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 - 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 - Fettke, Jörg A1 - Eckermann, Nora A1 - Poeste, Simon A1 - Steup, Martin T1 - The glycan substrate of the cytosolic (Pho 2) phosphorylase isozyme from Pisum sativum L. : identification, linkage analysis and subcellular localization N2 - The subcellular distribution of starch-related enzymes and the phenotype of Arabidopsis mutants defective in starch degradation suggest that the plastidial starch turnover is linked to a cytosolic glycan metabolism. In this communication, a soluble heteroglycan (SHG) from leaves of Pisum sativum L. has been studied. Major constituents of the SHG are galactose, arabinose and glucose. For subcellular location, the SHG was prepared from isolated protoplasts and chloroplasts. On a chlorophyll basis, protoplasts and chloroplasts yielded approximately 70% and less than 5%, respectively, of the amount of the leaf-derived SHG preparation. Thus, most of SHG resides inside the cell but outside the chloroplast. SHG is soluble and not membrane-associated. Using membrane filtration, the SHG was separated into a <10 kDa and a >10 kDa fraction. The latter was resolved into two subfractions (I and II) by field-flow fractionation. In the protoplast-derived >10 kDa SHG preparation the subfraction I was by far the most dominant compound. beta-Glucosyl Yariv reagent was reactive with subfraction II, but not with subfraction I. In in vitro assays the latter acted as glucosyl acceptor for the cytosolic (Pho 2) phosphorylase but not for rabbit muscle phosphorylase. Glycosidic linkage analyses of subfractions I and II and of the Yariv reagent reactive glycans revealed that all three glycans contain a high percentage of arabinogalactan-like linkages. However, SHG possesses a higher content of minor compounds, namely glucosyl, mannosyl, rhamnosyl and fucosyl residues. Based on glycosyl residues and glycosidic linkages, subfraction I possesses a more complex structure than subfraction II Y1 - 2004 SN - 0960-7412 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 - 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 - 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 - 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 - Eckermann, Nora A1 - Fettke, Jörg A1 - Pauly, Markus A1 - Bazant, Esther A1 - Steup, Martin T1 - Starch-metabolism related isozymes in higher plants Y1 - 2004 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 - Comparot-Moss, Sylviane A1 - Koetting, Oliver A1 - Stettler, Michaela A1 - Edner, Christoph A1 - Graf, Alexander A1 - Weise, Sean E. A1 - Streb, Sebastian A1 - Lue, Wei-Ling A1 - MacLean, Daniel A1 - Mahlow, Sebastian A1 - Ritte, Gerhard A1 - Steup, Martin A1 - Chen, Jychian A1 - Zeeman, Samuel C. A1 - Smith, Alison M. T1 - A putative phosphatase, LSF1, is required for normal starch turnover in Arabidopsis leaves N2 - A putative phosphatase, LSF1 (for LIKE SEX4; previously PTPKIS2), is closely related in sequence and structure to STARCH-EXCESS4 (SEX4), an enzyme necessary for the removal of phosphate groups from starch polymers during starch degradation in Arabidopsis (Arabidopsis thaliana) leaves at night. We show that LSF1 is also required for starch degradation: lsf1 mutants, like sex4 mutants, have substantially more starch in their leaves than wild-type plants throughout the diurnal cycle. LSF1 is chloroplastic and is located on the surface of starch granules. lsf1 and sex4 mutants show similar, extensive changes relative to wild-type plants in the expression of sugar-sensitive genes. However, although LSF1 and SEX4 are probably both involved in the early stages of starch degradation, we show that LSF1 neither catalyzes the same reaction as SEX4 nor mediates a sequential step in the pathway. Evidence includes the contents and metabolism of phosphorylated glucans in the single mutants. The sex4 mutant accumulates soluble phospho- oligosaccharides undetectable in wild-type plants and is deficient in a starch granule-dephosphorylating activity present in wild-type plants. The lsf1 mutant displays neither of these phenotypes. The phenotype of the lsf1/sex4 double mutant also differs from that of both single mutants in several respects. We discuss the possible role of the LSF1 protein in starch degradation. Y1 - 2010 UR - http://www.plantphysiol.org/ U6 - https://doi.org/10.1104/pp.109.148981 SN - 0032-0889 ER - TY - JOUR A1 - Fettke, Jörg A1 - Hejazi, Mahdi A1 - Smirnova, Julia A1 - Hoechel, Erik A1 - Stage, Marion A1 - Steup, Martin T1 - Eukaryotic starch degradation : integration of plastidial and cytosolic pathways N2 - Starch is an important plant product widely used as a nutrient, as a source of renewable energy, and for many technological applications. In plants, starch is the almost ubiquitous storage carbohydrate whereas most heterotrophic prokaryotes and eukaryotes rely on glycogen. Despite close similarities in basic chemical features, starch and glycogen differ in both structural and physicochemical properties. Glycogen is a hydrosoluble macromolecule with evenly distributed branching points. Starch exists as a water-insoluble particle having a defined (and evolutionary conserved) internal structure. The biochemistry of starch requires the co-operation of up to 40 distinct (iso)enzymes whilst approximately 10 (iso)enzymes permit glycogen metabolism. The biosynthesis and degradation of native starch include the transition of carbohydrates from the soluble to the solid phase and vice versa. In this review, two novel aspects of the eukaryotic plastidial starch degradation are discussed: Firstly, biochemical reactions that take place at the surface of particulate glucans and mediate the phase transition of carbohydrates. Secondly, processes that occur downstream of the export of starch-derived sugars into the cytosol. Degradation of transitory starch mainly results in the formation of neutral sugars, such as glucose and maltose, that are transported into the cytosol via the respective translocators. The cytosolic metabolism of the neutral sugars includes the action of a hexokinase, a phosphoglucomutase, and a transglucosidase that utilizes high molecular weight glycans as a transient glucosyl acceptor or donor. Data are included on the transglucosidase (disproportionating isozyme 2) in Cyanophora paradoxa that accumulates storage carbohydrates in the cytosol rather than in the plastid. Y1 - 2009 UR - http://jxb.oxfordjournals.org/ U6 - https://doi.org/10.1093/Jxb/Erp054 SN - 0022-0957 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 -