern revealed at least two expression networks, suggesting that functional redundancy may exist among subtilases with limited similarity. Furthermore, two hubs were identified, which may be involved in signalling or may represent higher-order regulatory factors involved in responses to environmental cues. A particular enrichment of co- regulated genes with metabolic functions was observed for four subtilases possibly representing late responsive elements of environmental stress. The kexin homologs show stronger associations with genes of transcriptional regulation context. Based on the analyses presented here and in accordance with previously characterized subtilases, we propose three main functions of subtilases: involvement in (i) control of development, (ii) protein turnover, and (iii) action as downstream components of signalling cascades
An artificial gene was constructed combining the T7 promoter and terminator with the EGFP-gene from the plasmid pEGFP. The functionality of the construct was shown by in vitro translation. The gene-construct was immobilised on a planar glass surface. The transcription was performed on the immobilised gene and mRNA was determined by RT-PCR. Multiple use of the immobilised gene was demonstrated
In silico identification of genes regulated by abscisic acid in Arabidopsis thaliana (L.) Heynh.
(2005)
Abscisic acid (ABA) is a major plant hormone that plays an important role during plant growth and development. During vegetative growth ABA mediates (in part) responses to various environmental stresses such as cold, drought and high salinity. The response triggered by ABA includes changes in the transcript level of genes involved in stress tolerance. The aim of this project was the In silico identification of genes putatively regulated by ABA in A. thaliana. In silico predictions were combined with experimental data in order to evaluate the reliability of computational predictions. Taking advantage of the genome sequence of A. thaliana publicly available since 2000, 1 kb upstream sequences were screened for combinations of cis-elements known to be involved in the regulation of ABA-responsive genes. It was found that around 10 to 20 percent of the genes of A. thaliana might be regulated by ABA. Further analyses of the predictions revealed that certain combinations of cis-elements that confer ABA-responsiveness were significantly over-represented compared with results in random sequences and with random expectations. In addition, it was observed that other combinations that confer ABA-responsiveness in monocotyledonous species might not be functional in A. thaliana. It is proposed that ABA-responsive genes in A. thaliana show pairs of ABRE (abscisic acid responsive element) with MYB binding sites, DRE (dehydration responsive element) or with itself. The analysis of the distances between pairs of cis-elements suggested that pairs of ABREs are bound by homodimers of ABRE binding proteins. In contrast, pairs between MYB binding sites and ABRE, or DRE and ABRE showed a distance between cis-elements that suggested that the binding proteins interact through protein complexes and not directly. The comparison of computational predictions with experimental data confirmed that the regulatory mechanisms leading to the induction or repression of genes by ABA is very incompletely understood. It became evident that besides the cis-elements proposed in this study to be present in ABA-responsive genes, other known and unknown cis-elements might play an important role in the transcriptional regulation of ABA-responsive genes. For example, auxin-related cis elements, or the cis-elements recognized by the NAM-family of transcription factors (Non-Apical meristem). This work documents the use of computational and experimental approaches to analyse possible interactions between cis-elements involved in the regulation of ABA-responsive genes. The computational predictions allowed the distinction between putatively relevant combinations of cis-elements from irrelevant combinations of cis-elements in ABA-responsive genes. The comparison with experimental data allowed to identify certain cis-elements that have not been previously associated to the ABA-mediated transcriptional regulation, but that might be present in ABA-responsive genes (e.g. auxin responsive elements). Moreover, the efforts to unravel the gene regulatory network associated with the ABA-signalling pathway revealed that NAM-transcription factors and their corresponding binding sequences are important components of this network.
Microarrays are new analytical devices that allow the parallel and simultaneous detection of thousands of target compounds. Microarrays, also called DNA chips, are widely used in gene expression, the genotyping of individuals, point mutations, detection of single nucleotide polymorphisms, and short tandem repeats. Microarrays have highly specific base-pair interactions with labeled complementary strands, which makes this technology to a powerful analytical device for monitoring whole genomes. In this article, we provide a survey of the common microarray manufacturing methods, from the selection of support material to surface structuring, immobilization and hybridization, and finally the detection with labeled complementary strands. Special attention is given to the immobilization of single strands, since fast chemical reactions, the creation of homogeneous surface functionalities as well as an oriented coupling are crucial pre-conditions for a good spot morphology and microarrays of high quality
Water-soluble heteroglycans (SHG) were isolated from leaves of wild-type Arabidopsis thaliana L. and from two starch-deficient mutants. Major constituents of the SHG are arabinose, galactose, rhamnose, and glucose. SHG was separated into low (< 10 kDa; SHG(S)) and high (> 10 kDa; SHG(L)) molecular weight compounds. SHG(S) was resolved into approximately 25 distinct oligoglycans by ion exchange chromatography. SHG(L) was further separated into two subfractions, designated as subfraction I and II, by field flow fractionation. For the intracellular localization of the various SHG compounds several approaches were chosen: first, leaf material was subjected to non-aqueous fractionation. The apolar gradient fractions were characterized by monitoring markers and were used as starting material for the SHG isolation. Subfraction I and SHG(S) exhibited a distribution similar to that of cytosolic markers whereas subfraction II cofractionated with crystalline cellulose. Secondly, intact organelles were isolated and used for SHG isolation. Preparations of intact organelles (mitochondria plus peroxisomes) contained no significant amount of any heteroglycan. In isolated intact microsomes a series of oligoglycans was recovered but neither subfraction I nor II. In in vitro assays using glucose 1-phosphate and recombinant cytosolic (Pho 2) phosphorylase both SHG(S) and subfraction I acted as glucosyl acceptor whereas subfraction II was essentially inactive. Rabbit muscle phosphorylase a did not utilize any of the plant glycans indicating a specific Pho 2-glycan interaction. As revealed by in vivo labeling experiments using (CO2)-C-14 carbon fluxes into subfraction I and II differed. Furthermore, in leaves the pool size of subfraction I varied during the light-dark regime
The comprehensive systems-biology database (CSB.DB) was used to reveal brassinosteroid (BR)-related genes from expression profiles based on co-response analyses. Genes exhibiting simultaneous changes in transcript levels are candidates of common transcriptional regulation. Combining numerous different experiments in data matrices allows ruling out outliers and conditional changes of transcript levels. CSB.DB was queried for transcriptional co-responses with the BR-signalling components BRI1 and BAK1: 301 out of 9694 genes represented in the nasc0271 database showed co-responses with both genes. As expected, these genes comprised pathway-involved genes (e.g. 72 BR-induced genes), because the BRI1 and BAK1 proteins are required for BR-responses. But transcript co-response takes the analysis a step further compared with direct approaches because BR-related non BR-responsive genes were identified. Insights into networks and the functional context of genes are provided, because factors determining expression patterns are reflected in correlations. Our findings demonstrate that transcript co-response analysis presents a valuable resource to uncover common regulatory patterns of genes. Different data matrices in CSB.DB allow examination of specific biological questions. All matrices are publicly available through CSB.DB. This work presents one possible roadmap to use the CSB.DB resources
The phosphorylation of amylopectin by the glucan, water dikinase (GWD; EC 2.7.9.4) is an essential step within starch metabolism. This is indicated by the starch excess phenotype of GWD-deficient plants, such as the sex1-3 mutant of Arabidopsis (Arabidopsis thaliana). To identify starch-related enzymes that rely on glucan-bound phosphate, we studied the binding of proteins extracted from Arabidopsis wild-type leaves to either phosphorylated or nonphosphorylated starch granules. Granules prepared from the sex1-3 mutant were prephosphorylated in vitro using recombinant potato (Solanum tuberosum) GWD. As a control, the unmodified, phosphate free granules were used. An as-yet uncharacterized protein was identified that preferentially binds to the phosphorylated starch. The C-terminal part of this protein exhibits similarity to that of GWD. The novel protein phosphorylates starch granules, but only following prephosphorylation with GWD. The enzyme transfers the beta-P of ATP to the phosphoglucan, whereas the gamma-P is released as orthophosphate. Therefore, the novel protein is designated as phosphoglucan, water dikinase (PWD). Unlike GWD that phosphorylates preferentially the C6 position of the glucose units, PWD phosphorylates predominantly (or exclusively) the C3 position. Western-blot analysis of protoplast and chloroplast fractions from Arabidopsis leaves reveals a plastidic location of PWD. Binding of PWD to starch granules strongly increases during net starch breakdown. Transgenic Arabidopsis plants in which the expression of PWD was reduced by either RNAi or a T-DNA insertion exhibit a starch excess phenotype. Thus, in Arabidopsis leaves starch turnover requires a close collaboration of PWD and GWD
The major aim of this work was the identification of new phloem sap proteins and a metabolic characterisation of this transport fluid. The experiments were performed on the three plant species C. sativus, C. maxima and B. napus. To characterise the phloem samples from B. napus, a new model plant for phloem analysis, western blot tests together with metabolite profiling were performed. GC-MS metabolite profiling and enzyme assays were used for measuring metabolites in the phloem of B. napus. Results from the phloem sap measurements showed, as expected, a typical sugar distribution for apoplasmic phloem loaders with sucrose being the predominant sugar. In stem extracts, the most abundant sugar was glucose with much lower fructose and sucrose levels. With the GC-MS approach it was possible to identify a number of metabolites which showed a differential distribution when phloem and stem tissue extracts were compared. For protein identification, two different approaches were employed (i) screening expression libraries with total phloem protein specific antisera and (ii) protein separation on 2 DE gels followed by ESI-MS/MS sequence analyses. For the first approach, three different phloem protein-specific antisera were produced and expression libraries were constructed. Phloem protein antisera were tested for specificity and some attempts to estimate specific epitopes were undertaken. Screening of the libraries resulted in the identification of 14 different proteins from all investigated species. Analyses of B. napus phloem sap proteins from 2 DE with ESI-MS/MS resulted in the identification of 5 different proteins. The phloem localisation of the identified proteins was additionally confirmed by western blot tests using specific antibodies. In order to functionally characterise some selected phloem proteins from B. napus, the group of potential calcium-binding polypeptides was analysed for functional Ca<sup>+2 binding properties and several Ca<sup>+2–binding proteins could be isolated. However, their sequences could as yet not be determined. Another approach used for functional protein characterisation was the analysis of Arabidopsis T-DNA insertion mutants. Four available mutants with insertions in phloem protein-specific genes were chosen from the SALK and GABI-Kat collections and selected homozygous lines were tested for the presence of the investigated proteins. In order to verify if the product of one of the mutated gene (GRP 7) is transported through the phloem, grafting experiments were performed followed by western blot analyses. Although the employed antiserum against GRP 7 protein did not allow distinguishing between the mutant and the wild type plants, successful Arabidopsis grafting could be established as a promising method for further studies on protein translocation through the phloem.
The acidic mining lakes of Eastern Germany are characterized by their extremely low pH and high iron concentrations. Low concentrations of CO2 in the epilimnion due to the low pH and reduced light transmission due to dissolved ferric iron potentially limit phytoplankton primary production (PP), whereas dissolved organic carbon (DOC) may promote heterotrophic production of bacteria (HP). We, therefore, tested whether HP exceeds PP in three lakes differing in pH and iron concentration (mean pH 2.3-3.0, 23-500 mg Fe L-1). Bacterial biomass and HP achieved highest values in the most acidic, most iron-rich lake, whereas PP was highest in the least acidic lake. HP was often higher than PP (ratio HP/PP up to 11), indicating that planktonic PP was not the main carbon source for the bacteria. HP was not related to PP and DOC, but HP as well as bacterial biomass increased with decreasing pH. Light stimulated the formation of ferrous iron, changed the DOC composition, and increased the HP in laboratory experiments, suggesting that iron photoreduction caused DOC degradation. This may explain why we found the highest HP in the most acidic and most rich lake. Overall, the importance of bacteria in the cycling of matter and as a basis for the whole food web seemed to increase in more acidic lakes with higher iron concentrations
National conservation planning should operate with measures of biodiversity similar to those applied globally in order to harmonize national and international conservation strategies. Here we suggest quantitative measures which enable two criteria of the global biodiversity hotspots to be applied on a national level for 74 large countries, and show how these measures can be applied to map national biodiversity hotspots. The plant endemism criteria of global hotspots are captured by quantitative measures of endemism, which are approximately scale-independent and can be corrected to account for a country's environmental conditions and conservation priorities. The flexible land use criteria for national biodiversity hotspots are defined from percentage of natural vegetation remaining in the global hotspots. The minimum-area-required approach is applied to define the borders of national biodiversity hotspots using data on vascular plants species richness. We show how national biodiversity hotspots can be mapped from the species- energy relationship for vascular plants using climate, topographical and land use data when spatial pattern of species richness is not known. This methodology to map national biodiversity hotspots from abiotic factors is applied to Russia as a case study. Three Russian biodiversity hotspots, North Caucasus, South Siberia and Far East were identified. The resulting hotspot maps cover national-scale environmental gradients across Russia and although they are also identified by Russian experts their actual geographical locations were hitherto unspecified. The large-scale national hotspots, identified for Russia, can be used for further fine scale and more detailed conservation planning. (c) 2005 Elsevier Ltd. All rights reserved
In this paper, habitat models were used to predict potential habitat for endangered species, which is an important question in landscape and conservation planning. Based on logistic regression, we developed habitat distribution models for the burnet moth Zygaena carniolica and the nymphalid butterfly Coenonympha arcania in Northern Bavaria, Germany. The relation between adult occurrence and habitat parameters, including the influence of landscape context, was analyzed on, 118 sites. Habitat connectivity analyses were carried out on the basis of (1) habitat suitability maps generated from these models and (2) dispersal data from mark recapture studies. Our results showed that (1) the presence of the burnet depended mainly on the presence of nectar plants and of nutrient-poor dry grasslands in direct vicinity, that of the nymphalid on larger areas of extensively used dry grasslands within 100 m vicinity in combination with small patches of higher shrubs and bushes. (2) Internal as well as external validation indicated the robustness and general applicability of the models. Transferability in time and space indicated their high potential relevance for applications in nature conservation, such as predicting possible effects of land use changes. (3) Habitat connectivity analyses revealed a high degree of habitat connectivity within the study area. Thus, we could show no effects of isolation or habitat size for both species. (c) 2005 Elsevier Ltd. All rights reserved
Two different methods for the quantification of human tissue inhibitor of metalloproteinases-2 (TIMP-2) were developed using surface plasmon resonance (SPR) and gold nanoparticles for signal enhancement. The first method, a competitive assay, used TIMP-2 immobilized to the sensor surface and the inactive form of matrix metalloproteinase-2 (proMMP-2) (EC 3.4.24.24) adsorbed to gold nanoparticles. The sensor signals resulting from the interaction of MMP-2- gold nanoparticles with immobilized TIMP-2 were inversely proportional to the amounts of TIMP-2 of the sample. The measuring range for TIMP-2 was about 15-180pM. The second method, a one-step sandwich assay, used proMMP-2 immobilized to the sensor surface and an anti-TIMP-2 monoclonal antibody coupled to gold nanoparticles. The lower detection limit of this assay format was 0.5 pM of TIMP-2. The binding signals were highly reproducible up to 100 pM of the inhibitor. The improvements obtained in TIMP-2 quantification over already existing tests could contribute to a better understanding and diagnosis of diseases like cancer. (c) 2005 Elsevier B.V. All rights reserved
Durch die anthropogene Nutzung sind viele Auen in Mitteleuropa verändert worden, wobei insbesondere die Retentionsflächen stark verringert wurden. Während Auen seit längerem im Fokus der wissenschaftlichen Bearbeitung stehen, gibt es bisher große Wissensdefizite in der Frage der Auenreaktivierungen. Zum einen sind derartige Projekte bisher kaum verwirklicht und zum anderen ist ein langfristiges Monitoring notwendig, um die Anpassung von Biozönosen an die veränderten Standortbedingungen beobachten zu können. Um die Folgen derartiger Eingriffe zu analysieren, bieten sich computergestützte Modellierungen der Landschaftsentwicklung an, wie sie in der vorliegenden Arbeit verwirklicht wurden. Ziel der Arbeit war, mit Hilfe eines Geografischen Informationssystems (GIS) das Entwicklungspotenzial der Landschaft bei verschiedenen Rückdeichungsvarianten auf der Ebene der Biotoptypen darzustellen. Dabei ging es nicht um die Erstellung eines allgemein gültigen Auenmodells sondern um die Erarbeitung eines Modells für einen konkreten Anwendungsfall. Der erarbeitete Ansatz sollte zudem für die landschaftsplanerische Praxis geeignet sein. Als Beispielgebiete wurden Flächen an der Mittleren Elbe bei Rogätz und Sandau, beide im nördlichen Teil von Sachsen-Anhalt, ausgewählt. Die vorliegende Arbeit gliedert sich in zwei Teile. Im ersten Teil werden Erhebungen und Auswertungen als Grundlage der Modellentwicklung dargestellt. Dazu wurden die Biotoptypen der Beispielgebiete flächendeckend erhoben und mit punktuellen Vegetationserhebungen ergänzt. Aus dem Forschungsprojekt "Rückgewinnung von Retentionsflächen und Altauenreaktivierung an der Mittleren Elbe in Sachsen-Anhalt" des Bundesministeriums für Bildung und Forschung (BMBF) standen standortökologische Daten der Hydrologie und Bodenkunde zur Verfügung. Ziel der Auswertung war, Schlüsselfaktoren für Hydrologie und Bodenbedingungen innerhalb der rezenten Aue zu identifizieren, die zur Ausprägung bestimmter Biotoptypen führen. Im zweiten Teil der Arbeit wurde ein Modell für Biotoptypenpotenziale auf den geplanten Rück–deichungsflächen entwickelt. Das Modell bearbeitet die Datenbank der verwendeten GIS-Dateien, die auf Daten zum Bestand beruht und um solche der Prognose der Standortökologie (Hydrologie und Boden) im Rückdeichungsfalle aus dem BMBF-Projekt erweitert wurde. Weitere Voraussetzung für die Modellierung war die Erarbeitung von Leitbildern, in denen unterschiedliche Nutzungsszenarios für die Landschaft nach Deichrückverlegung hypothetisch festgelegt wurden. Insbesondere die Nutzungsintensität wurde variiert, von einer Variante intensiver land- und forstwirtschaftlicher Nutzung über sogenannte integrierte Entwicklungsziele aus dem BMBF-Projekt bis hin zu einer Variante der Naturschutznutzung. Zusätzlich wurde eine zukünftige Potentielle Natürliche Vegetation modelliert. Eine Überprüfung des Modell fand für den Raum der rezenten Aue in der intensiven Nutzungsvariante statt, die der gegenwärtigen Nutzung am nächsten kommt. Werden Informationen des Bestandsbiotoptyps als Korrekturgröße in das Modell einbezogen, konnte für viele Biotoptypen eine Trefferquote von über 90 % erreicht werden. Bei flächenmäßig weniger bedeutenden Bio–toptypen lag dieser Wert aufgrund der schmaleren Datenbasis zwischen 20 und 40 %. Als Ergebnis liegt für unterschiedliche Deichvarianten und Leitbilder in den Beispielgebieten die Landschaftsentwicklung als Biotoppotenzial vor. Als eine vereinfachte Regionalisierung der punktuellen Vegetationsdaten wurde im Modell geprüft, inwieweit die modellierten Biotopflächen der Charakteristik der pflanzensoziologischen Aufnahmen aus der rezenten Aue entsprechen. In dem Falle wurde die Pflanzengesellschaft der jeweiligen ökologisch im Rahmen der Untersuchung einheitlichen Flächeneinheit zugeordnet. Anteilig lässt sich damit die Biotopprognosefläche pflanzensoziologisch konkretisieren. Die vorliegende Arbeit gehört zu den bisher wenigen Arbeiten, die sich mit den Folgen von Auenreaktivierung auf die Entwicklung der Landschaft auseinandersetzen. Sie zeigt eine Möglichkeit auf, Prognosemodelle für Biotoptypen und Vegetation anhand begrenzter Felduntersuchungen zu entwerfen. Derartige Modelle können zum Verständnis von Eingriffen in den Naturhaushalt, wie sie die Deichrückverlegungen darstellen, beitragen und eine Folgenabschätzung unterstützen.
Arabidopsis thaliana trichomes exhibit strong birefringence under polarized light, a characteristic of cell walls containing large amounts of highly ordered cellulose microfibrils. The tbr1 mutant of Arabidopsis lacks trichome birefringence and is deficient in secondary cell wall cellulose synthesis (Potikha and Delmer, 1995). The TBR gene was identified by recombinational mapping, candidate gene sequencing and molecular complementation using genomic cosmid clones, as well as a p35S:TBR genomic DNA construct, fully rescuing the mutant phenotype in both cases. The only mutant allele available (tbr-1) carries a substitution (G to E) in a conserved aminoacid domain of the protein. TBR gene structure was proved to have a longer size than the one found to be annotated at the time of identification in the data-base. A full cDNA clone containing the full transcript was available and also complementation experiments using different gene fragments (annotated and suggested) leaded to the result that TBR gene is indeed, longer. TBR encodes a novel plant-specific protein with predicted plasma membrane localization, therefore being consistent with idea that is required for-, or is a novel component of a functional cellulose synthase complex. TBR is part of an Arabidopsis gene/protein family, (TBL-trichome birefringence like) which, depending on homology, comprises up to 20 members, none of which has a biological or biochemical function attributed. T-DNA insertion lines in TBR gene and two close homologues have been screened by PCR, but no homozygous were found and no trichomes phenotype was identified. Promoter-GUS lines were produced for TBR, as well as for its two closest homologues (one being a segmentally duplicated gene on chromosome III), using 1.6-2 kb of promoter sequence upstream of the annotated start codons. The TBR promoter was the only one of the three that yielded trichome expression, this probably explaining the phenotype of the TBR mutant. Moreover, TBR is expressed in leaves, in growing lateral roots, and in vascular tissues of young Arabidopsis seedlings and plantlets. Later on, the expression appears in inflorescens, stems, flowers and green siliques. This expression pattern is largely overlapping with those of the two analyzed homologues and it corresponds with data of RT-PCR expression profiling performed for TBR and the two analyzed homologues in different tissues, at different developmental stages. Biochemical analysis of cell wall (leaves and trichomes), as GC and MALDI-TOF, were performed, but revealed no major differences between tbr1 and wild type plants. Scanning electron microscopy analysis and cell wall polysaccharides antibody labeling showed a clear difference in the trichomes cell wall structure between mutant plant and wild type.
During this PhD project three technical platforms were either improved or newly established in order to identify interesting genes involved in SNF, validate their expression and functionally characterise them. An existing 5.6K cDNA array (Colebatch et al., 2004) was extended to produce the 9.6K LjNEST array, while a second array, the 11.6K LjKDRI array, was also produced. Furthermore, the protocol for array hybridisation was substantially improved (Ott et al., in press). After functional classification of all clones according to the MIPS database and annotation of their corresponding tentative consensus sequence (TIGR) these cDNA arrays were used by several international collaborators and by our group (Krusell et al., 2005; in press). To confirm results obtained from the cDNA array analysis different sets of cDNA pools were generated that facilitate rapid qRT-PCR analysis of candidate gene expression. As stable transformation of Lotus japonicus takes several months, an Agrobacterium rhizogenes transformation system was established in the lab and growth conditions for screening transformants for symbiotic phenotypes were improved. These platforms enable us to identify genes, validate their expression and functionally characterise them in the minimum of time. The resources that I helped to establish, were used in collaboration with other people to characterise several genes like the potassium transporter LjKup and the sulphate transporter LjSst1, that were transcriptionally induced in nodules compared to uninfected roots, in more detail (Desbrosses et al., 2004; Krusell et al., 2005). Another gene that was studied in detail was LjAox1. This gene was identified during cDNA array experiments and detailed expression analysis revealed a strong and early induction of the gene during nodulation with high expression in young nodules which declines with the age of the nodule. Therefore, LjAox1 is an early nodulin. Promoter:gus fusions revealed an LjAox1 expression around the nodule endodermis. The physiological role of LjAox1 is currently being persued via RNAi. Using RNA interference, the synthesis of all symbiotic leghemoglobins was silenced simultaneously in Lotus japonicus. As a result, growth of LbRNAi lines was severely inhibited compared to wild-type plants when plants were grown under symbiotic conditions in the absence of mineral nitrogen. The nodules of these plants were arrested in growth 14 post inoculation and lacked the characteristic pinkish colour. Growing these transgenic plants in conditions where reduced nitrogen is available for the plant led to normal plant growth and development. This demonstrates that leghemoglobins are not required for plant development per se, and proves for the first time that leghemoglobins are indispensable for symbiotic nitrogen fixation. Absence of leghemoglobins in LbRNAi nodules led to significant increases in free-oxygen concentrations throughout the nodules, a decrease in energy status as reflected by the ATP/ADP ratio, and an absence of the bacterial nitrogenase protein. The bacterial population within nodules of LbRNAi plants was slightly reduced. Alterations of plant nitrogen and carbon metabolism in LbRNAi nodules was reflected in changes in amino acid composition and starch deposition (Ott et al., 2005). These data provide strong evidence that nodule leghemoglobins function as oxygen transporters that facilitate high flux rates of oxygen to the sites of respiration at low free oxygen concentrations within the infected cells.
The multidrug and toxic compounds extrusion (MATE) family includes hundreds of functionally uncharacterised proteins from bacteria and all eukaryotic kingdoms except the animal kingdom, that function as drug/toxin::Na<sup>+ or H<sup>+ antiporters. In Arabidopsis thaliana the MATE family comprises 56 members, one of which is NIC2 (Novel Ion Carrier 2). Using heterologous expression systems including Escherichia coli and Saccharomyces cerevisiae, and the homologous expression system of Arabidopsis thaliana, the functional characterisation of NIC2 was performed. It has been demonstrated that NIC2 confers resistance of E. coli towards the chemically diverse compounds such as tetraethylammonium chloride (TEACl), tetramethylammonium chloride (TMACl) and a toxic analogue of indole-3-acetic acid, 5-fluoro-indole-acetic acid (F-IAA). Therefore, NIC2 may be able to transport a broad range of drug and toxic compounds. In wild-type yeast the expression of NIC2 increased the tolerance towards lithium and sodium, but not towards potassium and calcium. In A. thaliana, the overexpression of NIC2 led to strong phenotypic changes. Under normal growth condtions overexpression caused an extremely bushy phenotype with no apical dominance but an enhanced number of lateral flowering shoots. The amount of rossette leaves and flowers with accompanying siliques were also much higher than in wild-type plants and the senescence occurred earlier in the transgenic plants. In contrast, RNA interference (RNAi) used to silence NIC2 expression, induced early flower stalk development and flowering compared with wild-type plants. In additon, the main flower stalks were not able to grow vertically, but instead had a strong tendency to bend towards the ground. While NIC2 RNAi seedlings produced many lateral roots outgrowing from the primary root and the root-shoot junction, NIC2 overexpression seedlings displayed longer primary roots that were characterised by a 2 to 4 h delay in the gravitropic response. In addition, these lines exhibited an enhanced resistance to exogenously applied auxins, i.e. indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) when compared with the wild-type roots. Based on these results, it is suggested that the NIC2 overexpression and NIC2 RNAi phenotypes were due to decreased or increased levels of auxin, respectively. The ProNIC2:GUS fusion gene revealed that NIC2 is expressed in the stele of the elongation zone, in the lateral root cap, in new lateral root primordia, and in pericycle cells of the root system. In the vascular tissue of rosette leaves and inflorescence stems, the expression was observed in the xylem parenchyma cells, while in siliques it was also in vascular tissue, but as well in the dehiscence and abscission zones. The organ- and tissue-specific expression sites of NIC2 correlate with the sites of auxin action in mature Arabidopsis plants. Further experiments using ProNIC2:GUS indicated that NIC2 is an auxin-inducible gene. Additionally, during the gravitropic response when an endogenous auxin gradient across the root tip forms, the GUS activity pattern of the ProNIC2:GUS fusion gene markedly changed at the upper side of the root tip, while at the lower side stayed unchanged. Finally, at the subcellular level NIC2-GFP fusion protein localised in the peroxisomes of Nicotana tabacum BY2 protoplasts. Considering the experimental results, it is proposed that the hypothetical function of NIC2 is the efflux transport which takes part in the auxin homeostasis in plant tissues probably by removing auxin conjugates from the cytoplasm into peroxisomes.