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Molecular and physiological analyses of diacylglycerol kinases from arabidopsis thaliana (L.) Heynh.
(2004)
Even though the structure of the plant cell wall is by and large quite well characterized, its synthesis and regulation remains largely obscure. However, it is accepted that the building blocks of the polysaccharidic part of the plant cell wall are nucleotide sugars. Thus to gain more insight into the cell wall biosynthesis, in the first part of this thesis, plant genes possibly involved in the nucleotide sugar interconversion pathway were identified using a bioinformatics approach and characterized in plants, mainly in Arabidopsis. For the computational identification profile hidden markov models were extracted from the Pfam and TIGR databases. Mainly with these, plant genes were identified facilitating the “hmmer” program. Several gene families were identified and three were further characterized, the UDP-rhamnose synthase (RHM), UDP-glucuronic acid epimerase (GAE) and the myo-inositol oxygenase (MIOX) families. For the three-membered RHM family relative ubiquitous expression was shown using variuos methods. For one of these genes, RHM2, T-DNA lines could be obtained. Moreover, the transcription of the whole family was downregulated facilitating an RNAi approach. In both cases a alteration of cell wall typic polysaccharides and developmental changes could be shown. In the case of the rhm2 mutant these were restricted to the seed or the seed mucilage, whereas the RNAi plants showed profound changes in the whole plant. In the case of the six-membered GAE family, the gene expressed to the highest level (GAE6) was cloned, expressed heterologously and its function was characterized. Thus, it could be shown that GAE6 encodes for an enzyme responsible for the conversion of UDP-glucuronic acid to UDP-galacturonic acid. However, a change in transcript level of variuos GAE family members achieved by T-DNA insertions (gae2, gae5, gae6), overexpression (GAE6) or an RNAi approach, targeting the whole family, did not reveal any robust changes in the cell wall. Contrary to the other two families the MIOX gene family had to be identified using a BLAST based approach due to the lack of enough suitable candidate genes for building a hidden markov model. An initial bioinformatic characterization was performed which will lead to further insights into this pathway. In total it was possible to identify the two gene families which are involved in the synthesis of the two pectin backbone sugars galacturonic acid and rhamnose. Moreover with the identification of the MIOX genes a genefamily, important for the supply of nucleotide sugar precursors was identified. In a second part of this thesis publicly available microarray datasets were analyzed with respect to co-responsive behavior of transcripts on a global basis using nearly 10,000 genes. The data has been made available to the community in form of a database providing additional statistical and visualization tools (http://csbdb.mpimp-golm.mpg.de). Using the framework of the database to identify nucleotide sugar converting genes indicated that co-response might be used for identification of novel genes involved in cell wall synthesis based on already known genes.
The past decades are characterized by various efforts to provide complete sequence information of genomes regarding various organisms. The availability of full genome data triggered the development of multiplex high-throughput assays allowing simultaneous measurement of transcripts, proteins and metabolites. With genome information and profiling technologies now in hand a highly parallel experimental biology is offering opportunities to explore and discover novel principles governing biological systems. Understanding biological complexity through modelling cellular systems represents the driving force which today allows shifting from a component-centric focus to integrative and systems level investigations. The emerging field of systems biology integrates discovery and hypothesis-driven science to provide comprehensive knowledge via computational models of biological systems. Within the context of evolving systems biology, investigations were made in large-scale computational analyses on transcript co-response data through selected prokaryotic and plant model organisms. CSB.DB - a comprehensive systems-biology database - (http://csbdb.mpimp-golm.mpg.de/) was initiated to provide public and open access to the results of biostatistical analyses in conjunction with additional biological knowledge. The database tool CSB.DB enables potential users to infer hypothesis about functional interrelation of genes of interest and may serve as future basis for more sophisticated means of elucidating gene function. The co-response concept and the CSB.DB database tool were successfully applied to predict operons in Escherichia coli by using the chromosomal distance and transcriptional co-responses. Moreover, examples were shown which indicate that transcriptional co-response analysis allows identification of differential promoter activities under different experimental conditions. The co-response concept was successfully transferred to complex organisms with the focus on the eukaryotic plant model organism Arabidopsis thaliana. The investigations made enabled the discovery of novel genes regarding particular physiological processes and beyond, allowed annotation of gene functions which cannot be accessed by sequence homology. GMD - the Golm Metabolome Database - was initiated and implemented in CSB.DB to integrated metabolite information and metabolite profiles. This novel module will allow addressing complex biological questions towards transcriptional interrelation and extent the recent systems level quest towards phenotyping.
Environmental stresses such as drought, high salt and low temperature affect plant growth and decrease crop productivity extremely. It is important to improve stress tolerance of the crop plant to increase crop yield under stress conditions. The Arabidopsis thaliana salt tolerance 1 gene (AtSTO1) was originally identified by Lippuner et al., (1996). In this study around 27 members of STO-like proteins were identified in Arabidopsis thaliana, rice and other plant species. The STO proteins have two consensus motifs (CCADEAAL and FCV(L)EDRA). The STO family members can be regarded as a distinct class of C2C2 proteins considering their low sequence similarity to other GATA like proteins and poor conservation in the C-terminus. AtSTO1 was found to be induced by salt, cold and drought in leaves and roots of 4-week-old Arabidopsis thaliana wild-type plants. The expression of AtSTO1 under salt and cold stress was more pronounced in roots than in leaves. The data provided here revealed that the AtSTO1 protein is localized in the nucleus. The observation that AtSTO1 localizes in the nucleus is consistent with its proposed function as a transcription factor. AtSTO1-dependent phenotypes were observed when plant were grown at 50 mM NaCl on agar plates. Leaves of AtSTO1 overexpression lines were bigger with dark green coloration, whereas stunted growth and yellowish leaves were observed in wild-type and RNAi plants. Also, the AtSTO1 overexpression plants when exposed to long-term cold stress had a red leaf coloration which was much stronger than in wild-type and RNAi lines. Growth of AtSTO1 overexpression lines in long term under salt and cold stress was always associated with long roots which was more pronounced than in wild-type and RNAi lines. Proline accumulation increased more strongly in leaves and roots of AtSTO1 overexpression lines than in tissues of wild-type and RNAi lines when treated with 200 mM NaCl, exposed to cold stress or when watering was prevented for one day or two weeks. Also, soluble sugar content increased to higher levels under salt, cold and drought stress in AtSTO1 overexpression lines when compared to wild-type and RNAi lines. The increase in soluble sugar content was detected in AtSTO1 overexpression lines after long-term (2 weeks) growth of plants under these stresses. Anthocyanins accumulated in leaves of AtSTO1 overexpression lines when exposed to long term salt stress (200 mM NaCl for 2 weeks) or to 4°C for 6 and 8 weeks. Also, anthocyanin content was increased in flowers of AtSTO1 overexpression plants kept at 4°C for 8 weeks. Taken together these data indicate that overexpression of AtSTO1 enhances abiotic stress toleranc via a more pronounced accumulation of compatible solutes under stress.
The overall objective of the study is an elaboration of quantitative methods for national conservation planning, coincident with the international approach ('hotspots' approach). This objective requires a solution of following problems: 1) How to estimate large scale vegetation diversity from abiotic factors only? 2) How to adopt 'global hotspots' approach for bordering of national biodiversity hotspots? 3) How to set conservation targets, accounting for difference in environmental conditions and human threats between national biodiversity hotspots? 4) How to design large scale national conservation plan reflecting hierarchical nature of biodiversity? The case study for national conservation planning is Russia. Conclusions: · Large scale vegetation diversity can be predicted to a major extent by climatically determined latent heat for evaporation and geometrical structure of landscape, described as an altitudinal difference. The climate based model reproduces observed species number of vascular plant for different areas of the world with an average error 15% · National biodiversity hotspots can be mapped from biotic or abiotic data using corrected for a country the quantitative criteria for plant endemism and land use from the 'global hotspots' approach · Quantitative conservation targets, accounting for difference in environmental conditions and human threats between national biodiversity hotspots can be set using national data for Red Data book species · Large scale national conservation plan reflecting hierarchical nature of biodiversity can be designed by combination of abiotic method at national scale (identification of large scale hotspots) and biotic method at regional scale (analysis of species data from Red Data book)
In this work different approaches are undertaken to improve the understanding of the sucrose-to-starch pathway in developing potato tubers. At first an inducible gene expression system from fungal origin is optimised for the use of studying metabolism in the potato tuber. It is found that the alc system from Aspergillus nidulans responds more rapidly to acetaldehyde than ethanol, and that acetaldehyde has less side-effects on metabolism. The optimal induction conditions then are used to study the effects of temporally controlled cytosolic expression of a yeast invertase on metabolism of potato tubers. The observed differences between induced and constitutive expression of the invertase lead to the conclusion that glycolysis is induced after an ATP demand has been created by an increase in sucrose cycling. Furthermore, the data suggest that in the potato tuber maltose is a product of glucose condensation rather than starch degradation. In the second part of the work it is shown that the expression of a yeast invertase in the vacuole of potato tubers has similar effects on metabolism than the expression of the same enzyme in the apoplast. These observations give further evidence to the presence of a mechanism by which sucrose is taken up via endocytosis to the vacuole rather than via transporters directly to the cytosol. Finally, a kinetic in silico model of sucrose breakdown is presented that is able to simulate this part of potato tuber metabolism on a quantitative level. Furthermore, it can predict the metabolic effects of the introduction of a yeast invertase in the cytosol of potato tubers with an astonishing precision. In summary, these data prove that inducible gene expression and kinetic computer models of metabolic pathways are useful tools to greatly improve the understanding of plant metabolism.
For recombinant production of proteins for structural and functional analyses, the E. coli expression system is the most widely used due to high yields and straightforward processing. However, particularly the expression of eukaryotic proteins in E. coli is often problematic, e.g. when the protein is not folded correctly and is deposited in insoluble inclusion bodies. In some cases it is favourable to analyse deletion constructs of a protein or an individual protein domain instead of the full-length protein. This implies the generation of a set of expression constructs that need to be characterised. In this work methods to optimise and evaluate in vitro folding of inclusion body proteins as well as high-throughput characterisation of expression constructs were developed. Transferring inclusion body proteins to their native state involves two steps: (a) solubilisation with a chaotropic reagent or a strong ionic detergent and (b) folding of the protein by removal of the chaotrop accompanied by the transfer into an appropriate buffer. The yield of natively folded protein is often substantially reduced due to aggregation or misfolding; it may, however, be improved by certain additives to the folding buffer. These additives need to be identified empirically. In this thesis a screening procedure for folding conditions was developed. To reduce the number of possible combinations of screening additives, empirical observations documented in the literature as well as well known properties of certain screening additives were considered. To decrease the amount of protein and work invested, the screen was miniaturised and automated using a pipetting robot. Twenty rapid dilution conditions for the denatured protein are tested and two conditions for folding of proteins using the detergent/cyclodextrin protein folding system of Rozema et al. (1996). 100 µg protein is used per condition. In addition, eight conditions can be tested for folding of His-tagged proteins (approx. 200 µg) immobilised on metal chelate resins. The screen was successfully applied to fold a human protein, the p22 subunit of dynactin that is expressed in inclusion bodies in E. coli. For p22 dynactin – as is the case for many proteins – there was no biological assay available to assess the success of the folding screen. Protein solubility can not be used as a stringent criterion because beside natively folded protein, soluble misfolded species and microaggregates may occur. This work evaluates methods to detect small amounts of natively folded protein after automated folding screening. Before folding screening with p22 dynactin, two model enzymes, bovine carbonic anhydrase II (CAB) and pig heart mitochondrial malate dehydrogenase, were used for evaluation. Recovered activity after refolding was correlated to different biophysical methods. 8-anilino-1-naphtalenesulfonic acid binding-experiments gave no useful information when refolding CAB, due to low sensitivity and because misfolded protein could not be readily distinguished from native protein. Tryptophan fluorescence spectra of refolded CAB were used to assess the success of refolding. The shift of the intensity maximum to a shorter wavelength, compared to the denaturant unfolded protein, as well as the fluorescence intensity correlated to recovered enzymatic activity. For both model enzymes, analytical hydrophobic interaction chromatography (HIC) was useful to identify refolded samples that contain active enzyme. Compactly folded, active enzyme eluted in a distinct peak in a decreasing ammonium sulfate gradient. The detection limit of analytical HIC was approx. 5 µg. In case of CAB, tryptophan fluorescence spectroscopy and analytical HIC showed that both methods in combination can be useful to rule out false positives or false negatives obtained with one method. These two methods were also useful to identify conditions for folding of p22 dynactin. However, tryptophan fluorescence spectroscopy can lead to false positives because in some cases spectra of soluble microaggregates are not well distinguishable from spectra of natively folded protein. In summary, a fast and reliable screening procedure was developed to make inclusion body proteins accessible to structural or functional analyses. In a separate project, 88 different E. coli expression constructs for 17 human protein domains that had been identified by sequence analysis were analysed using high-throughput purification and folding analysis in order to obtain candidates suitable for structural analysis. After 96 deep-well microplate expression and automated protein purification, solubly expressed protein domains were directly analysed using 1D ¹H-NMR spectroscopy. It was found that isolated methyl group signals below 0.5 ppm are particularly sensitive and reliable probes for folded protein. In addition – similar to the evaluation of a folding screen – analytical HIC proved to be an efficient tool for identifying constructs that yield compactly folded protein. Both methods, 1D ¹H-NMR spectroscopy and analytical HIC, provided complementary results. Six constructs, representing two domains, could be quickly identified as targets that are well suitable for structural analysis. The structure of one of these domains was solved recently by co-workers, the other structure was published by another group during this project.
Recent high-throughput technologies enable the acquisition of a variety of complementary data and imply regulatory networks on the systems biology level. A common approach to the reconstruction of such networks is the cluster analysis which is based on a similarity measure. We use the information theoretic concept of the mutual information, that has been originally defined for discrete data, as a measure of similarity and propose an extension to a commonly applied algorithm for its calculation from continuous biological data. We compare our approach to previously existing algorithms. We develop a performance optimised software package for the application of the mutual information to large-scale datasets. Furthermore, we design and implement a web-based service for the analysis of integrated data measured with different technologies. Application to biological data reveals biologically relevant groupings and reconstructed signalling networks show agreements with physiological findings.
Red cell development in adult humans results in the mean daily production of 2x1011 erythrocytes. Mature hemoglobinized and enucleated erythrocytes develop from multipotent hematopoietic stem/progenitor cells through more committed progenitor cell types such as BFU-E and CFU-E. The studies on the molecular mechanisms of erythropoiesis in the human system require a sufficient number of purified erythroid progenitors of the different stages of erythropoiesis. Primary human erythroid progenitors are difficult to obtain as a homogenous population in sufficiently high cell numbers. Various culture conditions for the in vitro cell culture of primary human erythroid progenitors have been previously described. Mainly, the culture resulted in the generation of rather mature stages of Epo-dependent erythroid progenitors. In this study our efforts were directed towards the isolation and characterization of more early red cell progenitors that are Epo-independent. To identify such progenitors, CD34+ cells were purified from cord blood and cultured under serum free conditions in the presence of the growth factors SCF, IL-3 and hyper-IL-6, referred to as SI2 culture conditions. By immunomagnetic bead selection of E-cadherin (E-cad) positive cells, E-cad+ progenitors were isolated. These Epo-independent E-cad+ progenitors have been amplified under SI2 conditions to large cell numbers. The E-cad+ progenitors were characterized for surface antigen expression by flow cytometry, response to growth factors in proliferation assay and for their differentiation potential into mature red cells. Additionally, the properties of E-cad+ progenitors were compared to those of two other erythroid progenitors: Epo-dependent progenitors described by Panzenböck et al. (referred to as SCF/Epo progenitor), and CD36+ progenitors described by Freyssinier et al. (Panzenböck et al., 1998; Freyssinier et al., 1999). Finally, the gene expression profile of E-cad+ progenitors was compared to the profiles of SCF/Epo progenitors and CD36+ progenitors using the DNA microarray technique. Based on our studies we propose that Epo-independent E-cad+ progenitors are early stage, BFU-E like progenitors. They respond to Epo, despite the fact that they were generated in the absence of Epo, and can completely undergo erythroid differentiation. Furthermore, we demonstrate that the growth properties, the growth factor response and the surface marker expression of E-cad+ progenitors are similar to those of the SCF/Epo progenitors and the CD36+ progenitors. By the comparison of gene profiles, we were also able to demonstrate that the Epo-dependent and Epo-independent red cell progenitors are very similar. Analyzing the molecular differences between E-cad+ and SCF/Epo progenitors revealed several candidate genes such as galectin-3, cyclin D1, AMHR, PDF and IGFBP4, which are potential regulators involved in red cell development. We also demonstrate that the CD36+ progenitors, isolated by immunomagentic bead selection, are a heterogeneous progenitor population containing an E-cad+ and an E-cad- subpopulation. Based on their gene expression profile, CD36+ progenitors seem to exhibit both erythroid and megakaryocytic features. These studies led to a more updated model of erythroid cell development that should pave the way for further studies on molecular mechanisms of erythropoiesis.