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Background
Natural accessions of Arabidopsis thaliana are a well-known system to measure levels of intraspecific genetic variation. Leaf starch content correlates negatively with biomass. Starch is synthesized by the coordinated action of many (iso)enzymes. Quantitatively dominant is the repetitive transfer of glucosyl residues to the non-reducing ends of α-glucans as mediated by starch synthases. In the genome of A. thaliana, there are five classes of starch synthases, designated as soluble starch synthases (SSI, SSII, SSIII, and SSIV) and granule-bound synthase (GBSS). Each class is represented by a single gene. The five genes are homologous in functional domains due to their common origin, but have evolved individual features as well. Here, we analyze the extent of genetic variation in these fundamental protein classes as well as possible functional implications on transcript and protein levels.
Findings
Intraspecific sequence variation of the five starch synthases was determined by sequencing the entire loci including promoter regions from 30 worldwide distributed accessions of A. thaliana. In all genes, a considerable number of nucleotide polymorphisms was observed, both in non-coding and coding regions, and several amino acid substitutions were identified in functional domains. Furthermore, promoters possess numerous polymorphisms in potentially regulatory cis-acting regions. By realtime experiments performed with selected accessions, we demonstrate that DNA sequence divergence correlates with significant differences in transcript levels.
Conclusions
Except for AtSSII, all starch synthase classes clustered into two or three groups of haplotypes, respectively. Significant difference in transcript levels among haplotype clusters in AtSSIV provides evidence for cis-regulation. By contrast, no such correlation was found for AtSSI, AtSSII, AtSSIII, and AtGBSS, suggesting trans-regulation. The expression data presented here point to a regulation by common trans-regulatory transcription factors which ensures a coordinated action of the products of these four genes during starch granule biosynthesis. The apparent cis-regulation of AtSSIV might be related to its role in the initiation of de novo biosynthesis of granules.
Saccharomyces cerevisiae possesses two glycogenin isoforms (designated as Glg1p and Glg2p) that both contain a conserved tyrosine residue, Tyr232. However, Glg2p possesses an additional tyrosine residue, Tyr230 and therefore two potential autoglucosylation sites. Glucosylation of Glg2p was studied using both matrix-assisted laser desorption ionization and electrospray quadrupole time of flight mass spectrometry. Glg2p, carrying a C-terminal (His(6)) tag, was produced in Escherichia coli and purified. By tryptic digestion and reversed phase chromatography a peptide (residues 219-246 of the complete Glg2p sequence) was isolated that contained 4-25 glucosyl residues. Following incubation of Glg2p with UDPglucose, more than 36 glucosyl residues were covalently bound to this peptide. Using a combination of cyanogen bromide cleavage of the protein backbone, enzymatic hydrolysis of glycosidic bonds and reversed phase chromatography, mono- and diglucosylated peptides having the sequence PNYGYQSSPAM were generated. MS/MS spectra revealed that glucosyl residues were attached to both Tyr232 and Tyr230 within the same peptide. The formation of the highly glucosylated eukaryotic Glg2p did not favour the bacterial glycogen accumulation. Under various experimental conditions Glg2p-producing cells accumulated approximately 30% less glycogen than a control transformed with a Glg2p lacking plasmid. The size distribution of the glycogen and extractable activities of several glycogen-related enzymes were essentially unchanged. As revealed by high performance anion exchange chromatography, the intracellular maltooligosaccharide pattern of the bacterial cells expressing the functional eukaryotic transgene was significantly altered. Thus, the eukaryotic glycogenin appears to be incompatible with the bacterial initiation of glycogen biosynthesis
The nature of the periplastidial pathway of starch biosynthesis was investigated with the model cryptophyte Guillardia theta. The storage polysaccharide granules were shown to be composed of both amylose and amylopectin fractions with a chain length distribution and crystalline organization very similar to those of starch from green algae and land plants. Most starch granules displayed a shape consistent with biosynthesis occurring around the pyrenoid through the rhodoplast membranes. A protein with significant similarity to the amylose-synthesizing granule-bound starch syntbase 1 from green plants was found as the major polypeptide bound to the polysaccharide matrix. N-terminal sequencing of the mature protein proved that the precursor protein carries a nonfunctional transit peptide in its bipartite topogenic signal sequence which is cleaved without yielding transport of the enzyme across the two inner plastid membranes. The enzyme was shown to display similar affinities for ADP and UDP-glucose, while the V-max measured with UDP-glucose was twofold higher. The granule-bound starch synthase from Guillardia theta was demonstrated to be responsible for the synthesis of long glucan chains and therefore to be the functional equivalent of the amylose- synthesizing enzyme of green plants. Preliminary characterization of the starch pathway suggests that Guillardia theta utilizes a UDP-glucose-based pathway to synthesize starch
The recently characterized cytosolic transglucosidase DPE2 (EC 2.4.1.25) is essential for the cytosolic metabolism of maltose, an intermediate on the pathway by which starch is converted to sucrose at night. In in vitro assays, the enzyme utilizes glycogen as a glucosyl acceptor but the in vivo acceptor molecules remained unknown. In this communication we present evidence that DPE2 acts on the recently identified cytosolic water-soluble heteroglycans (SHG) as does the cytosolic phosphorylase (EC 2.4.1.1) isoform. By using in vitro two-step C-14 labeling assays we demonstrate that the two transferases can utilize the same acceptor sites of the SHG. Cytosolic heteroglycans from a DPE2-deficient Arabidopsis mutant were characterized. Compared with the wild type the glucose content of the heteroglycans was increased. Most of the additional glucosyl residues were found in the outer chains of SHG that are released by an endo- alpha-arabinanase (EC 3.2.1.99). Additional starch-related mutants were characterized for further analysis of the increased glucosyl content. Based on these data, the cytosolic metabolism of starch-derived carbohydrates is discussed
Die nun begonnene Reihe „studieren++“ resultiert aus einer von der Universität Potsdam angebotenen Vorlesungsreihe. Das Besondere an dieser Vorlesungsreihe ist der multidisziplinäre Anspruch und die konsequent umgesetzte Zusammenarbeit über Disziplingrenzen hinweg. Die nicht nur über Instituts-, sondern über Fakultätsgrenzen praktizierte Interdisziplinarität erlaubt die Betrachtung eines Problems oder Sachverhalts aus unterschiedlichen Blickwinkeln. Wissenschaftliche Fragestellungen sind komplex und nicht immer auf eine Disziplin beschränkt. Sie in ihrer Gänze erfassen und nachhaltige Lösungsstrategien oder Konzepte entwickeln zu können gelingt oft nur durch eine multidisziplinäre Kooperation. Eine Lehrveranstaltung wie die vorliegende ist nicht nur für die Studierenden einer Universität eine hervorragende Möglichkeit, um über die Grenzen der eigenen Disziplin hinaus zu blicken und die Zusammenarbeit mit Wissenschaftlerinnen und Wissenschaftlern aus anderen Bereichen zu pflegen. So lernt man, sich in andere Sichtweisen hineinzuversetzen und sich zwischen den Disziplinen zu bewegen – eine Kompetenz, die in der hochkomplexen Arbeitswelt von heute von hohem Nutzen ist.
Der vorliegende erste Band der Reihe hat „Raum und Zahl“ zum Thema und ist aus einer Ringvorlesung aus dem Wintersemester 2013/2014 entstanden. Drei der fünf Fakultäten, insgesamt neun Institute der Universität Potsdam, haben sich an der Vorlesung beteiligt und sich dieses spannenden Themas angenommen. Als jemand, der sich jahrelang wissenschaftlich mit algorithmischer Geometrie sowie mit raumbezogenen Datenbanken und Navigationssystemen beschäftigt hat, kann ich nur bekräftigen, dass die Bezüge zwischen Raum und Zahl, zwischen Räumen und Zahlen, noch viel stärker im öffentlichen Bewusstsein verankert gehören. Räume auch quantitativ zu erfassen und zu verstehen ist eine Kulturtechnik, die an Wichtigkeit eher noch zunimmt, vor allem vor dem Hintergrund, dass wir genetisch nicht allzu gut auf derartige Herausforderungen vorbereitet sind. Denn viele unserer einschlägigen Gene entstammen noch aus der Zeit der Savanne, einer Zeit, zu der das Raumkonzept sich fast ausschließlich auf die unmittelbare räumliche Umgebung bezog und Zahlen jenseits von 10 nur wenig Relevanz für das eigene Überleben hatten.
Als Präsident der Universität Potsdam freut es mich ganz besonders, dass sich die hier vertretenen Wissenschaftler bereit erklärt haben, ihre Überlegungen mit den Studierenden und ihren Kolleginnen und Kollegen zu teilen. Herrn Kollegen Hans-Joachim Petsche möchte ich für sein Engagement danken und ihm zu dieser gelungenen Reihe gratulieren. Der Geist der Wissenschaft, der nicht nur einsam im Büro oder Labor gelebt wird, sondern gerade an einer Universität auch aktiv nach außen getragen werden sollte, wird hier in besonderer Weise sichtbar. Ich wünsche Ihnen viel Freude bei der Lektüre des Bandes und freue mich auf weitere Veröffentlichungen in dieser Reihe.
A novel method for the encapsulation of biomacromolecules, such as nucleic acids and proteins, into polyelectrolyte microcapsules is described. Fluorescence-labelled double-stranded DNA and human serum albumin (HSA) are used as model substances for encapsulation in hollow microcapsules templated on human erythrocytes. The encapsulation procedure involves an intermediate drying C, step. The accumulation of DNA and HSA in the capsules is observed by confocal laser scanning microscopy, UV spectroscopy, and flourimetry. The mechanism of encapsulation is discussed