TY - JOUR A1 - Albrecht, Tanja A1 - Greve, Burkhard A1 - Pusch, Kerstin A1 - Koßmann, Jens A1 - Buchner, Peter A1 - Wobus, Ulrich A1 - Steup, Martin T1 - Homo- and Heterodimers of Pho1-Type Phosphorylase Isoforms in Solanum tuberosum L. as Revealed by Sequence- Specific Antibodies Y1 - 1998 ER - TY - JOUR A1 - Albrecht, Tanja A1 - Haebel, Sophie A1 - Koch, Anke A1 - Krause, Ulrike A1 - Eckermann, Nora A1 - Steup, Martin T1 - Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosin residues but results in a reduced bacterial glycogen accumulation N2 - Saccharomyces cerevisiae possesses two glycogenin isoforms (designated as Glg1p and Glg2p) that both contain a conserved tyrosine residue, Tyr232. However, Glg2p possesses an additional tyrosine residue, Tyr230 and therefore two potential autoglucosylation sites. Glucosylation of Glg2p was studied using both matrix-assisted laser desorption ionization and electrospray quadrupole time of flight mass spectrometry. Glg2p, carrying a C-terminal (His(6)) tag, was produced in Escherichia coli and purified. By tryptic digestion and reversed phase chromatography a peptide (residues 219-246 of the complete Glg2p sequence) was isolated that contained 4-25 glucosyl residues. Following incubation of Glg2p with UDPglucose, more than 36 glucosyl residues were covalently bound to this peptide. Using a combination of cyanogen bromide cleavage of the protein backbone, enzymatic hydrolysis of glycosidic bonds and reversed phase chromatography, mono- and diglucosylated peptides having the sequence PNYGYQSSPAM were generated. MS/MS spectra revealed that glucosyl residues were attached to both Tyr232 and Tyr230 within the same peptide. The formation of the highly glucosylated eukaryotic Glg2p did not favour the bacterial glycogen accumulation. Under various experimental conditions Glg2p-producing cells accumulated approximately 30% less glycogen than a control transformed with a Glg2p lacking plasmid. The size distribution of the glycogen and extractable activities of several glycogen-related enzymes were essentially unchanged. As revealed by high performance anion exchange chromatography, the intracellular maltooligosaccharide pattern of the bacterial cells expressing the functional eukaryotic transgene was significantly altered. Thus, the eukaryotic glycogenin appears to be incompatible with the bacterial initiation of glycogen biosynthesis Y1 - 2004 ER - TY - JOUR A1 - Albrecht, Tanja A1 - Koch, Anke A1 - Lode, Anja A1 - Greve, Burkhard A1 - Schneider-Mergener, Jens A1 - Steup, Martin T1 - Plastidic (Pho1-type) phosphorylase isoforms in potato (Solanum tuberosum L.) plants : expression analysis and immunochemical characterization Y1 - 2001 ER - TY - JOUR A1 - Ball, Steven G. A1 - Liénard, Luc A1 - Wattebled, Fabrice A1 - Steup, Martin A1 - Hicks, Glenn A1 - d'Hulst, Christophe T1 - Defining the functions of maltodextrin active enzymes in starch metabolism in the unicellular alga Chlamydomonas reinhardtii Y1 - 2003 ER - TY - JOUR A1 - Cencil, Ugo A1 - Nitschke, Felix A1 - Steup, Martin A1 - Minassian, Berge A. A1 - Colleoni, Christophe A1 - Ball, Steven G. T1 - Transition from glycogen to starch metabolism in Archaeplastida JF - Trends in plant science N2 - In this opinion article we propose a scenario detailing how two crucial components have evolved simultaneously to ensure the transition of glycogen to starch in the cytosol of the Archaeplastida last common ancestor: (i) the recruitment of an enzyme from intracellular Chlamydiae pathogens to facilitate crystallization of alpha-glucan chains; and (ii) the evolution of novel types of polysaccharide (de)phosphorylating enzymes from preexisting glycogen (de)phosphorylation host pathways to allow the turnover of such crystals. We speculate that the transition to starch benefitted Archaeplastida in three ways: more carbon could be packed into osmotically inert material; the host could resume control of carbon assimilation from the chlamydial pathogen that triggered plastid endosymbiosis; and cyanobacterial photosynthate export could be integrated in the emerging Archaeplastida. KW - evolution of plastids KW - starch and glycogen metabolism KW - polyglucan debranching reactions KW - starch and glycogen (de)phosphorylation KW - Chlamydia-like bacteria KW - Lafora disease Y1 - 2014 U6 - https://doi.org/10.1016/j.tplants.2013.08.004 SN - 1360-1385 VL - 19 IS - 1 SP - 18 EP - 28 PB - Elsevier CY - London ER - TY - JOUR A1 - Cisek, Richard A1 - Tokarz, Danielle A1 - Kontenis, Lukas A1 - Barzda, Virginijus A1 - Steup, Martin T1 - Polarimetric second harmonic generation microscopy BT - an analytical tool for starch bioengineering JF - Starch-Starke N2 - Second harmonic generation (SHG) is a nonlinear optical process that inherently generates signal in non-centrosymmetric materials, such as starch granules, and therefore can be used for label-free imaging. Both intensity and polarization of SHG are determined by material properties that are characterized by the nonlinear susceptibility tensor, ((2)). Examination of the tensor is performed for each focal volume of the image by measuring the outgoing polarization state of the SHG signal for a set of incoming laser beam polarizations. Mapping of nonlinear properties expressed as the susceptibility ratio reveals structural features including the organization of crystalline material within a single starch granule, and the distribution of structural properties in a population of granules. Isolated granules, as well as in situ starch, can be analyzed using polarimetric SHG microscopy. Due to the fast sample preparation and short imaging times, polarimetric SHG microscopy allows for a quick assessment of starch structure and permits rapid feedback for bioengineering applications. This article presents the basics of SHG theory and microscopy applications for starch-containing materials. Quantification of ultrastructural features within individual starch granules is described. New results obtained by polarization resolved SHG microscopy of starch granules are presented for various maize genotypes revealing heterogeneity within a single starch particle and between various granules. KW - Determination of crystallinity KW - Determination of hydration KW - Label-free imaging KW - Nonlinear optical microscopy KW - Structural determination Y1 - 2017 U6 - https://doi.org/10.1002/star.201700031 SN - 0038-9056 SN - 1521-379X VL - 70 IS - 1-2 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Cisek, Richard A1 - Tokarz, Danielle A1 - Krouglov, Serguei A1 - Steup, Martin A1 - Emes, Michael J. A1 - Tetlow, Ian J. A1 - Barzda, Virginijus T1 - Second harmonic generation mediated by aligned water in starch granules JF - The journal of physical chemistry : B, Condensed matter, materials, surfaces, interfaces & biophysical chemistry N2 - The origin of second harmonic generation (SHG) in starch granules was investigated using ab initio quantum mechanical modeling and experimentally examined using polarization-in, polarization-out (PIPO) second harmonic generation microscopy. Ab initio calculations revealed that the largest contribution to the SHG signal from A- and B-type allomorphs of starch originates from the anisotropic organization of hydroxide and hydrogen bonds mediated by aligned water found in the polymers. The hypothesis was experimentally tested by imaging maize starch granules under various hydration and heat treatment conditions that alter the hydrogen bond network. The highest SHG intensity was found in fully hydrated starch granules, and heat treatment diminished the SHG intensity. The PIPO SHG imaging showed that dried starch granules have a much higher nonlinear optical susceptibility component ratio than fully hydrated granules. In contrast, deuterated starch granules showed a smaller susceptibility component ratio demonstrating that SHG is highly sensitive to the organization of the hydroxyl and hydrogen bond network. The polarization SHG imaging results of potato starch granules, representing starch allomorph B, were compared to those of maize starch granules representing allomorph A. The results showed that the amount of aligned water was higher in the maize granules. Nonlinear microscopy of starch granules provides evidence that varying hydration conditions leads to significant changes in the nonlinear susceptibility ratio as well as the SHG intensity, supporting the hypothesis from ab initio calculations that the dominant contribution to SHG is due to the ordered hydroxide and hydrogen bond network. Y1 - 2014 U6 - https://doi.org/10.1021/jp508751s SN - 1520-6106 VL - 118 IS - 51 SP - 14785 EP - 14794 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Cisek, Richard A1 - Tokarz, Danielle A1 - Steup, Martin A1 - Tetlow, Ian J. A1 - Emes, Michael J. A1 - Hebelstrup, Kim H. A1 - Blennow, Andreas A1 - Barzda, Virginijus T1 - Second harmonic generation microscopy investigation of the crystalline ultrastructure of three barley starch lines affected by hydration JF - Biomedical optics express N2 - Second harmonic generation (SHG) microscopy is employed to study changes in crystalline organization due to altered gene expression and hydration in barley starch granules. SHG intensity and susceptibility ratio values (R'(SHG)) are obtained using reduced Stokes-Mueller polarimetric microscopy. The maximum R'(SHG) values occur at moderate moisture indicating the narrowest orientation distribution of nonlinear dipoles from the cylindrical axis of glucan helices. The maximum SHG intensity occurs at the highest moisture and amylopectin content. These results support the hypothesis that SHG is caused by ordered hydrogen and hydroxyl bond networks which increase with hydration of starch granules. (C) 2015 Optical Society of America Y1 - 2015 U6 - https://doi.org/10.1364/BOE.6.003694 SN - 2156-7085 VL - 6 IS - 10 SP - 3694 EP - 3700 PB - Optical Society of America CY - Washington 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 - Dauvillee, David A1 - Chochois, Vincent A1 - Steup, Martin A1 - Haebel, Sophie A1 - Eckermann, Nora A1 - Ritte, Gerhard A1 - Ral, Jean-Philippe A1 - Colleoni, Christophe A1 - Hicks, Glenn A1 - Wattebled, Fabrice A1 - Deschamps, Philippe A1 - Lienard, Luc A1 - Cournac, Laurent A1 - Putaux, Jean-Luc A1 - Dupeyre, Danielle A1 - Ball, Steven G. T1 - Plastidial phosphorylase is required for normal starch synthesis in Chlamydomonas reinhardtii JF - The plant journal N2 - Among the three distinct starch phosphorylase activities detected in Chlamydomonas reinhardtii, two distinct plastidial enzymes (PhoA and PhoB) are documented while a single extraplastidial form (PhoC) displays a higher affinity for glycogen as in vascular plants. The two plastidial phosphorylases are shown to function as homodimers containing two 91-kDa (PhoA) subunits and two 110-kDa (PhoB) subunits. Both lack the typical 80-amino-acid insertion found in the higher plant plastidial forms. PhoB is exquisitely sensitive to inhibition by ADP-glucose and has a low affinity for malto-oligosaccharides. PhoA is more similar to the higher plant plastidial phosphorylases: it is moderately sensitive to ADP-glucose inhibition and has a high affinity for unbranched malto-oligosaccharides. Molecular analysis establishes that STA4 encodes PhoB. Chlamydomonas reinhardtii strains carrying mutations at the STA4 locus display a significant decrease in amounts of starch during storage that correlates with the accumulation of abnormally shaped granules containing a modified amylopectin structure and a high amylose content. The wild-type phenotype could be rescued by reintroduction of the cloned wild-type genomic DNA, thereby demonstrating the involvement of phosphorylase in storage starch synthesis. KW - Chlamydomonas KW - starch KW - amylopectin KW - (glycogen) starch phosphorylase Y1 - 2006 U6 - https://doi.org/10.1111/j.1365-313X.2006.02870.x SN - 0960-7412 VL - 48 IS - 2 SP - 274 EP - 285 PB - Blackwell CY - Oxford ER -