@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{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} } @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{MalinovaSteupFettke2013, author = {Malinova, Irina and Steup, Martin and Fettke, J{\"o}rg}, title = {Carbon transitions from either Calvin cycle or transitory starch to heteroglycans as revealed by 14-C-labeling experiments using protoplasts from Arabidopsis}, series = {Physiologia plantarum}, volume = {149}, journal = {Physiologia plantarum}, number = {1}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {0031-9317}, doi = {10.1111/ppl.12033}, pages = {25 -- 44}, year = {2013}, abstract = {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.}, language = {en} } @article{SchmiederNitschkeSteupetal.2013, author = {Schmieder, Peter and Nitschke, Felix and Steup, Martin and Mallow, Keven and Specker, Edgar}, title = {Determination of glucan phosphorylation using heteronuclear H-1,C-13 double and H-1,C-13,P-31 triple-resonance NMR spectra}, series = {Magnetic resonance in chemistry}, volume = {51}, journal = {Magnetic resonance in chemistry}, number = {10}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {0749-1581}, doi = {10.1002/mrc.3996}, pages = {655 -- 661}, year = {2013}, abstract = {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.}, language = {en} } @article{RuzanskiSmirnovaRejzeketal.2013, author = {Ruzanski, Christian and Smirnova, Julia and Rejzek, Martin and Cockburn, Darrell and Pedersen, Henriette L. and Pike, Marilyn and Willats, William G. T. and Svensson, Birte and Steup, Martin and Ebenh{\"o}h, Oliver and Smith, Alison M. and Field, Robert A.}, title = {A bacterial glucanotransferase can replace the complex maltose metabolism required for starch to sucrose conversion in leaves at night}, series = {The journal of biological chemistry}, volume = {288}, journal = {The journal of biological chemistry}, number = {40}, publisher = {American Society for Biochemistry and Molecular Biology}, address = {Bethesda}, issn = {0021-9258}, doi = {10.1074/jbc.M113.497867}, pages = {28581 -- 28598}, year = {2013}, abstract = {Controlled conversion of leaf starch to sucrose at night is essential for the normal growth of Arabidopsis. The conversion involves the cytosolic metabolism of maltose to hexose phosphates via an unusual, multidomain protein with 4-glucanotransferase activity, DPE2, believed to transfer glucosyl moieties to a complex heteroglycan prior to their conversion to hexose phosphate via a cytosolic phosphorylase. The significance of this complex pathway is unclear; conversion of maltose to hexose phosphate in bacteria proceeds via a more typical 4-glucanotransferase that does not require a heteroglycan acceptor. It has recently been suggested that DPE2 generates a heterogeneous series of terminal glucan chains on the heteroglycan that acts as a glucosyl buffer to ensure a constant rate of sucrose synthesis in the leaf at night. Alternatively, DPE2 and/or the heteroglycan may have specific properties important for their function in the plant. To distinguish between these ideas, we compared the properties of DPE2 with those of the Escherichia coli glucanotransferase MalQ. We found that MalQ cannot use the plant heteroglycan as an acceptor for glucosyl transfer. However, experimental and modeling approaches suggested that it can potentially generate a glucosyl buffer between maltose and hexose phosphate because, unlike DPE2, it can generate polydisperse malto-oligosaccharides from maltose. Consistent with this suggestion, MalQ is capable of restoring an essentially wild-type phenotype when expressed in mutant Arabidopsis plants lacking DPE2. In light of these findings, we discuss the possible evolutionary origins of the complex DPE2-heteroglycan pathway.}, language = {en} } @article{MartinsHejaziFettkeetal.2013, author = {Martins, Marina Camara Mattos and Hejazi, Mahdi and Fettke, J{\"o}rg and Steup, Martin and Feil, Regina and Krause, Ursula and Arrivault, Stephanie and Vosloh, Daniel and Figueroa, Carlos Maria and Ivakov, Alexander and Yadav, Umesh Prasad and Piques, Maria and Metzner, Daniela and Stitt, Mark and Lunn, John Edward}, title = {Feedback inhibition of starch degradation in arabidopsis leaves mediated by trehalose 6-phosphate}, series = {Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants}, volume = {163}, journal = {Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants}, number = {3}, publisher = {American Society of Plant Physiologists}, address = {Rockville}, issn = {0032-0889}, doi = {10.1104/pp.113.226787}, pages = {1142 -- 1163}, year = {2013}, abstract = {Many plants accumulate substantial starch reserves in their leaves during the day and remobilize them at night to provide carbon and energy for maintenance and growth. In this paper, we explore the role of a sugar-signaling metabolite, trehalose-6-phosphate (Tre6P), in regulating the accumulation and turnover of transitory starch in Arabidopsis (Arabidopsis thaliana) leaves. Ethanol-induced overexpression of trehalose-phosphate synthase during the day increased Tre6P levels up to 11-fold. There was a transient increase in the rate of starch accumulation in the middle of the day, but this was not linked to reductive activation of ADP-glucose pyrophosphorylase. A 2- to 3-fold increase in Tre6P during the night led to significant inhibition of starch degradation. Maltose and maltotriose did not accumulate, suggesting that Tre6P affects an early step in the pathway of starch degradation in the chloroplasts. Starch granules isolated from induced plants had a higher orthophosphate content than granules from noninduced control plants, consistent either with disruption of the phosphorylation-dephosphorylation cycle that is essential for efficient starch breakdown or with inhibition of starch hydrolysis by beta-amylase. Nonaqueous fractionation of leaves showed that Tre6P is predominantly located in the cytosol, with estimated in vivo Tre6P concentrations of 4 to 7 mu M in the cytosol, 0.2 to 0.5 mu M in the chloroplasts, and 0.05 mu M in the vacuole. It is proposed that Tre6P is a component in a signaling pathway that mediates the feedback regulation of starch breakdown by sucrose, potentially linking starch turnover to demand for sucrose by growing sink organs at night.}, language = {en} } @article{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}, doi = {10.1186/1756-0500-6-84}, year = {2013}, language = {en} }