TY - JOUR A1 - Yu, Tien-Shin A1 - Kofler, Heike A1 - Häusler, Rainer E. A1 - Hille, Diana A1 - Flügge, Ulf-Ingo A1 - Zeeman, Samuel C. A1 - Smith, Alison M. A1 - Kossmann, Jens A1 - Lloyd, James R. A1 - Ritte, Gerhard A1 - Steup, Martin A1 - Lue, Wei-Ling A1 - Chen, Jychian A1 - Weber, Andreas P. M. T1 - The Arabidopsis sex1 mutant is defective in the R1 protein, a general regulator of starch degradation in plants, and not in the chloroplast hexose transporter Y1 - 2001 SN - 1040-4651 ER - TY - JOUR A1 - Werner, Deljana A1 - Behrsing, Olaf A1 - Scharte, Gudrun A1 - Woller, Jochen A1 - Steup, Martin A1 - Micheel, Burkhard T1 - Monoclonal anti-diuron antibodies prevent inhibition of photosynthesis by diuron Y1 - 2002 ER - TY - JOUR A1 - Tenenboim, Hezi A1 - Smirnova, Julia A1 - Willmitzer, Lothar A1 - Steup, Martin A1 - Brotman, Yariv T1 - VMP1-deficient Chlamydomonas exhibits severely aberrant cell morphology and disrupted cytokinesies JF - BMC plant biology N2 - Background: The versatile Vacuole Membrane Protein 1 (VMP1) has been previously investigated in six species. It has been shown to be essential in macroautophagy, where it takes part in autophagy initiation. In addition, VMP1 has been implicated in organellar biogenesis; endo-, exo- and phagocytosis, and protein secretion; apoptosis; and cell adhesion. These roles underly its proven involvement in pancreatitis, diabetes and cancer in humans. Results: In this study we analyzed a VMP1 homologue from the green alga Chlamydomonas reinhardtii. CrVMP1 knockdown lines showed severe phenotypes, mainly affecting cell division as well as the morphology of cells and organelles. We also provide several pieces of evidence for its involvement in macroautophagy. KW - VMP1 KW - Autophagy KW - Cytokinesis Y1 - 2014 U6 - https://doi.org/10.1186/1471-2229-14-121 SN - 1471-2229 VL - 14 PB - BioMed Central CY - London ER - TY - JOUR A1 - Steup, Martin T1 - Raum und Zahl in der Pflanzenphysiologie JF - Raum und Zahl Y1 - 2015 SN - 978-3-86464-082-7 SP - 77 EP - 109 PB - Trafo CY - Berlin ER - TY - JOUR A1 - Steinfath, Matthias A1 - Strehmel, Nadine A1 - Peters, Rolf A1 - Schauer, Nicolas A1 - Groth, Detlef A1 - Hummel, Jan A1 - Steup, Martin A1 - Selbig, Joachim A1 - Kopka, Joachim A1 - Geigenberger, Peter A1 - Dongen, Joost T. van T1 - Discovering plant metabolic biomarkers for phenotype prediction using an untargeted approach N2 - Biomarkers are used to predict phenotypical properties before these features become apparent and, therefore, are valuable tools for both fundamental and applied research. Diagnostic biomarkers have been discovered in medicine many decades ago and are now commonly applied. While this is routine in the field of medicine, it is of surprise that in agriculture this approach has never been investigated. Up to now, the prediction of phenotypes in plants was based on growing plants and assaying the organs of interest in a time intensive process. For the first time, we demonstrate in this study the application of metabolomics to predict agronomic important phenotypes of a crop plant that was grown in different environments. Our procedure consists of established techniques to screen untargeted for a large amount of metabolites in parallel, in combination with machine learning methods. By using this combination of metabolomics and biomathematical tools metabolites were identified that can be used as biomarkers to improve the prediction of traits. The predictive metabolites can be selected and used subsequently to develop fast, targeted and low-cost diagnostic biomarker assays that can be implemented in breeding programs or quality assessment analysis. The identified metabolic biomarkers allow for the prediction of crop product quality. Furthermore, marker-assisted selection can benefit from the discovery of metabolic biomarkers when other molecular markers come to its limitation. The described marker selection method was developed for potato tubers, but is generally applicable to any crop and trait as it functions independently of genomic information. Y1 - 2010 UR - http://www3.interscience.wiley.com/cgi-bin/issn?DESCRIPTOR=PRINTISSN&VALUE=1467-7644 U6 - https://doi.org/10.1111/j.1467-7652.2010.00516.x SN - 1467-7644 ER - TY - JOUR A1 - Stahl, Bernd A1 - Thurl, Stephan A1 - Zeng, Jianru A1 - Karas, Michael A1 - Hillenkamp, Franz A1 - Steup, Martin A1 - Sawatzki, Günther T1 - Oligosaccharides from human milk as revealed by matrix-assisted laser desorption : ionization mass spectrometry Y1 - 1994 ER - TY - JOUR A1 - Stahl, Bernd A1 - Linos, Alexandros A1 - Karas, Michael A1 - Hillenkamp, Franz A1 - Steup, Martin T1 - Analysis of fructans from higher plants by matrix-assisted laser desorption/ionization mass spectrometry Y1 - 1997 ER - TY - JOUR A1 - Stahl, Bernd A1 - Klabunde, Thomas A1 - Witzel, Herbert A1 - Krebs, Bernt A1 - Steup, Martin A1 - Karas, Michael A1 - Hillenkamp, Franz T1 - The oligosaccharides of the Fe(III)-Zn(II) purple acid phosphatase of the red kidney bean : determination of the structure by a combination of matrix-assisted laser desorption-ionization mass spectrometry and selective enzymic degradation Y1 - 1994 ER - TY - JOUR A1 - Sonnewald, Uwe A1 - Basner, Astrid A1 - Greve, Burkhard A1 - Steup, Martin T1 - A second L-type isozyme of potato glucan phosphorylase : cloning, antisense inhibition and expression analysis Y1 - 1995 ER - TY - JOUR A1 - Smirnova, Julia A1 - Fernie, Alisdair R. A1 - Spahn, Christian M. T. A1 - Steup, Martin T1 - Photometric assay of maltose and maltose-forming enzyme activity by using 4-alpha-glucanotransferase (DPE2) from higher plants JF - Analytical biochemistry : methods in the biological sciences N2 - Maltose frequently occurs as intermediate of the central carbon metabolism of prokaryotic and eukaryotic cells. Various mutants possess elevated maltose levels. Maltose exists as two anomers, (alpha- and beta-form) which are rapidly interconverted without requiring enzyme-mediated catalysis. As maltose is often abundant together with other oligoglucans, selective quantification is essential. In this communication, we present a photometric maltose assay using 4-alpha-glucanotransferase (AtDPE2) from Arabidopsis thaliana. Under in vitro conditions, AtDPE2 utilizes maltose as glucosyl donor and glycogen as acceptor releasing the other hexosyl unit as free glucose which is photometrically quantified following enzymatic phosphorylation and oxidation. Under the conditions used, DPE2 does not noticeably react with other di- or oligosaccharides. Selectivity compares favorably with that of maltase frequently used in maltose assays. Reducing end interconversion of the two maltose anomers is in rapid equilibrium and, therefore, the novel assay measures total maltose contents. Furthermore, an AtDPE2-based continuous photometric assay is presented which allows to quantify beta-amylase activity and was found to be superior to a conventional test. Finally, the AtDPE2-based maltose assay was used to quantify leaf maltose contents of both Arabidopsis wild type and AtDPE2-deficient plants throughout the light-dark cycle. These data are presented together with assimilatory starch levels. (C) 2017 Published by Elsevier Inc. KW - Arabidopsis thaliana KW - beta-amylase assay KW - Disproportionating isozyme 2 (DPE2) dpe2-deficient plants KW - Maltose assay KW - Leaf maltose content Y1 - 2017 U6 - https://doi.org/10.1016/j.ab.2017.05.026 SN - 0003-2697 SN - 1096-0309 VL - 532 SP - 72 EP - 82 PB - Elsevier CY - San Diego ER - TY - JOUR A1 - Schwarte, Sandra A1 - Wegner, Fanny A1 - Havenstein, Katja A1 - Groth, Detlef A1 - Steup, Martin A1 - Tiedemann, Ralph T1 - Sequence variation, differential expression, and divergent evolution in starch-related genes among accessions of Arabidopsis thaliana JF - Plant molecular biology : an international journal of fundamental research and genetic engineering N2 - Transitory starch metabolism is a nonlinear and highly regulated process. It originated very early in the evolution of chloroplast-containing cells and is largely based on a mosaic of genes derived from either the eukaryotic host cell or the prokaryotic endosymbiont. Initially located in the cytoplasm, starch metabolism was rewired into plastids in Chloroplastida. Relocation was accompanied by gene duplications that occurred in most starch-related gene families and resulted in subfunctionalization of the respective gene products. Starch-related isozymes were then evolutionary conserved by constraints such as internal starch structure, posttranslational protein import into plastids and interactions with other starch-related proteins. 25 starch-related genes in 26 accessions of Arabidopsis thaliana were sequenced to assess intraspecific diversity, phylogenetic relationships, and modes of selection. Furthermore, sequences derived from additional 80 accessions that are publicly available were analyzed. Diversity varies significantly among the starch-related genes. Starch synthases and phosphorylases exhibit highest nucleotide diversities, while pyrophosphatases and debranching enzymes are most conserved. The gene trees are most compatible with a scenario of extensive recombination, perhaps in a Pleistocene refugium. Most genes are under purifying selection, but disruptive selection was inferred for a few genes/substitutiones. To study transcript levels, leaves were harvested throughout the light period. By quantifying the transcript levels and by analyzing the sequence of the respective accessions, we were able to estimate whether transcript levels are mainly determined by genetic (i.e., accession dependent) or physiological (i.e., time dependent) parameters. We also identified polymorphic sites that putatively affect pattern or the level of transcripts. KW - Arabidopsis thaliana KW - Divergent evolution KW - Intraspecific genetic variation KW - Positive selection KW - Starch metabolizing enzymes KW - Transcript levels Y1 - 2015 U6 - https://doi.org/10.1007/s11103-015-0293-2 SN - 0167-4412 SN - 1573-5028 VL - 87 IS - 4-5 SP - 489 EP - 519 PB - Springer CY - Dordrecht ER - TY - JOUR A1 - Schwarte, Sandra A1 - Brust, Henrike A1 - Steup, Martin A1 - Tiedemann, Ralph T1 - Intraspecific sequence variation and differential expression in starch synthase genes of Arabidopsis thaliana Y1 - 2013 UR - http://www.biomedcentral.com/content/pdf/1756-0500-6-84.pdf U6 - https://doi.org/10.1186/1756-0500-6-84 ER - TY - JOUR A1 - Schmieder, Peter A1 - Nitschke, Felix A1 - Steup, Martin A1 - Mallow, Keven A1 - Specker, Edgar T1 - Determination of glucan phosphorylation using heteronuclear H-1,C-13 double and H-1,C-13,P-31 triple-resonance NMR spectra JF - Magnetic resonance in chemistry N2 - Phosphorylation and dephosphorylation of starch and glycogen are important for their physicochemical properties and also their physiological functions. It is therefore desirable to reliably determine the phosphorylation sites. Heteronuclear multidimensional NMR-spectroscopy is in principle a straightforward analytical approach even for complex carbohydrate molecules. With heterogeneous samples from natural sources, however, the task becomes more difficult because a full assignment of the resonances of the carbohydrates is impossible to obtain. Here, we show that the combination of heteronuclear H-1,C-13 and H-1,C-13,P-31 techniques and information derived from spectra of a set of reference compounds can lead to an unambiguous determination of the phosphorylation sites even in heterogeneous samples. KW - heteronuclear NMR KW - triple resonance KW - phosphorylation KW - starch Y1 - 2013 U6 - https://doi.org/10.1002/mrc.3996 SN - 0749-1581 VL - 51 IS - 10 SP - 655 EP - 661 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Schmalzlin, E. A1 - van Dongen, J. T. A1 - Klimant, I. A1 - Marmodee, Bettina A1 - Steup, Martin A1 - Fisahn, Joachim A1 - Geigenberger, Peter Ludwig A1 - Löhmannsröben, Hans-Gerd T1 - An optical multifrequency phase-modulation method using microbeads for measuring intracellular oxygen concentrations in plants N2 - A technique has been developed to measure absolute intracellular oxygen concentrations in green plants. Oxygen- sensitive phosphorescent microbeads were injected into the cells and an optical multifrequency phase-modulation technique was used to discriminate the sensor signal from the strong auto fluorescence of the plant tissue. The method was established using photosynthesis- competent cells of the giant algae Chara corallina L., and was validated by application to various cell types of other plant species Y1 - 2005 SN - 0006-3495 ER - TY - JOUR A1 - Ruzanski, Christian A1 - Smirnova, Julia A1 - Rejzek, Martin A1 - Cockburn, Darrell A1 - Pedersen, Henriette L. A1 - Pike, Marilyn A1 - Willats, William G. T. A1 - Svensson, Birte A1 - Steup, Martin A1 - Ebenhöh, Oliver A1 - Smith, Alison M. A1 - Field, Robert A. T1 - A bacterial glucanotransferase can replace the complex maltose metabolism required for starch to sucrose conversion in leaves at night JF - The journal of biological chemistry N2 - Controlled conversion of leaf starch to sucrose at night is essential for the normal growth of Arabidopsis. The conversion involves the cytosolic metabolism of maltose to hexose phosphates via an unusual, multidomain protein with 4-glucanotransferase activity, DPE2, believed to transfer glucosyl moieties to a complex heteroglycan prior to their conversion to hexose phosphate via a cytosolic phosphorylase. The significance of this complex pathway is unclear; conversion of maltose to hexose phosphate in bacteria proceeds via a more typical 4-glucanotransferase that does not require a heteroglycan acceptor. It has recently been suggested that DPE2 generates a heterogeneous series of terminal glucan chains on the heteroglycan that acts as a glucosyl buffer to ensure a constant rate of sucrose synthesis in the leaf at night. Alternatively, DPE2 and/or the heteroglycan may have specific properties important for their function in the plant. To distinguish between these ideas, we compared the properties of DPE2 with those of the Escherichia coli glucanotransferase MalQ. We found that MalQ cannot use the plant heteroglycan as an acceptor for glucosyl transfer. However, experimental and modeling approaches suggested that it can potentially generate a glucosyl buffer between maltose and hexose phosphate because, unlike DPE2, it can generate polydisperse malto-oligosaccharides from maltose. Consistent with this suggestion, MalQ is capable of restoring an essentially wild-type phenotype when expressed in mutant Arabidopsis plants lacking DPE2. In light of these findings, we discuss the possible evolutionary origins of the complex DPE2-heteroglycan pathway. KW - Carbohydrate Metabolism KW - Computer Modeling KW - Metabolic Regulation KW - Oligosaccharide KW - Plant Biochemistry KW - Glucanotransferase KW - Leaf Cell KW - Maltose Metabolism KW - Starch Degradation Y1 - 2013 U6 - https://doi.org/10.1074/jbc.M113.497867 SN - 0021-9258 SN - 1083-351X VL - 288 IS - 40 SP - 28581 EP - 28598 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER - TY - JOUR A1 - Ritte, Gerhard A1 - Steup, Martin A1 - Kossmann, Jens A1 - Lloyd, James R. T1 - Determination of the starch phosphorylating enzyme activity in plant extracts Y1 - 2003 ER - TY - JOUR A1 - Ritte, Gerhard A1 - Scharf, Anke A1 - Eckermann, Nora A1 - Haebel, Sophie A1 - Steup, Martin T1 - Phosphorylation of transitory starch is increased during degradation N2 - 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 Y1 - 2004 ER - TY - JOUR A1 - Ritte, Gerhard A1 - Ruth, Lorberth A1 - Steup, Martin T1 - Reversible binding of the starch-related R1 protein to the surface of transitory starch granules Y1 - 2000 ER - TY - JOUR A1 - Ritte, Gerhard A1 - Lloyd, James R. A1 - Eckermann, Nora A1 - Rottmann, Antje A1 - Kossmann, Jens A1 - Steup, Martin T1 - The starch-related R1 protein is an a-glucan, water dikinase Y1 - 2002 SN - 0027-8424 ER - TY - JOUR A1 - Ritte, Gerhard A1 - Heydenreich, Matthias A1 - Mahlow, Sebastian A1 - Haebel, Sophie A1 - Koetting, Oliver A1 - Steup, Martin T1 - Phosphorylation of C6- and C3-positions of glucosyl residues in starch is catalysed by distinct dikinases JF - FEBS letters : the journal for rapid publication of short reports in molecular biosciences N2 - Glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) are required for normal starch metabolism. We analysed starch phosphorylation in Arabidopsis wildtype plants and mutants lacking either GWD or PWD using P-31 NMR. Phosphorylation at both C6- and C3-positions of glucose moieties in starch was drastically decreased in GWD-deficient mutants. In starch from PWD-deficient plants C3-bound phosphate was reduced to levels close to the detection limit. The latter result contrasts with previous reports according to which GWD phosphorylates both C6- and C3-positions. In these studies, phosphorylation had been analysed by HPLC of acid-hydrolysed glucans. We now show that maltose-6-phosphate, a product of incomplete starch hydrolysis, co-eluted with glucose-3-phosphate under the chromatographic conditions applied. Re-examination of the specificity of the dikinases using an improved method demonstrates that C6- and C3-phosphorylation is selectively catalysed by GWD and PWD, respectively. KW - starch phosphorylation KW - GWD KW - PWD KW - P-31 NMR Y1 - 2006 U6 - https://doi.org/10.1016/j.febslet.2006.07.085 SN - 0014-5793 VL - 580 IS - 20 SP - 4872 EP - 4876 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Ritte, Gerhard A1 - Eckermann, Nora A1 - Haebel, Sophie A1 - Lorberth, Ruth A1 - Steup, Martin T1 - Compartmentation of the starch-related R1 protein in higher plants Y1 - 2000 ER - TY - JOUR A1 - Reimann, Rezarta A1 - Ritte, Gerhard A1 - Steup, Martin A1 - Appenroth, Klaus-J. T1 - Association of a-amylase and the R1 protein with starch granules precedes the initiation of net starch degradation in turions of Spirodela polyrhiza Y1 - 2002 SN - 0031-9317 ER - TY - JOUR A1 - Rading, M. Michael A1 - Sandmann, Michael A1 - Steup, Martin A1 - Chiarugi, Davide A1 - Valleriani, Angelo T1 - Weak correlation of starch and volume in synchronized photosynthetic cells JF - Physical review : E, Statistical, nonlinear and soft matter physics N2 - In cultures of unicellular algae, features of single cells, such as cellular volume and starch content, are thought to be the result of carefully balanced growth and division processes. Single-cell analyses of synchronized photoautotrophic cultures of the unicellular alga Chlamydomonas reinhardtii reveal, however, that the cellular volume and starch content are only weakly correlated. Likewise, other cell parameters, e.g., the chlorophyll content per cell, are only weakly correlated with cell size. We derive the cell size distributions at the beginning of each synchronization cycle considering growth, timing of cell division and daughter cell release, and the uneven division of cell volume. Furthermore, we investigate the link between cell volume growth and starch accumulation. This work presents evidence that, under the experimental conditions of light-dark synchronized cultures, the weak correlation between both cell features is a result of a cumulative process rather than due to asymmetric partition of biomolecules during cell division. This cumulative process necessarily limits cellular similarities within a synchronized cell population. Y1 - 2015 U6 - https://doi.org/10.1103/PhysRevE.91.012711 SN - 1539-3755 SN - 1550-2376 VL - 91 IS - 1 PB - American Physical Society CY - College Park ER - TY - JOUR A1 - Paparelli, Eleonora A1 - Gonzali, Silvia A1 - Parlanti, Sandro A1 - Novi, Giacomo A1 - Giorgi, Federico M. A1 - Licausi, Francesco A1 - Kosmacz, Monika A1 - Feil, Regina A1 - Lunn, John Edward A1 - Brust, Henrike A1 - van Dongen, Joost T. A1 - Steup, Martin A1 - Perata, Pierdomenico T1 - Misexpression of a chloroplast aspartyl protease leads to severe growth defects and alters carbohydrate metabolism in arabidopsis JF - Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants N2 - The crucial role of carbohydrate in plant growth and morphogenesis is widely recognized. In this study, we describe the characterization of nana, a dwarf Arabidopsis (Arabidopsis thaliana) mutant impaired in carbohydrate metabolism. We show that the nana dwarf phenotype was accompanied by altered leaf morphology and a delayed flowering time. Our genetic and molecular data indicate that the mutation in nana is due to a transfer DNA insertion in the promoter region of a gene encoding a chloroplast-located aspartyl protease that alters its pattern of expression. Overexpression of the gene (oxNANA) phenocopies the mutation. Both nana and oxNANA display alterations in carbohydrate content, and the extent of these changes varies depending on growth light intensity. In particular, in low light, soluble sugar levels are lower and do not show the daily fluctuations observed in wild-type plants. Moreover, nana and oxNANA are defective in the expression of some genes implicated in sugar metabolism and photosynthetic light harvesting. Interestingly, some chloroplast-encoded genes as well as genes whose products seem to be involved in retrograde signaling appear to be down-regulated. These findings suggest that the NANA aspartic protease has an important regulatory function in chloroplasts that not only influences photosynthetic carbon metabolism but also plastid and nuclear gene expression. Y1 - 2012 U6 - https://doi.org/10.1104/pp.112.204016 SN - 0032-0889 VL - 160 IS - 3 SP - 1237 EP - 1250 PB - American Society of Plant Physiologists CY - Rockville ER - TY - JOUR A1 - Nitschke, Felix A1 - Wang, Peixiang A1 - Schmieder, Peter A1 - Girard, Jean-Marie A1 - Awrey, Donald E. A1 - Wang, Tony A1 - Israelian, Johan A1 - Zhao, XiaoChu A1 - Turnbull, Julie A1 - Heydenreich, Matthias A1 - Kleinpeter, Erich A1 - Steup, Martin A1 - Minassian, Berge A. T1 - Hyperphosphorylation of glucosyl C6 carbons and altered structure of glycogen in the neurodegenerative epilepsy lafora disease JF - Cell metabolism N2 - Laforin or malin deficiency causes Lafora disease, characterized by altered glycogen metabolism and teenage-onset neurodegeneration with intractable and invariably fatal epilepsy. Plant starches possess small amounts of metabolically essential monophosphate esters. Glycogen contains similar phosphate amounts, which are thought to originate from a glycogen synthase error side reaction and therefore lack any specific function. Glycogen is also believed to lack monophosphates at glucosyl carbon C6, an essential phosphorylation site in plant starch metabolism. We now show that glycogen phosphorylation is not due to a glycogen synthase side reaction, that C6 is a major glycogen phosphorylation site, and that C6 monophosphates predominate near centers of glycogen molecules and positively correlate with glycogen chain lengths. Laforin or malin deficiency causes C6 hyperphosphorylation, which results in malformed long-chained glycogen that accumulates in many tissues, causing neurodegeneration in brain. Our work advances the understanding of Lafora disease pathogenesis and suggests that glycogen phosphorylation has important metabolic function. Y1 - 2013 U6 - https://doi.org/10.1016/j.cmet.2013.04.006 SN - 1550-4131 SN - 1932-7420 VL - 17 IS - 5 SP - 756 EP - 767 PB - Cell Press CY - Cambridge ER - TY - JOUR A1 - Neuhaus, Heinz Eckhard A1 - Thom, E. A1 - Steup, Martin A1 - Kampfenkel, Karlheinz T1 - Characterization of a novel eukaryotic ATP/ADP translocator located in the plastid envelope of Arabidopsis thaliana L. Y1 - 1997 ER - TY - JOUR A1 - Nakamura, Yasunori A1 - Steup, Martin A1 - Colleoni, Christophe A1 - Iglesias, Alberto A. A1 - Bao, Jinsong A1 - Fujita, Naoko A1 - Tetlow, Ian T1 - Molecular regulation of starch metabolism JF - Plant molecular biology : an international journal of fundamental research and genetic engineering Y1 - 2022 U6 - https://doi.org/10.1007/s11103-022-01253-0 SN - 0167-4412 SN - 1573-5028 VL - 108 IS - 4-5 SP - 289 EP - 290 PB - Springer CY - Dordrecht ER - TY - JOUR A1 - Nakamura, Yasunori A1 - Ono, Masami A1 - Utsumi, Chikako A1 - Steup, Martin T1 - Functional interaction between plastidial starch phosphorylase and starch branching enzymes from rice during the synthesis of branched maltodextrins JF - Plant & cell physiology N2 - The present study established the way in which plastidial alpha-glucan phosphorylase (Pho1) synthesizes maltodextrin (MD) which can be the primer for starch biosynthesis in rice endosperm. The synthesis of MD by Pho1 was markedly accelerated by branching enzyme (BE) isozymes, although the greatest effect was exhibited by the presence of branching isozyme I (BEI) rather than by isozyme IIa (BEIIa) or isozyme IIb (BEIIb). The enhancement of the activity of Pho1 by BE was not merely due to the supply of a non-reducing ends. At the same time, Pho1 greatly enhanced the BE activity, possibly by generating a branched carbohydrate substrate which is used by BE with a higher affinity. The addition of isoamylase to the reaction mixture did not prevent the concerted action of Pho1 and BEI. Furthermore, in the product, the branched structure was, at least to some extent, maintained. Based on these results we propose that the interaction between Pho1 and BE is not merely due to chain-elongating and chain-branching reactions, but occurs in a physically and catalytically synergistic manner by each activating the mutual capacity of the other, presumably forming a physical association of Pho1, BEI and branched MDs. This close interaction might play a crucial role in the synthesis of branched MDs and the branched MDs can act as a primer for the biosynthesis of amylopectin molecules. KW - Amylopectin KW - Glucan KW - Phosphorylase KW - Rice KW - Starch KW - Starch branching enzyme Y1 - 2012 U6 - https://doi.org/10.1093/pcp/pcs030 SN - 0032-0781 VL - 53 IS - 5 SP - 869 EP - 878 PB - Oxford Univ. Press CY - Oxford ER - TY - JOUR A1 - Nakamura, Yasunori A1 - Ono, Masami A1 - Sawada, Takayuki A1 - Crofts, Naoko A1 - Fujita, Naoko A1 - Steup, Martin T1 - Characterization of the functional interactions of plastidial starch phosphorylase and starch branching enzymes from rice endosperm during reserve starch biosynthesis JF - Plant science : an international journal of experimental plant biology N2 - Functional interactions of plastidial phosphorylase (Phol) and starch branching enzymes (BEs) from the developing rice endosperm are the focus of this study. In the presence of both Phol and BE, the same branched primer molecule is elongated and further branched almost simultaneously even at very low glucan concentrations present in the purified enzyme preparations. By contrast, in the absence of any BE, glucans are not, to any significant extent, elongated by Phol. Based on our in vitro data, in the developing rice endosperm, Phol appears to be weakly associated with any of the BE isozymes. By using fluorophore-labeled malto-oligosaccharides, we identified maltose as the smallest possible primer for elongation by Phol. Linear dextrins act as carbohydrate substrates for BEs. By functionally interacting with a BE, Phol performs two essential functions during the initiation of starch biosynthesis in the rice endosperm: First, it elongates maltodextrins up to a degree of polymerization of at least 60. Second, by closely interacting with BEs, Phol is able to elongate branched glucans efficiently and thereby synthesizes branched carbohydrates essential for the initiation of amylopectin biosynthesis. Y1 - 2017 U6 - https://doi.org/10.1016/j.plantsci.2017.09.002 SN - 0168-9452 VL - 264 SP - 83 EP - 95 PB - Elsevier CY - Clare ER - TY - JOUR A1 - Martins, Marina Camara Mattos A1 - Hejazi, Mahdi A1 - Fettke, Jörg A1 - Steup, Martin A1 - Feil, Regina A1 - Krause, Ursula A1 - Arrivault, Stephanie A1 - Vosloh, Daniel A1 - Figueroa, Carlos Maria A1 - Ivakov, Alexander A1 - Yadav, Umesh Prasad A1 - Piques, Maria A1 - Metzner, Daniela A1 - Stitt, Mark A1 - Lunn, John Edward T1 - Feedback inhibition of starch degradation in arabidopsis leaves mediated by trehalose 6-phosphate JF - Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants N2 - Many plants accumulate substantial starch reserves in their leaves during the day and remobilize them at night to provide carbon and energy for maintenance and growth. In this paper, we explore the role of a sugar-signaling metabolite, trehalose-6-phosphate (Tre6P), in regulating the accumulation and turnover of transitory starch in Arabidopsis (Arabidopsis thaliana) leaves. Ethanol-induced overexpression of trehalose-phosphate synthase during the day increased Tre6P levels up to 11-fold. There was a transient increase in the rate of starch accumulation in the middle of the day, but this was not linked to reductive activation of ADP-glucose pyrophosphorylase. A 2- to 3-fold increase in Tre6P during the night led to significant inhibition of starch degradation. Maltose and maltotriose did not accumulate, suggesting that Tre6P affects an early step in the pathway of starch degradation in the chloroplasts. Starch granules isolated from induced plants had a higher orthophosphate content than granules from noninduced control plants, consistent either with disruption of the phosphorylation-dephosphorylation cycle that is essential for efficient starch breakdown or with inhibition of starch hydrolysis by beta-amylase. Nonaqueous fractionation of leaves showed that Tre6P is predominantly located in the cytosol, with estimated in vivo Tre6P concentrations of 4 to 7 mu M in the cytosol, 0.2 to 0.5 mu M in the chloroplasts, and 0.05 mu M in the vacuole. It is proposed that Tre6P is a component in a signaling pathway that mediates the feedback regulation of starch breakdown by sucrose, potentially linking starch turnover to demand for sucrose by growing sink organs at night. Y1 - 2013 U6 - https://doi.org/10.1104/pp.113.226787 SN - 0032-0889 SN - 1532-2548 VL - 163 IS - 3 SP - 1142 EP - 1163 PB - American Society of Plant Physiologists CY - Rockville ER - TY - JOUR A1 - Malinova, Irina A1 - Steup, Martin A1 - Fettke, Jörg T1 - Starch-related cytosolic heteroglycans in roots from Arabidopsis thaliana JF - Journal of plant physiology : biochemistry, physiology, molecular biology and biotechnology of plants N2 - Both photoautotrophic and heterotrophic plant cells are capable of accumulating starch inside the plastid. However, depending on the metabolic state of the respective cell the starch-related carbon fluxes are different. The vast majority of the transitory starch biosynthesis relies on the hexose phosphate pools derived from the reductive pentose phosphate cycle and, therefore, is restricted to ongoing photosynthesis. Transitory starch is usually degraded in the subsequent dark period and mainly results in the formation of neutral sugars, such as glucose and maltose, that both are exported into the cytosol. The cytosolic metabolism of the two carbohydrates includes reversible glucosyl transfer reactions to a heteroglycan that are mediated by two glucosyl transferases. DPE2 and PHS2 (or, in all other species, Pho2). In heterotrophic cells, accumulation of starch mostly depends on the long distance transport of reduced carbon compounds from source to sink organs and, therefore, includes as an essential step the import of carbohydrates from the cytosol into the starch forming plastids. In this communication, we focus on starch metabolism in heterotrophic tissues from Arabidopsis thaliana wild type plants (and in various starch-related mutants as well). By using hydroponically grown A. thaliana plants, we were able to analyse starch-related biochemical processes in leaves and roots from the same plants. Within the roots we determined starch levels and the morphology of native starch granules. Cytosolic and apoplastic heteroglycans were analysed in roots and compared with those from leaves of the same plants. A. thaliana mutants lacking functional enzymes either inside the plastid (such as phosphoglucomutase) or in the cytosol (disproportionating isoenzyme 2 or the phosphorylase isozyme, PHS2) were included in this study. In roots and leaves from the three mutants (and from the respective wild type organ as well), starch and heteroglycans as well as enzyme patterns were analysed. KW - Cytosolic heteroglycans KW - Cytosolic glucosyl transferases KW - Photoautotrophic tissues KW - Heterotrophic tissues KW - Starch metabolism Y1 - 2011 U6 - https://doi.org/10.1016/j.jplph.2010.12.008 SN - 0176-1617 VL - 168 IS - 12 SP - 1406 EP - 1414 PB - Elsevier CY - Jena ER - TY - JOUR A1 - Malinova, Irina A1 - Steup, Martin A1 - Fettke, Jörg T1 - Carbon transitions from either Calvin cycle or transitory starch to heteroglycans as revealed by 14-C-labeling experiments using protoplasts from Arabidopsis JF - Physiologia plantarum N2 - Plants metabolize transitory starch by precisely coordinated plastidial and cytosolic processes. The latter appear to include the action of water-soluble heteroglycans (SHG(in)) whose monosaccharide pattern is similar to that of apoplastic glycans (SHG(ex)) but, unlike SHG(ex), SHG(in) strongly interacts with glucosyl transferases. In this study, we analyzed starch metabolism using mesophyll protoplasts from wild-type plants and two knock-out mutants [deficient in the cytosolic transglucosidase, disproportionating isoenzyme 2 (DPE2) or the plastidial phosphoglucomutase (PGM1)] from Arabidopsis thaliana. Protoplasts prelabeled by photosynthetic (CO2)-C-14 fixation were transferred to an unlabeled medium and were darkened or illuminated. Carbon transitions from the Calvin cycle or from starch to both SHG(in) and SHG(ex) were analyzed. In illuminated protoplasts, starch turn-over was undetectable but darkened protoplasts continuously degraded starch. During illumination, neither the total C-14 content nor the labeling patterns of the sugar residues of SHG(in) were significantly altered but both the total amount and the labeling of the constituents of SHG(ex) increased with time. In darkened protoplasts, the C-14-content of most of the sugar residues of SHG(in) transiently and strongly increased and then declined. This effect was not observed in any SHG(ex) constituent. In darkened DPE2-deficient protoplasts, none of the SHG(in) constituents exhibited an essential transient increase in labeling. In contrast, some residues of SHG(in) from the PGM1 mutant exhibited a transient increase in label but this effect significantly differed from that of the wild type. Two conclusions are reached: first, SHG(in) and SHG(ex) exert different metabolic functions and second, SHG(in) is directly involved in starch degradation. Y1 - 2013 U6 - https://doi.org/10.1111/ppl.12033 SN - 0031-9317 VL - 149 IS - 1 SP - 25 EP - 44 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Malinova, Irina A1 - Mahlow, Sebastian A1 - Alseekh, Saleh A1 - Orawetz, Tom A1 - Fernie, Alisdair R. A1 - Baumann, Otto A1 - Steup, Martin A1 - Fettke, Jörg T1 - Double knockout mutants of arabidopsis grown under normal conditions reveal that the plastidial phosphorylase isozyme participates in transitory starch metabolism JF - Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants N2 - In leaves of two starch-related single-knockout lines lacking either the cytosolic transglucosidase (also designated as disproportionating enzyme 2, DPE2) or the maltose transporter (MEX1), the activity of the plastidial phosphorylase isozyme (PHS1) is increased. In both mutants, metabolism of starch-derived maltose is impaired but inhibition is effective at different subcellular sites. Two constitutive double knockout mutants were generated (designated as dpe2-1 x phs1a and mex1 x phs1b) both lacking functional PHS1. They reveal that in normally grown plants, the plastidial phosphorylase isozyme participates in transitory starch degradation and that the central carbon metabolism is closely integrated into the entire cell biology. All plants were grown either under continuous illumination or in a light-dark regime. Both double mutants were compromised in growth and, compared with the single knockout plants, possess less average leaf starch when grown in a light-dark regime. Starch and chlorophyll contents decline with leaf age. As revealed by transmission electron microscopy, mesophyll cells degrade chloroplasts, but degradation is not observed in plants grown under continuous illumination. The two double mutants possess similar but not identical phenotypes. When grown in a light-dark regime, mesophyll chloroplasts of dpe2-1 x phs1a contain a single starch granule but under continuous illumination more granules per chloroplast are formed. The other double mutant synthesizes more granules under either growth condition. In continuous light, growth of both double mutants is similar to that of the parental single knockout lines. Metabolite profiles and oligoglucan patterns differ largely in the two double mutants. Y1 - 2014 U6 - https://doi.org/10.1104/pp.113.227843 SN - 0032-0889 SN - 1532-2548 VL - 164 IS - 2 SP - 907 EP - 921 PB - American Society of Plant Physiologists CY - Rockville ER - TY - JOUR A1 - Li, Jing A1 - Francisco, Perigio A1 - Zhou, Wenxu A1 - Edner, Christoph A1 - Steup, Martin A1 - Ritte, Gerhard A1 - Bond, Charles S. A1 - Smith, Steven M. T1 - Catalytically-inactive beta-amylase BAM4 required for starch breakdown in Arabidopsis leaves is a starch- binding-protein N2 - Of the four chloroplast beta-amylase (BAM) proteins identified in Arabidopsis, BAM3 and BAM4 were previously shown to play the major roles in leaf starch breakdown, although BAM4 apparently lacks key active site residues and beta- amylase activity. Here we tested multiple BAM4 proteins with different N-terminal sequences with a range of glucan substrates and assay methods, but detected no alpha-1,4-glucan hydrolase activity. BAM4 did not affect BAM1, BAM2 or BAM3 activity even when added in 10-fold excess, nor the BAM3-catalysed release of maltose from isolated starch granules in the presence of glucan water dikinase. However, BAM4 binds to amylopectin and to amylose-Sepharose whereas BAM2 has very low beta-amylase activity and poor glucan binding. The low activity of BAM2 may be explained by poor glucan binding but absence of BAM4 activity is not. These results suggest that BAM4 facilitates starch breakdown by a mechanism involving direct interaction with starch or other alpha-1,4-glucan. Y1 - 2009 UR - http://www.sciencedirect.com/science/journal/00039861 U6 - https://doi.org/10.1016/j.abb.2009.07.024 SN - 0003-9861 ER - TY - JOUR A1 - Kötting, Oliver A1 - Pusch, Kerstin A1 - Tiessen, Axel A1 - Geigenberger, Peter Ludwig A1 - Steup, Martin A1 - Ritte, Gerhard T1 - Identification of a novel enzyme required for starch metabolism in Arabidopsis leaves : the phosphoglucan, water dikinase N2 - 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 Y1 - 2005 ER - TY - JOUR A1 - Kreft, Oliver A1 - Georgieva, Radostina A1 - Bäumler, Hans A1 - Steup, Martin A1 - Müller-Röber, Bernd A1 - Sukhorukov, Gleb B. A1 - Möhwald, Helmuth T1 - Red blood cell templated polyelectrolyte capsules : a novel vehicle for the stable encapsulation of DNA and proteins N2 - 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 Y1 - 2006 UR - http://www3.interscience.wiley.com/cgi-bin/jhome/10003270 U6 - https://doi.org/10.1002/marc.200500777 SN - 1022-1336 ER - TY - JOUR A1 - Koetting, Oliver A1 - Santelia, Diana A1 - Edner, Christoph A1 - Eicke, Simona A1 - Marthaler, Tina A1 - Gentry, Matthew S. A1 - Comparot-Moss, Sylviane A1 - Chen, Jychian A1 - Smith, Alison M. A1 - Steup, Martin A1 - Ritte, Gerhard A1 - Zeeman, Samuel C. T1 - STARCH-EXCESS4 is a laforin-like phosphoglucan phosphatase required for starch degradation in Arabidopsis thaliana N2 - Starch is the major storage carbohydrate in plants. It is comprised of glucans that form semicrystalline granules. Glucan phosphorylation is a prerequisite for normal starch breakdown, but phosphoglucan metabolism is not understood. A putative protein phosphatase encoded at the Starch Excess 4 (SEX4) locus of Arabidopsis thaliana was recently shown to be required for normal starch breakdown. Here, we show that SEX4 is a phosphoglucan phosphatase in vivo and define its role within the starch degradation pathway. SEX4 dephosphorylates both the starch granule surface and soluble phosphoglucans in vitro, and sex4 null mutants accumulate phosphorylated intermediates of starch breakdown. These compounds are linear alpha-1,4-glucans esterified with one or two phosphate groups. They are released from starch granules by the glucan hydrolases alpha-amylase and isoamylase. In vitro experiments show that the rate of starch granule degradation is increased upon simultaneous phosphorylation and dephosphorylation of starch. We propose that glucan phosphorylating enzymes and phosphoglucan phosphatases work in synergy with glucan hydrolases to mediate efficient starch catabolism. Y1 - 2009 UR - http://www.plantcell.org/ U6 - https://doi.org/10.1105/tpc.108.064360 SN - 1040-4651 ER - TY - JOUR A1 - Kehr, Julia A1 - Haebel, Sophie A1 - Blechschmidt-Schneider, Sabine A1 - Willmitzer, Lothar A1 - Steup, Martin A1 - Fisahn, Joachim T1 - Analysis of phloem protein patterns from different organs of Cucurbita maxima Duch. by matrix-assisted laser desorption/ionization time of flight mass spectroscopy combined with sodium dodecyl sufate-polyacryilamide gel electrophoresis Y1 - 1999 ER - TY - JOUR A1 - Jüppner, Jessica A1 - Mubeen, Umarah A1 - Leisse, Andrea A1 - Caldana, Camila A1 - Brust, Henrike A1 - Steup, Martin A1 - Herrmann, Marion A1 - Steinhauser, Dirk A1 - Giavalisco, Patrick T1 - Dynamics of lipids and metabolites during the cell cycle of Chlamydomonas reinhardtii JF - The plant journal N2 - Metabolites and lipids are the final products of enzymatic processes, distinguishing the different cellular functions and activities of single cells or whole tissues. Understanding these cellular functions within a well-established model system requires a systemic collection of molecular and physiological information. In the current report, the green alga Chlamydomonas reinhardtii was selected to establish a comprehensive workflow for the detailed multi-omics analysis of a synchronously growing cell culture system. After implementation and benchmarking of the synchronous cell culture, a two-phase extraction method was adopted for the analysis of proteins, lipids, metabolites and starch from a single sample aliquot of as little as 10-15million Chlamydomonas cells. In a proof of concept study, primary metabolites and lipids were sampled throughout the diurnal cell cycle. The results of these time-resolved measurements showed that single compounds were not only coordinated with each other in different pathways, but that these complex metabolic signatures have the potential to be used as biomarkers of various cellular processes. Taken together, the developed workflow, including the synchronized growth of the photoautotrophic cell culture, in combination with comprehensive extraction methods and detailed metabolic phenotyping has the potential for use in in-depth analysis of complex cellular processes, providing essential information for the understanding of complex biological systems. KW - Chlamydomonas reinhardtii KW - synchronized cell cultures KW - photoautotrophic growth KW - cell cycle KW - metabolomics KW - lipidomics KW - systems biology KW - two-phase extraction KW - diurnal cycle KW - technical advance Y1 - 2017 U6 - https://doi.org/10.1111/tpj.13642 SN - 0960-7412 SN - 1365-313X VL - 92 SP - 331 EP - 343 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Hemme, Dorothea A1 - Veyel, Daniel A1 - Muehlhaus, Timo A1 - Sommer, Frederik A1 - Jueppner, Jessica A1 - Unger, Ann-Katrin A1 - Sandmann, Michael A1 - Fehrle, Ines A1 - Schoenfelder, Stephanie A1 - Steup, Martin A1 - Geimer, Stefan A1 - Kopka, Joachim A1 - Giavalisco, Patrick A1 - Schroda, Michael T1 - Systems-wide analysis of acclimation responses to long-term heat stress and recovery in the photosynthetic model organism Chlamydomonas reinhardtii JF - The plant cell N2 - We applied a top-down systems biology approach to understand how Chlamydomonas reinhardtii acclimates to long-term heat stress (HS) and recovers from it. For this, we shifted cells from 25 to 42 degrees C for 24 h and back to 25 degrees C for >= 8 h and monitored abundances of 1856 proteins/protein groups, 99 polar and 185 lipophilic metabolites, and cytological and photosynthesis parameters. Our data indicate that acclimation of Chlamydomonas to long-term HS consists of a temporally ordered, orchestrated implementation of response elements at various system levels. These comprise (1) cell cycle arrest; (2) catabolism of larger molecules to generate compounds with roles in stress protection; (3) accumulation of molecular chaperones to restore protein homeostasis together with compatible solutes; (4) redirection of photosynthetic energy and reducing power from the Calvin cycle to the de novo synthesis of saturated fatty acids to replace polyunsaturated ones in membrane lipids, which are deposited in lipid bodies; and (5) when sinks for photosynthetic energy and reducing power are depleted, resumption of Calvin cycle activity associated with increased photorespiration, accumulation of reactive oxygen species scavengers, and throttling of linear electron flow by antenna uncoupling. During recovery from HS, cells appear to focus on processes allowing rapid resumption of growth rather than restoring pre-HS conditions. Y1 - 2014 U6 - https://doi.org/10.1105/tpc.114.130997 SN - 1040-4651 SN - 1532-298X VL - 26 IS - 11 SP - 4270 EP - 4297 PB - American Society of Plant Physiologists CY - Rockville ER - TY - JOUR A1 - Hejazi, Mahdi A1 - Steup, Martin A1 - Fettke, Jörg T1 - The plastidial glucan, water dikinase (GWD) catalyses multiple phosphotransfer reactions JF - The FEBS journal N2 - The plant genome encodes at least two distinct and evolutionary conserved plastidial starch-related dikinases that phosphorylate a low percentage of glucosyl residues at the starch granule surface. Esterification of starch favours the transition of highly ordered a-glucans to a less ordered state and thereby facilitates the cleavage of interglucose bonds by hydrolases. Metabolically most important is the phosphorylation at position C6, which is catalysed by the glucan, water dikinase (GWD). The reactions mediated by recombinant wild-type GWD from Arabidopsis thaliana (AtGWD) and from Solanum tuberosum (StGWD) were studied. Two mutated proteins lacking the conserved histidine residue that is indispensible for glucan phosphorylation were also included. The wild-type GWDs consume approximately 20% more ATP than is required for glucan phosphorylation. Similarly, although incapable of phosphorylating a-glucans, the two mutated dikinase proteins are capable of degrading ATP. Thus, consumption of ATP and phosphorylation of a-glucans are not strictly coupled processes but, to some extent, occur as independent phosphotransfer reactions. As revealed by incubation of the GWDs with [gamma-33P]ATP, the consumption of ATP includes the transfer of the gamma-phosphate group to the GWD protein but this autophosphorylation does not require the conserved histidine residue. Thus, the GWD proteins possess two vicinal phosphorylation sites, both of which are transiently phosphorylated. Following autophosphorylation at both sites, native dikinases flexibly use various terminal phosphate acceptors, such as water, alpha-glucans, AMP and ADP. A model is presented describing the complex phosphotransfer reactions of GWDs as affected by the availability of the various acceptors. KW - glucan phosphorylation KW - glucan KW - water dikinase KW - protein autophosphorylation KW - starch metabolism KW - starch phosphorylation Y1 - 2012 U6 - https://doi.org/10.1111/j.1742-4658.2012.08576.x SN - 1742-464X VL - 279 IS - 11 SP - 1953 EP - 1966 PB - Wiley-Blackwell CY - Malden ER - TY - JOUR A1 - Hejazi, Mahdi A1 - Fettke, Jörg A1 - Paris, Oskar A1 - Steup, Martin T1 - The two plastidial starch-related dikinases sequentially phosphorylate glucosyl residues at the surface of both the a- and b-type allomorphs of crystallized maltodextrins but the mode of action differs N2 - In this study, two crystallized maltodextrins were generated that consist of the same oligoglucan pattern but differ strikingly in the physical order of double helices. As revealed by x-ray diffraction, they represent the highly ordered A- and B-type allomorphs. Both crystallized maltodextrins were similar in size distribution and birefringence. They were used as model substrates to study the consecutive action of the two starch-related dikinases, the glucan, water dikinase and the phosphoglucan, water dikinase. The glucan, water dikinase and the phosphoglucan, water dikinase selectively esterify glucosyl residues in the C6 and C3 positions, respectively. Recombinant glucan, water dikinase phosphorylated both allomorphs with similar rates and caused complete glucan solubilization. Soluble neutral maltodextrins inhibited the glucan, water dikinase-mediated phosphorylation of crystalline particles. Recombinant phosphoglucan, water dikinase phosphorylated both the A- and B-type allomorphs only following a prephosphorylation by the glucan, water dikinase, and the activity increased with the extent of prephosphorylation. The action of the phosphoglucan, water dikinase on the prephosphorylated A- and B-type allomorphs differed. When acting on the B-type allomorph, by far more phosphoglucans were solubilized as compared with the A type. However, with both allomorphs, the phosphoglucan, water dikinase formed significant amounts of mono-phosphorylated phosphoglucans. Thus, the enzyme is capable of acting on neutral maltodextrins. It is concluded that the actual carbohydrate substrate of the phosphoglucan, water dikinase is defined by physical rather than by chemical parameters. A model is proposed that explains, at the molecular level, the consecutive action of the two starch-related dikinases. Y1 - 2009 UR - http://www.plantphysiol.org/ U6 - https://doi.org/10.1104/pp.109.138750 SN - 0032-0889 ER - TY - JOUR A1 - Hejazi, Mahdi A1 - Fettke, Jörg A1 - Koetting, Oliver A1 - Zeeman, Samuel C. A1 - Steup, Martin T1 - The Laforin-like dual-specificity phosphatase SEX4 from Arabidopsis hydrolyzes both C6-and C3-phosphate esters introduced by starch-related dikinases and thereby affects phase transition of alpha-glucans N2 - The biochemical function of the Laforin-like dual-specific phosphatase AtSEX4 (EC 3.1.3.48) has been studied. Crystalline maltodextrins representing the A- or the B-type allomorph were prephosphorylated using recombinant glucan, water dikinase (StGWD) or the successive action of both plastidial dikinases (StGWD and AtPWD). AtSEX4 hydrolyzed carbon 6-phosphate esters from both the prephosphorylated A- and B-type allomorphs and the kinetic constants are similar. The phosphatase also acted on prelabeled carbon-3 esters from both crystalline maltodextrins. Similarly, native starch granules prelabeled in either the carbon-6 or carbon-3 position were also dephosphorylated by AtSEX4. The phosphatase did also hydrolyze phosphate esters of both prephosphorylated maltodextrins when the (phospho)glucans had been solubilized by heat treatment. Submillimolar concentrations of nonphosphorylated maltodextrins inhibited AtSEX4 provided they possessed a minimum of length and had been solubilized. As opposed to the soluble phosphomaltodextrins, the AtSEX4- mediated dephosphorylation of the insoluble substrates was incomplete and at least 50% of the phosphate esters were retained in the pelletable (phospho) glucans. The partial dephosphorylation of the insoluble glucans also strongly reduced the release of nonphosphorylated chains into solution. Presumably, this effect reflects fast structural changes that following dephosphorylation occur near the surface of the maltodextrin particles. A model is proposed defining distinct stages within the phosphorylation/dephosphorylation-dependent transition of alpha-glucans from the insoluble to the soluble state. Y1 - 2010 UR - http://www.plantphysiol.org/ U6 - https://doi.org/10.1104/pp.109.149914 SN - 0032-0889 ER - TY - JOUR A1 - Haebel, Sophie A1 - Albrecht, Tanja A1 - Sparbier, Katrin A1 - Walden, Peter A1 - Körner, Roman A1 - Steup, Martin T1 - Electrophoresis-related protein modification: alkylation of carboxy residues revealed by mass spectrometry Y1 - 1998 ER - TY - BOOK A1 - Günther, Oliver A1 - Petsche, Hans-Joachim A1 - Fischer, Martin H. A1 - Franz, Norbert P. A1 - Steup, Martin A1 - Sixtus, Elena A1 - Heimann, Heinz-Dieter A1 - Pröve, Ralf ED - Petsche, Hans-Joachim T1 - Raum und Zahl im Fokus der Wissenschaften BT - eine multidisziplinäre Vorlesungsreihe T3 - Studieren ++ : Konzepte, Perspektiven, Kompetenzen ; 1 N2 - 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. KW - Raum KW - Zahl KW - Aufsatzsammlung Y1 - 2015 SN - 978-3-86464-082-7 PB - Trafo CY - Berlin ER - TY - JOUR A1 - Grunwaldt, Gisela A1 - Haebel, Sophie A1 - Spitz, Christian A1 - Steup, Martin A1 - Menzel, Ralf T1 - Multiple binding sites of fluorescein isothiocyanate moieties on myoglobin : photophysical heterogeneity as revealed by ground- and excited-state spectroscopy Y1 - 2002 SN - 1011-1344 ER - TY - JOUR A1 - Garz, Andreas A1 - Sandmann, Michael A1 - Rading, Michael A1 - Ramm, Sascha A1 - Menzel, Ralf A1 - Steup, Martin T1 - Cell-to-cell diversity in a synchronized chlamydomonas culture as revealed by single-cell analyses JF - Biophysical journal N2 - In a synchronized photoautotrophic culture of Chlamydomonas reinhardtii, cell size, cell number, and the averaged starch content were determined throughout the light-dark cycle. For single-cell analyses, the relative cellular starch was quantified by measuring the second harmonic generation (SHG). In destained cells, amylopectin essentially represents the only biophotonic structure. As revealed by various validation procedures, SHG signal intensities are a reliable relative measure of the cellular starch content. During photosynthesis-driven starch biosynthesis, synchronized Chlamydomonas cells possess an unexpected cell-to-cell diversity both in size and starch content, but the starch-related heterogeneity largely exceeds that of size. The cellular volume, starch content, and amount of starch/cell volume obey lognormal distributions. Starch degradation was initiated by inhibiting the photosynthetic electron transport in illuminated cells or by darkening. Under both conditions, the averaged rate of starch degradation is almost constant, but it is higher in illuminated than in darkened cells. At the single-cell level, rates of starch degradation largely differ but are unrelated to the initial cellular starch content. A rate equation describing the cellular starch degradation Y1 - 2012 U6 - https://doi.org/10.1016/j.bpj.2012.07.026 SN - 0006-3495 VL - 103 IS - 5 SP - 1078 EP - 1086 PB - Cell Press CY - Cambridge ER - TY - JOUR A1 - Fettke, Jörg A1 - Poeste, Simon A1 - Eckermann, Nora A1 - Tiessen, Axel A1 - Pauly, Markus A1 - Geigenberger, Peter Ludwig A1 - Steup, Martin T1 - Analysis of cytosolic heteroglycans from leaves of transgenic potato (Solanum tuberosum L.) plants that under- or overexpress the Pho 2 phosphorylase isozyme N2 - 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 Y1 - 2005 ER - TY - JOUR A1 - Fettke, Jörg A1 - Nunes-Nesi, Adriano A1 - Fernie, Alisdair R. A1 - Steup, Martin T1 - Identification of a novel heteroglycan-interacting protein, HIP 1.3, from Arabidopsis thaliana JF - Journal of plant physiology : biochemistry, physiology, molecular biology and biotechnology of plants N2 - Plastidial degradation of transitory starch yields mainly maltose and glucose. Following the export into the cytosol, maltose acts as donor for a glucosyl transfer to cytosolic heteroglycans as mediated by a cytosolic transglucosidase (DPE2; EC 2.4.1.25) and the second glucosyl residue is liberated as glucose. The cytosolic phosphorylase (Pho2/PHS2; EC 2.4.1.1) also interacts with heteroglycans using the same intramolecular sites as DPE2. Thus, the two glucosyl transferases interconnect the cytosolic pools of glucose and glucose 1-phosphate. Due to the complex monosaccharide pattern, other heteroglycan-interacting proteins (Hips) are expected to exist. Identification of those proteins was approached by using two types of affinity chromatography. Heteroglycans from leaves of Arabidopsis thaliana (Col-0) covalently bound to Sepharose served as ligands that were reacted with a complex mixture of buffer-soluble proteins from Arabidopsis leaves. Binding proteins were eluted by sodium chloride. For identification, SDS-PAGE, tryptic digestion and MALDI-TOF analyses were applied. A strongly interacting polypeptide (approximately 40 kDa; designated as HIP1.3) was observed as product of locus At1g09340. Arabidopsis mutants deficient in HIP1.3 were reduced in growth and contained heteroglycans displaying an altered monosaccharide pattern. Wild type plants express HIP1.3 most strongly in leaves. As revealed by immuno fluorescence, HIP1.3 is located in the cytosol of mesophyll cells but mostly associated with the cytosolic surface of the chloroplast envelope membranes. In an HIP1.3-deficient mutant the immunosignal was undetectable. Metabolic profiles from leaves of this mutant and wild type plants as well were determined by GC-MS. As compared to the wild type control, more than ten metabolites, such as ascorbic acid, fructose, fructose bisphosphate, glucose, glycine, were elevated in darkness but decreased in the light. Although the biochemical function of HIP1.3 has not yet been elucidated, it is likely to possess an important function in the central carbon metabolism of higher plants. KW - Arabidopsis thaliana KW - Carbohydrate binding proteins KW - Cytosolic heteroglycans KW - Maltose metabolism KW - Starch metabolism Y1 - 2011 U6 - https://doi.org/10.1016/j.jplph.2010.09.008 SN - 0176-1617 VL - 168 IS - 12 SP - 1415 EP - 1425 PB - Elsevier CY - Jena ER - TY - JOUR A1 - Fettke, Jörg A1 - Malinova, Irina A1 - Eckermann, Nora A1 - Steup, Martin T1 - Cytosolic heteroglycans in photoautotrophic and in heterotrophic plant cells N2 - 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. Y1 - 2009 UR - http://www.sciencedirect.com/science/journal/00319422 U6 - https://doi.org/10.1016/j.phytochem.2009.03.016 SN - 0031-9422 ER -