@article{FettkeLeifelsBrustetal.2012, author = {Fettke, J{\"o}rg and Leifels, Lydia and Brust, Henrike and Herbst, Karoline and Steup, Martin}, title = {Two carbon fluxes to reserve starch in potato (Solanum tuberosum L.) tuber cells are closely interconnected but differently modulated by temperature}, series = {Journal of experimental botany}, volume = {63}, journal = {Journal of experimental botany}, number = {8}, publisher = {Oxford Univ. Press}, address = {Oxford}, issn = {0022-0957}, doi = {10.1093/jxb/ers014}, pages = {3011 -- 3029}, year = {2012}, abstract = {Parenchyma cells from tubers of Solanum tuberosum L. convert several externally supplied sugars to starch but the rates vary largely. Conversion of glucose 1-phosphate to starch is exceptionally efficient. In this communication, tuber slices were incubated with either of four solutions containing equimolar [U-C-14]glucose 1-phosphate, [U-C-14]sucrose, [U-C-14]glucose 1-phosphate plus unlabelled equimolar sucrose or [U-C-14]sucrose plus unlabelled equimolar glucose 1-phosphate. C-14-incorporation into starch was monitored. In slices from freshly harvested tubers each unlabelled compound strongly enhanced C-14 incorporation into starch indicating closely interacting paths of starch biosynthesis. However, enhancement disappeared when the tubers were stored. The two paths (and, consequently, the mutual enhancement effect) differ in temperature dependence. At lower temperatures, the glucose 1-phosphate-dependent path is functional, reaching maximal activity at approximately 20 degrees C but the flux of the sucrose-dependent route strongly increases above 20 degrees C. Results are confirmed by in vitro experiments using [U-C-14]glucose 1-phosphate or adenosine-[U-C-14]glucose and by quantitative zymograms of starch synthase or phosphorylase activity. In mutants almost completely lacking the plastidial phosphorylase isozyme(s), the glucose 1-phosphate-dependent path is largely impeded. Irrespective of the size of the granules, glucose 1-phosphate-dependent incorporation per granule surface area is essentially equal. Furthermore, within the granules no preference of distinct glucosyl acceptor sites was detectable. Thus, the path is integrated into the entire granule biosynthesis. In vitro C-14-incorporation into starch granules mediated by the recombinant plastidial phosphorylase isozyme clearly differed from the in situ results. Taken together, the data clearly demonstrate that two closely but flexibly interacting general paths of starch biosynthesis are functional in potato tuber cells.}, language = {en} } @article{HejaziFettkeParisetal.2009, author = {Hejazi, Mahdi and Fettke, J{\"o}rg and Paris, Oskar and Steup, Martin}, title = {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}, issn = {0032-0889}, doi = {10.1104/pp.109.138750}, year = {2009}, abstract = {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.}, language = {en} } @article{HejaziSteupFettke2012, author = {Hejazi, Mahdi and Steup, Martin and Fettke, J{\"o}rg}, title = {The plastidial glucan, water dikinase (GWD) catalyses multiple phosphotransfer reactions}, series = {The FEBS journal}, volume = {279}, journal = {The FEBS journal}, number = {11}, publisher = {Wiley-Blackwell}, address = {Malden}, issn = {1742-464X}, doi = {10.1111/j.1742-4658.2012.08576.x}, pages = {1953 -- 1966}, year = {2012}, abstract = {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.}, language = {en} } @article{HejaziFettkeKoettingetal.2010, author = {Hejazi, Mahdi and Fettke, J{\"o}rg and Koetting, Oliver and Zeeman, Samuel C. and Steup, Martin}, title = {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}, issn = {0032-0889}, doi = {10.1104/pp.109.149914}, year = {2010}, abstract = {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.}, language = {en} } @article{FettkeEckermannPoesteetal.2004, author = {Fettke, J{\"o}rg and Eckermann, Nora and Poeste, Simon and Steup, Martin}, title = {The glycan substrate of the cytosolic (Pho 2) phosphorylase isozyme from Pisum sativum L. : identification, linkage analysis and subcellular localization}, issn = {0960-7412}, year = {2004}, abstract = {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}, language = {en} } @phdthesis{Fettke2006, author = {Fettke, J{\"o}rg}, title = {St{\"a}rkerelevante cytosolische Heteroglykane: Identifizierung und funftionelle Analyse}, address = {Potsdam}, pages = {131 S. : graph. Darst.}, year = {2006}, language = {de} } @article{MalinovaSteupFettke2011, author = {Malinova, Irina and Steup, Martin and Fettke, J{\"o}rg}, title = {Starch-related cytosolic heteroglycans in roots 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.2010.12.008}, pages = {1406 -- 1414}, year = {2011}, abstract = {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.}, language = {en} } @article{EckermannFettkePaulyetal.2004, author = {Eckermann, Nora and Fettke, J{\"o}rg and Pauly, Markus and Bazant, Esther and Steup, Martin}, title = {Starch-metabolism related isozymes in higher plants}, year = {2004}, language = {en} } @article{CompartLiFettke2021, author = {Compart, Julia and Li, Xiaoping and Fettke, J{\"o}rg}, title = {Starch-A complex and undeciphered biopolymer}, series = {Journal of plant physiology : biochemistry, physiology, molecular biology and biotechnology of plants}, volume = {258}, journal = {Journal of plant physiology : biochemistry, physiology, molecular biology and biotechnology of plants}, publisher = {Elsevier}, address = {M{\"u}nchen}, issn = {0176-1617}, doi = {10.1016/j.jplph.2021.153389}, pages = {258 -- 259}, year = {2021}, abstract = {Starch is a natural storage carbohydrate in plants and algae. It consists of two relatively simple homo-biopolymers, amylopectin and amylose, with only alpha-1,4 and alpha-1,6 linked glucosyl units. Starch is an essential source of nutrition and animal food, as well as an important raw material for industry. However, despite increasing knowledge, detailed information about its structure and turnover are largely lacking. In the last decades, most data were generated using bulk experiments, a method which obviously presents limitations regarding a deeper understanding of the starch metabolism. Here, we discuss some unavoidable questions arising from the existing data. We focus on a few examples related to starch biosynthesis, degradation, and structure where these limitations strongly emerge. Closing these knowledge gaps will also be extremely important for taking the necessary steps in order to set up starch-providing crops for the challenges of the ongoing climate changes, as well as for increasing the usability of starches for industrial applications by biotechnology.}, language = {en} } @article{MalinovaAlseekhFeiletal.2017, author = {Malinova, Irina and Alseekh, Saleh and Feil, Regina and Fernie, Alisdair R. and Baumann, Otto and Schoettler, Mark Aurel and Lunn, John Edward and Fettke, J{\"o}rg}, title = {Starch Synthase 4 and Plastidal Phosphorylase Differentially Affect Starch Granule Number and Morphology}, series = {Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants}, volume = {174}, journal = {Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants}, publisher = {American Society of Plant Physiologists}, address = {Rockville}, issn = {0032-0889}, doi = {10.1104/pp.16.01859}, pages = {73 -- 85}, year = {2017}, abstract = {The process of starch granule formation in leaves of Arabidopsis ( Arabidopsis thaliana) is obscure. Besides STARCH SYNTHASE4 (SS4), the PLASTIDIAL PHOSPHORYLASE (PHS1) also seems to be involved, since dpe2-1/phs1a double mutants lacking both PHS1 and the cytosolic DISPROPORTIONATING ENZYME2 (DPE2) displayed only one starch granule per chloroplast under normal growth conditions. For further studies, a dpe2-1/phs1a/ss4 triple mutant and various combinations of double mutants were generated and metabolically analyzed with a focus on starch metabolism. The dpe2-1/phs1a/ ss4 mutant revealed a massive starch excess phenotype. Furthermore, these plants grown under 12 h of light/12 h of dark harbored a single large and spherical starch granule per plastid. The number of starch granules was constant when the light/dark regime was altered, but this was not observed in the parental lines. With regard to growth, photosynthetic parameters, and metabolic analyses, the triple mutant additionally displayed alterations in comparison with ss4 and dpe21/phs1a. The results clearly illustrate that PHS1 and SS4 are differently involved in starch granule formation and do not act in series. However, SS4 appears to exert a stronger influence. In connection with the characterized double mutants, we discuss the generation of starch granules and the observed formation of spherical starch granules.}, language = {en} }