@inproceedings{EbenhoehKartalSkupinetal.2013, author = {Ebenh{\"o}h, O. and Kartal, Oe. and Skupin, A. and Mahlow, S. and Steup, Martin}, title = {The role of mixing entropy in carbohydrate metabolism}, series = {European biophysics journal : with biophysics letters ; an international journal of biophysics}, volume = {42}, booktitle = {European biophysics journal : with biophysics letters ; an international journal of biophysics}, publisher = {Springer}, address = {New York}, issn = {0175-7571}, pages = {S73 -- S73}, year = {2013}, language = {en} } @article{SchwarteWegnerHavensteinetal.2015, author = {Schwarte, Sandra and Wegner, Fanny and Havenstein, Katja and Groth, Detlef and Steup, Martin and Tiedemann, Ralph}, title = {Sequence variation, differential expression, and divergent evolution in starch-related genes among accessions of Arabidopsis thaliana}, series = {Plant molecular biology : an international journal of fundamental research and genetic engineering}, volume = {87}, journal = {Plant molecular biology : an international journal of fundamental research and genetic engineering}, number = {4-5}, publisher = {Springer}, address = {Dordrecht}, issn = {0167-4412}, doi = {10.1007/s11103-015-0293-2}, pages = {489 -- 519}, year = {2015}, abstract = {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.}, language = {en} } @article{RadingSandmannSteupetal.2015, author = {Rading, M. Michael and Sandmann, Michael and Steup, Martin and Chiarugi, Davide and Valleriani, Angelo}, title = {Weak correlation of starch and volume in synchronized photosynthetic cells}, series = {Physical review : E, Statistical, nonlinear and soft matter physics}, volume = {91}, journal = {Physical review : E, Statistical, nonlinear and soft matter physics}, number = {1}, publisher = {American Physical Society}, address = {College Park}, issn = {1539-3755}, doi = {10.1103/PhysRevE.91.012711}, pages = {11}, year = {2015}, abstract = {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.}, language = {en} } @misc{CencilNitschkeSteupetal.2014, author = {Cencil, Ugo and Nitschke, Felix and Steup, Martin and Minassian, Berge A. and Colleoni, Christophe and Ball, Steven G.}, title = {Transition from glycogen to starch metabolism in Archaeplastida}, series = {Trends in plant science}, volume = {19}, journal = {Trends in plant science}, number = {1}, publisher = {Elsevier}, address = {London}, issn = {1360-1385}, doi = {10.1016/j.tplants.2013.08.004}, pages = {18 -- 28}, year = {2014}, abstract = {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.}, language = {en} } @article{TenenboimSmirnovaWillmitzeretal.2014, author = {Tenenboim, Hezi and Smirnova, Julia and Willmitzer, Lothar and Steup, Martin and Brotman, Yariv}, title = {VMP1-deficient Chlamydomonas exhibits severely aberrant cell morphology and disrupted cytokinesies}, series = {BMC plant biology}, volume = {14}, journal = {BMC plant biology}, publisher = {BioMed Central}, address = {London}, issn = {1471-2229}, doi = {10.1186/1471-2229-14-121}, pages = {13}, year = {2014}, abstract = {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.}, language = {en} } @article{CisekTokarzSteupetal.2015, author = {Cisek, Richard and Tokarz, Danielle and Steup, Martin and Tetlow, Ian J. and Emes, Michael J. and Hebelstrup, Kim H. and Blennow, Andreas and Barzda, Virginijus}, title = {Second harmonic generation microscopy investigation of the crystalline ultrastructure of three barley starch lines affected by hydration}, series = {Biomedical optics express}, volume = {6}, journal = {Biomedical optics express}, number = {10}, publisher = {Optical Society of America}, address = {Washington}, issn = {2156-7085}, doi = {10.1364/BOE.6.003694}, pages = {3694 -- 3700}, year = {2015}, abstract = {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}, language = {en} } @misc{CisekTokarzKontenisetal.2018, author = {Cisek, Richard and Tokarz, Danielle and Kontenis, Lukas and Barzda, Virginijus and Steup, Martin}, title = {Polarimetric second harmonic generation microscopy}, series = {Starch-Starke}, volume = {70}, journal = {Starch-Starke}, number = {1-2}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {0038-9056}, doi = {10.1002/star.201700031}, pages = {15}, year = {2018}, abstract = {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.}, language = {en} } @misc{SullivanNitschkeSteupetal.2017, author = {Sullivan, Mitchell A. and Nitschke, Silvia and Steup, Martin and Minassian, Berge A. and Nitschke, Felix}, title = {Pathogenesis of Lafora disease}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {1080}, issn = {1866-8372}, doi = {10.25932/publishup-47462}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-474622}, pages = {18}, year = {2017}, abstract = {Lafora disease (LD, OMIM \#254780) is a rare, recessively inherited neurodegenerative disease with adolescent onset, resulting in progressive myoclonus epilepsy which is fatal usually within ten years of symptom onset. The disease is caused by loss-of-function mutations in either of the two genes EPM2A (laforin) or EPM2B (malin). It characteristically involves the accumulation of insoluble glycogen-derived particles, named Lafora bodies (LBs), which are considered neurotoxic and causative of the disease. The pathogenesis of LD is therefore centred on the question of how insoluble LBs emerge from soluble glycogen. Recent data clearly show that an abnormal glycogen chain length distribution, but neither hyperphosphorylation nor impairment of general autophagy, strictly correlates with glycogen accumulation and the presence of LBs. This review summarizes results obtained with patients, mouse models, and cell lines and consolidates apparent paradoxes in the LD literature. Based on the growing body of evidence, it proposes that LD is predominantly caused by an impairment in chain-length regulation affecting only a small proportion of the cellular glycogen. A better grasp of LD pathogenesis will further develop our understanding of glycogen metabolism and structure. It will also facilitate the development of clinical interventions that appropriately target the underlying cause of LD.}, language = {en} } @misc{SchwarteBrustSteupetal.2013, author = {Schwarte, Sandra and Brust, Henrike and Steup, Martin and Tiedemann, Ralph}, title = {Intraspecific sequence variation and differential expression in starch synthase genes of Arabidopsis thaliana}, series = {BMC Research Notes}, journal = {BMC Research Notes}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-401128}, pages = {14}, year = {2013}, abstract = {Background Natural accessions of Arabidopsis thaliana are a well-known system to measure levels of intraspecific genetic variation. Leaf starch content correlates negatively with biomass. Starch is synthesized by the coordinated action of many (iso)enzymes. Quantitatively dominant is the repetitive transfer of glucosyl residues to the non-reducing ends of α-glucans as mediated by starch synthases. In the genome of A. thaliana, there are five classes of starch synthases, designated as soluble starch synthases (SSI, SSII, SSIII, and SSIV) and granule-bound synthase (GBSS). Each class is represented by a single gene. The five genes are homologous in functional domains due to their common origin, but have evolved individual features as well. Here, we analyze the extent of genetic variation in these fundamental protein classes as well as possible functional implications on transcript and protein levels. Findings Intraspecific sequence variation of the five starch synthases was determined by sequencing the entire loci including promoter regions from 30 worldwide distributed accessions of A. thaliana. In all genes, a considerable number of nucleotide polymorphisms was observed, both in non-coding and coding regions, and several amino acid substitutions were identified in functional domains. Furthermore, promoters possess numerous polymorphisms in potentially regulatory cis-acting regions. By realtime experiments performed with selected accessions, we demonstrate that DNA sequence divergence correlates with significant differences in transcript levels. Conclusions Except for AtSSII, all starch synthase classes clustered into two or three groups of haplotypes, respectively. Significant difference in transcript levels among haplotype clusters in AtSSIV provides evidence for cis-regulation. By contrast, no such correlation was found for AtSSI, AtSSII, AtSSIII, and AtGBSS, suggesting trans-regulation. The expression data presented here point to a regulation by common trans-regulatory transcription factors which ensures a coordinated action of the products of these four genes during starch granule biosynthesis. The apparent cis-regulation of AtSSIV might be related to its role in the initiation of de novo biosynthesis of granules.}, language = {en} } @article{AlbrechtHaebelKochetal.2004, author = {Albrecht, Tanja and Haebel, Sophie and Koch, Anke and Krause, Ulrike and Eckermann, Nora and Steup, Martin}, title = {Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosin residues but results in a reduced bacterial glycogen accumulation}, year = {2004}, abstract = {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}, language = {en} } @article{DeschampsHaferkampDauvilleeetal.2006, author = {Deschamps, Philippe and Haferkamp, Ilka and Dauvillee, David and Haebel, Sophie and Steup, Martin and Buleon, Alain and Putaux, Jean-Luc and Colleoni, Christophe and d'Hulst, Christophe and Plancke, Charlotte and Gould, Sven and Maier, Uwe and Neuhaus, Heinz Eckhard and Ball, Steven G.}, title = {Nature of the periplastidial pathway of starch synthesis in the cryptophyte Guillardia theta}, issn = {1535-9778}, doi = {10.1128/Ec.00380-05}, year = {2006}, abstract = {The nature of the periplastidial pathway of starch biosynthesis was investigated with the model cryptophyte Guillardia theta. The storage polysaccharide granules were shown to be composed of both amylose and amylopectin fractions with a chain length distribution and crystalline organization very similar to those of starch from green algae and land plants. Most starch granules displayed a shape consistent with biosynthesis occurring around the pyrenoid through the rhodoplast membranes. A protein with significant similarity to the amylose-synthesizing granule-bound starch syntbase 1 from green plants was found as the major polypeptide bound to the polysaccharide matrix. N-terminal sequencing of the mature protein proved that the precursor protein carries a nonfunctional transit peptide in its bipartite topogenic signal sequence which is cleaved without yielding transport of the enzyme across the two inner plastid membranes. The enzyme was shown to display similar affinities for ADP and UDP-glucose, while the V-max measured with UDP-glucose was twofold higher. The granule-bound starch synthase from Guillardia theta was demonstrated to be responsible for the synthesis of long glucan chains and therefore to be the functional equivalent of the amylose- synthesizing enzyme of green plants. Preliminary characterization of the starch pathway suggests that Guillardia theta utilizes a UDP-glucose-based pathway to synthesize starch}, language = {en} } @article{FettkeChiaEckermannetal.2006, author = {Fettke, J{\"o}rg and Chia, Tansy and Eckermann, Nora and Smith, Alison M. and Steup, Martin}, title = {A transglucosidase necessary for starch degradation and maltose metabolism in leaves at night acts on cytosolic heteroglycans (SHG)}, issn = {0960-7412}, doi = {10.1111/j.1365-313X.2006.02732.x}, year = {2006}, abstract = {The recently characterized cytosolic transglucosidase DPE2 (EC 2.4.1.25) is essential for the cytosolic metabolism of maltose, an intermediate on the pathway by which starch is converted to sucrose at night. In in vitro assays, the enzyme utilizes glycogen as a glucosyl acceptor but the in vivo acceptor molecules remained unknown. In this communication we present evidence that DPE2 acts on the recently identified cytosolic water-soluble heteroglycans (SHG) as does the cytosolic phosphorylase (EC 2.4.1.1) isoform. By using in vitro two-step C-14 labeling assays we demonstrate that the two transferases can utilize the same acceptor sites of the SHG. Cytosolic heteroglycans from a DPE2-deficient Arabidopsis mutant were characterized. Compared with the wild type the glucose content of the heteroglycans was increased. Most of the additional glucosyl residues were found in the outer chains of SHG that are released by an endo- alpha-arabinanase (EC 3.2.1.99). Additional starch-related mutants were characterized for further analysis of the increased glucosyl content. Based on these data, the cytosolic metabolism of starch-derived carbohydrates is discussed}, language = {en} } @article{KreftGeorgievaBaeumleretal.2006, author = {Kreft, Oliver and Georgieva, Radostina and B{\"a}umler, Hans and Steup, Martin and M{\"u}ller-R{\"o}ber, Bernd and Sukhorukov, Gleb B. and M{\"o}hwald, Helmuth}, title = {Red blood cell templated polyelectrolyte capsules : a novel vehicle for the stable encapsulation of DNA and proteins}, issn = {1022-1336}, doi = {10.1002/marc.200500777}, year = {2006}, abstract = {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}, language = {en} } @article{NitschkeWangSchmiederetal.2013, author = {Nitschke, Felix and Wang, Peixiang and Schmieder, Peter and Girard, Jean-Marie and Awrey, Donald E. and Wang, Tony and Israelian, Johan and Zhao, XiaoChu and Turnbull, Julie and Heydenreich, Matthias and Kleinpeter, Erich and Steup, Martin and Minassian, Berge A.}, title = {Hyperphosphorylation of glucosyl C6 carbons and altered structure of glycogen in the neurodegenerative epilepsy lafora disease}, series = {Cell metabolism}, volume = {17}, journal = {Cell metabolism}, number = {5}, publisher = {Cell Press}, address = {Cambridge}, issn = {1550-4131}, doi = {10.1016/j.cmet.2013.04.006}, pages = {756 -- 767}, year = {2013}, abstract = {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.}, language = {en} } @article{SmirnovaFernieSpahnetal.2017, author = {Smirnova, Julia and Fernie, Alisdair and Spahn, Christian M. T. and Steup, Martin}, title = {Photometric assay of maltose and maltose-forming enzyme activity by using 4-alpha-glucanotransferase (DPE2) from higher plants}, series = {Analytical biochemistry : methods in the biological sciences}, volume = {532}, journal = {Analytical biochemistry : methods in the biological sciences}, publisher = {Elsevier}, address = {San Diego}, issn = {0003-2697}, doi = {10.1016/j.ab.2017.05.026}, pages = {72 -- 82}, year = {2017}, abstract = {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.}, language = {en} } @article{FettkeNunesNesiFernieetal.2011, author = {Fettke, J{\"o}rg and Nunes-Nesi, Adriano and Fernie, Alisdair and Steup, Martin}, title = {Identification of a novel heteroglycan-interacting protein, HIP 1.3, from Arabidopsis thaliana}, series = {Journal of plant physiology : biochemistry, physiology, molecular biology and biotechnology of plants}, volume = {168}, journal = {Journal of plant physiology : biochemistry, physiology, molecular biology and biotechnology of plants}, number = {12}, publisher = {Elsevier}, address = {Jena}, issn = {0176-1617}, doi = {10.1016/j.jplph.2010.09.008}, pages = {1415 -- 1425}, year = {2011}, abstract = {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.}, language = {en} } @article{MalinovaMahlowAlseekhetal.2014, author = {Malinova, Irina and Mahlow, Sebastian and Alseekh, Saleh and Orawetz, Tom and Fernie, Alisdair and Baumann, Otto and Steup, Martin and Fettke, J{\"o}rg}, title = {Double knockout mutants of arabidopsis grown under normal conditions reveal that the plastidial phosphorylase isozyme participates in transitory starch metabolism}, series = {Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants}, volume = {164}, journal = {Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants}, number = {2}, publisher = {American Society of Plant Physiologists}, address = {Rockville}, issn = {0032-0889}, doi = {10.1104/pp.113.227843}, pages = {907 -- 921}, year = {2014}, abstract = {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.}, language = {en} }