@article{FettkeChiaEckermannetal.2006, author = {Fettke, J{\"o}rg and Chia, Tansy and Eckermann, Nora and Smith, Alison M. and Steup, Martin}, title = {A transglucosidase necessary for starch degradation and maltose metabolism in leaves at night acts on cytosolic heteroglycans (SHG)}, issn = {0960-7412}, doi = {10.1111/j.1365-313X.2006.02732.x}, year = {2006}, abstract = {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}, language = {en} } @article{FettkePoesteEckermannetal.2005, author = {Fettke, J{\"o}rg and Poeste, Simon and Eckermann, Nora and Tiessen, Axel and Pauly, Markus and Geigenberger, Peter Ludwig and Steup, Martin}, title = {Analysis of cytosolic heteroglycans from leaves of transgenic potato (Solanum tuberosum L.) plants that under- or overexpress the Pho 2 phosphorylase isozyme}, year = {2005}, abstract = {During starch degradation, chloroplasts export neutral sugars into the cytosol where they appear to enter a complex glycan metabolism. Interactions between glycans and glucosyl transferases residing in the cytosol were studied by analyzing transgenic potato (Solanum tuberosum L.) plants that possess either decreased or elevated levels of the cytosolic (Pho 2) phosphorylase isoform. Water-soluble heteroglycans (SHGs) were isolated from these plants and were characterized. SHG contains, as major constituents, arabinose, rhamnose, galactose and glucose. Non-aqueous fractionation combined with other separation techniques revealed a distinct pool of the SHG that is located in the cytosol. Under in vitro conditions, the cytosolic heteroglycans act as glucosyl acceptor selectively for Pho 2. Acceptor sites were characterized by a specific hydrolytic degradation following the Pho 2-catalyzed glucosyl transfer. The size distribution of the cytosolic SHG increased during the dark period, indicating a distinct metabolic activity related to net starch degradation. Antisense inhibition of Pho 2 resulted in increased glucosyl and rhamnosyl contents of the glycans. Overexpression of Pho 2 decreased the content of both residues. Compared with the wild type, in both types of transgenic plants the size of the cytosolic glycans was increased}, language = {en} } @article{RitteEckermannHaebeletal.2000, author = {Ritte, Gerhard and Eckermann, Nora and Haebel, Sophie and Lorberth, Ruth and Steup, Martin}, title = {Compartmentation of the starch-related R1 protein in higher plants}, year = {2000}, language = {en} } @article{FettkeMalinovaEckermannetal.2009, author = {Fettke, J{\"o}rg and Malinova, Irina and Eckermann, Nora and Steup, Martin}, title = {Cytosolic heteroglycans in photoautotrophic and in heterotrophic plant cells}, issn = {0031-9422}, doi = {10.1016/j.phytochem.2009.03.016}, year = {2009}, abstract = {In plants several 'starch-related' enzymes exist as plastid- and cytosol-specific isoforms and in some cases the extraplastidial isoforms represent the majority of the enzyme activity. Due to the compartmentation of the plant cells, these extraplastidial isozymes have no access to the plastidial starch granules and, therefore, their in vivo function remained enigmatic. Recently, cytosolic heteroglycans have been identified that possess a complex pattern of the monomer composition and glycosidic bonds. The glycans act both as acceptors and donors for cytosolic glucosyl transferases. In autotrophic tissues the heteroglycans are essential for the nocturnal starch-sucrose conversion. In this review we summarize the current knowledge of these glycans, their interaction with glucosyl transferases and their possible cellular functions. We include data on the heteroglycans in heterotrophic plant tissues and discuss their role in intracellular carbon fluxes that originate from externally supplied carbohydrates.}, language = {en} } @article{BaumannEckermannKrauseetal.1994, author = {Baumann, Ingrid and Eckermann, Nora and Krause, Udo and Baumann, Guido}, title = {Effects of sucrose in the culture medium on cytological characteristics, pigments and photosynthetic activity of green callus cultures of sugar beet}, year = {1994}, language = {en} } @article{EckermannBaumann1995, author = {Eckermann, Nora and Baumann, Guido}, title = {Enzymatic changes in callus cultures of sugar beet during the transition from photoheterotrophic to photoautotrophic growth}, year = {1995}, language = {en} } @article{EckermannFettkeSteup2002, author = {Eckermann, Nora and Fettke, J{\"o}rg and Steup, Martin}, title = {Identification of polysaccharide binding proteins by affinity electrophoresis in inhomogeneous polyacrylamide gels and subsequent SDS-PAGE/MALDI-TOF analysis}, year = {2002}, language = {en} } @article{FettkeEckermannTiessenetal.2005, author = {Fettke, J{\"o}rg and Eckermann, Nora and Tiessen, Axel and Geigenberger, Peter Ludwig and Steup, Martin}, title = {Identification, subcellular localization and biochemical characterization of water-soluble heteroglycans (SHG) in leaves of Arabidopsis thaliana L. : distinct SHG reside in the cytosol and in the apoplast}, issn = {0960-7412}, year = {2005}, abstract = {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}, language = {en} } @article{RitteScharfEckermannetal.2004, author = {Ritte, Gerhard and Scharf, Anke and Eckermann, Nora and Haebel, Sophie and Steup, Martin}, title = {Phosphorylation of transitory starch is increased during degradation}, year = {2004}, abstract = {The starch excess phenotype of Arabidopsis mutants defective in the starch phosphorylating enzyme glucan, water dikinase (EC 2.7.9.4) indicates that phosphorylation of starch is required for its degradation. However, the underlying mechanism has not yet been elucidated. In this study, two in vivo systems have been established that allow the analysis of phosphorylation of transitory starch during both biosynthesis in the light and degradation in darkness. First, a photoautotrophic culture of the unicellular green alga Chlamydomonas reinhardtii was used to monitor the incorporation of exogenously supplied P-32 orthophosphate into starch. Illuminated cells incorporated P-32 into starch with a constant rate during 2 h. By contrast, starch phosphorylation in darkened cells exceeded that in illuminated cells within the first 30 min, but subsequently phosphate incorporation declined. Pulse-chase experiments performed with P-32/P-31 orthophosphate revealed a high turnover of the starch-bound phosphate esters in darkened cells but no detectable turnover in illuminated cells. Secondly, leaf starch granules were isolated from potato (Solanum tuberosum) plants grown under controlled conditions and glucan chains from the outer granule layer were released by isoamylase. Phosphorylated chains were purified and analyzed using high performance anion-exchange chromatography and matrix-assisted laser desorption/ionization mass spectrometry. Glucans released from the surface of starch granules that had been isolated from darkened leaves possessed a considerably higher degree of phosphorylation than those prepared from leaves harvested during the light period. Thus, in the unicellular alga as well as in potato leaves, net starch degradation is accompanied with an increased phosphorylation of starch}, language = {en} } @article{DauvilleeChochoisSteupetal.2006, author = {Dauvillee, David and Chochois, Vincent and Steup, Martin and Haebel, Sophie and Eckermann, Nora and Ritte, Gerhard and Ral, Jean-Philippe and Colleoni, Christophe and Hicks, Glenn and Wattebled, Fabrice and Deschamps, Philippe and Lienard, Luc and Cournac, Laurent and Putaux, Jean-Luc and Dupeyre, Danielle and Ball, Steven G.}, title = {Plastidial phosphorylase is required for normal starch synthesis in Chlamydomonas reinhardtii}, series = {The plant journal}, volume = {48}, journal = {The plant journal}, number = {2}, publisher = {Blackwell}, address = {Oxford}, issn = {0960-7412}, doi = {10.1111/j.1365-313X.2006.02870.x}, pages = {274 -- 285}, year = {2006}, abstract = {Among the three distinct starch phosphorylase activities detected in Chlamydomonas reinhardtii, two distinct plastidial enzymes (PhoA and PhoB) are documented while a single extraplastidial form (PhoC) displays a higher affinity for glycogen as in vascular plants. The two plastidial phosphorylases are shown to function as homodimers containing two 91-kDa (PhoA) subunits and two 110-kDa (PhoB) subunits. Both lack the typical 80-amino-acid insertion found in the higher plant plastidial forms. PhoB is exquisitely sensitive to inhibition by ADP-glucose and has a low affinity for malto-oligosaccharides. PhoA is more similar to the higher plant plastidial phosphorylases: it is moderately sensitive to ADP-glucose inhibition and has a high affinity for unbranched malto-oligosaccharides. Molecular analysis establishes that STA4 encodes PhoB. Chlamydomonas reinhardtii strains carrying mutations at the STA4 locus display a significant decrease in amounts of starch during storage that correlates with the accumulation of abnormally shaped granules containing a modified amylopectin structure and a high amylose content. The wild-type phenotype could be rescued by reintroduction of the cloned wild-type genomic DNA, thereby demonstrating the involvement of phosphorylase in storage starch synthesis.}, language = {en} }