@article{MalinovaFettke2017, author = {Malinova, Irina and Fettke, J{\"o}rg}, title = {Reduced starch granule number per chloroplast in the dpe2/phs1 mutant is dependent on initiation of starch degradation}, series = {PLoS one}, volume = {12}, journal = {PLoS one}, publisher = {PLoS}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0187985}, pages = {11}, year = {2017}, abstract = {An Arabidopsis double knock-out mutant lacking cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) revealed a dwarf-growth phenotype, reduced starch content, an uneven distribution of starch within the plant rosette, and a reduced number of starch granules per chloroplast under standard growth conditions. In contrast, the wild type contained 5-7 starch granules per chloroplast. Mature and old leaves of the double mutant were essentially starch free and showed plastidial disintegration. Several analyses revealed that the number of starch granules per chloroplast was affected by the dark phase. So far, it was unclear if it was the dark phase per se or starch degradation in the dark that was connected to the observed decrease in the number of starch granules per chloroplast. Therefore, in the background of the double mutant dpe2/phs1, a triple mutant was generated lacking the initial starch degrading enzyme glucan, water dikinase (GWD). The triple mutant showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to wild type. However, starch granule morphology was only slightly affected by the lack of GWD as in the triple mutant and, like in dpe2/phs1, more spherical starch granules were observed. The characterized triple mutant was discussed in the context of the generation of starch granules and the formation of starch granule morphology.}, 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} } @article{OrawetzMalinovaOrzechowskietal.2016, author = {Orawetz, Tom and Malinova, Irina and Orzechowski, Slawomir and Fettke, J{\"o}rg}, title = {Reduction of the plastidial phosphorylase in potato (Solanum tuberosum L.) reveals impact on storage starch structure during growth at low temperature}, series = {Plant physiology and biochemistry : an official journal of the Federation of European Societies of Plant Physiology}, volume = {100}, journal = {Plant physiology and biochemistry : an official journal of the Federation of European Societies of Plant Physiology}, publisher = {Elsevier}, address = {Paris}, issn = {0981-9428}, doi = {10.1016/j.plaphy.2016.01.013}, pages = {141 -- 149}, year = {2016}, abstract = {Tubers of potato (Solanum tuberosum L.), one of the most important crops, are a prominent example for an efficient production of storage starch. Nevertheless, the synthesis of this storage starch is not completely understood. The plastidial phosphorylase (Phol; EC 2.4.11) catalyzes the reversible transfer of glucosyl residues from glucose-1-phosphate to the non-reducing end of alpha-glucans with the release of orthophosphate. Thus, the enzyme is in principle able to act during starch synthesis. However, so far under normal growth conditions no alterations in tuber starch metabolism were observed. Based on analyses of other species and also from in vitro experiments with potato tuber slices it was supposed, that Phol has a stronger impact on starch metabolism, when plants grow under low temperature conditions. Therefore, we analyzed the starch content, granule size, as well as the internal structure of starch granules isolated from potato plants grown under low temperatures. Besides wild type, transgenic potato plants with a strong reduction in the Phol activity were analyzed. No significant alterations in starch content and granule size were detected. In contrast, when plants were cultivated at low temperatures the chain length distributions of the starch granules were altered. Thus, the granules contained more short glucan chains. That was not observed in the transgenic plants, revealing that Pho1 in wild type is involved in the formation of the short glucan chains, at least at low temperatures. (C) 2016 Elsevier Masson SAS. All rights reserved.}, language = {en} } @article{GietlerNykielOrzechowskietal.2016, author = {Gietler, Marta and Nykiel, Malgorzata and Orzechowski, Slawomir and Zagdanska, Barbara and Fettke, J{\"o}rg}, title = {Proteomic analysis of S-nitrosylated and S-glutathionylated proteins in wheat seedlings with different dehydration tolerances}, series = {Plant physiology and biochemistry : an official journal of the Federation of European Societies of Plant Physiology}, volume = {108}, journal = {Plant physiology and biochemistry : an official journal of the Federation of European Societies of Plant Physiology}, publisher = {Elsevier}, address = {Paris}, issn = {0981-9428}, doi = {10.1016/j.plaphy.2016.08.017}, pages = {507 -- 518}, year = {2016}, abstract = {A loss of dehydration tolerance in wheat seedlings on the fifth day following imbibition is associated with a disturbance in cellular redox homeostasis, as documented by a shift of the reduced/oxidized glutathione ratio to a more oxidized state and a significant increase in the ratio of protein thiols to the total thiol group content. Therefore, the identification and characterization of redox-sensitive proteins are important steps toward understanding the molecular mechanisms of the loss of dehydration tolerance. In the present study, proteins that were differentially expressed between fully turgid (control), dehydrated tolerant (four-day-old) and dehydrated sensitive (six-day-old) wheat seedlings were analysed. Protein spots having at least a significant (p < 0.05) two-fold change in protein abundance were selected by Delta2D as differentially expressed, identified by MALDI-TOF and LC-MS/MS, and classified according to their function. The observed changes in the proteomic patterns of the differentially S-nitrosylated and S-glutathionylated proteins were highly specific in dehydration-tolerant and-sensitive wheat seedlings. The metabolic function of these proteins indicates that dehydration tolerance is mainly related to nucleic acids, protein metabolism, and energy metabolism. It has been proven that leaf-specific thionins BTH6 and DB4, chloroplastic 50S ribosomal protein L16, phospholipase A1-II delta, and chloroplastic thioredoxin M2 are both S-nitrosylated and S-glutathionylated upon water deficiency. Our results revealed the existence of interplay between S-nitrosylation and S-glutathionylation, two redox-regulated protein posttranslational modifications that could enhance plant defence mechanisms and/or facilitate the acclimation of plants to unfavourable environmental conditions. (C) 2016 Elsevier Masson SAS. All rights reserved.}, language = {en} } @misc{MahlowOrzechowskiFettke2016, author = {Mahlow, Sebastian and Orzechowski, Slawomir and Fettke, J{\"o}rg}, title = {Starch phosphorylation: insights and perspectives}, series = {Cellular and molecular life sciences}, volume = {73}, journal = {Cellular and molecular life sciences}, publisher = {Springer}, address = {Basel}, issn = {1420-682X}, doi = {10.1007/s00018-016-2248-4}, pages = {2753 -- 2764}, year = {2016}, abstract = {During starch metabolism, the phosphorylation of glucosyl residues of starch, to be more precise of amylopectin, is a repeatedly observed process. This phosphorylation is mediated by dikinases, the glucan, water dikinase (GWD) and the phosphoglucan, water dikinase (PWD). The starch-related dikinases utilize ATP as dual phosphate donor transferring the terminal gamma-phosphate group to water and the beta-phosphate group selectively to either C6 position or C3 position of a glucosyl residue within amylopectin. By the collaborative action of both enzymes, the initiation of a transition of alpha-glucans from highly ordered, water-insoluble state to a less order state is realized and thus the initial process of starch degradation. Consequently, mutants lacking either GWD or PWD reveal a starch excess phenotype as well as growth retardation. In this review, we focus on the increased knowledge collected over the last years related to enzymatic properties, the precise definition of the substrates, the physiological implications, and discuss ongoing questions.}, language = {en} } @article{KrasuskaCiackaOrzechowskietal.2016, author = {Krasuska, Urszula and Ciacka, Katarzyna and Orzechowski, Slawomir and Fettke, J{\"o}rg and Bogatek, Renata and Gniazdowska, Agnieszka}, title = {Modification of the endogenous NO level influences apple embryos dormancy by alterations of nitrated and biotinylated protein patterns}, series = {Planta}, volume = {244}, journal = {Planta}, publisher = {Springer}, address = {New York}, issn = {0032-0935}, doi = {10.1007/s00425-016-2553-z}, pages = {877 -- 891}, year = {2016}, abstract = {NO donors and Arg remove dormancy of apple embryos and stimulate germination. Compounds lowering NO level (cPTIO, L -NAME, CAN) strengthen dormancy. Embryo transition from dormancy state to germination is linked to increased nitric oxide synthase (NOS)-like activity. Germination of embryos is associated with declined level of biotin containing proteins and nitrated proteins in soluble protein fraction of root axis. Pattern of nitrated proteins suggest that storage proteins are putative targets of nitration. Nitric oxide (NO) acts as a key regulatory factor in removal of seed dormancy and is a signal necessary for seed transition from dormant state into germination. Modulation of NO concentration in apple (Malus domestica Borkh.) embryos by NO fumigation, treatment with NO donor (S-nitroso-N-acetyl-d,l-penicillamine, SNAP), application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), N (omega)-nitro-l-arginine methyl ester (l-NAME), canavanine (CAN) or arginine (Arg) allowed us to investigate the NO impact on seed dormancy status. Arg analogs and NO scavenger strengthened embryo dormancy by lowering reactive nitrogen species level in embryonic axes. This effect was accompanied by strong inhibition of NOS-like activity, without significant influence on tissue NO2 (-) concentration. Germination sensu stricto of apple embryos initiated by dormancy breakage via short term NO treatment or Arg supplementation were linked to a reduced level of biotinylated proteins in root axis. Decrease of total soluble nitrated proteins was observed at the termination of germination sensu stricto. Also modulation of NO tissue status leads to modification in nitrated protein pattern. Among protein bands that correspond to molecular mass of approximately 95 kDa, storage proteins (legumin A-like and seed biotin-containing protein) were identified, and can be considered as good markers for seed dormancy status. Moreover, pattern of nitrated proteins suggest that biotin containing proteins are also targets of nitration.}, language = {en} } @article{SchopperMuhlenbockSorenssonetal.2015, author = {Schopper, S. and Muhlenbock, P. and Sorensson, C. and Hellborg, L. and Lenman, M. and Widell, S. and Fettke, J{\"o}rg and Andreasson, Erik}, title = {Arabidopsis cytosolic alpha-glycan phosphorylase, PHS2, is important during carbohydrate imbalanced conditions}, series = {Plant biology}, volume = {17}, journal = {Plant biology}, number = {1}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {1435-8603}, doi = {10.1111/plb.12190}, pages = {74 -- 80}, year = {2015}, abstract = {Arabidopsis thaliana has two isoforms of alpha-glycan phosphorylase (EC 2.4.1.1), one residing in the plastid and the other in the cytosol. The cytosolic phosphorylase, PHS2, acts on soluble heteroglycans that constitute a part of the carbohydrate pool in a plant. This study aimed to define a physiological role for PHS2. Under standard growth conditions phs2 knock-out mutants do not show any clear growth phenotype, and we hypothesised that during low-light conditions where carbohydrate imbalance is perturbed, this enzyme is important. Soil-grown phs2 mutant plants developed leaf lesions when placed in very low light. Analysis of soluble heteroglycan (SHG) levels showed that the amount of glucose residues in SHG was higher in the phs2 mutant compared to wild-type plants. Furthermore, a standard senescence assay from soil-grown phs2 mutant plants showed that leaves senesced significantly faster in darkness than the wild-type leaves. We also found decreased hypocotyl extension in in vitro-grown phs2 mutant seedlings when grown for long time in darkness at 6 degrees C. We conclude that PHS2 activity is important in the adult stage during low-light conditions and senescence, as well as during prolonged seedling development when carbohydrate levels are unbalanced.}, language = {en} } @misc{FettkeFernie2015, author = {Fettke, J{\"o}rg and Fernie, Alisdair R.}, title = {Intracellular and cell-to-apoplast compartmentation of carbohydrate metabolism}, series = {Trends in plant science}, volume = {20}, journal = {Trends in plant science}, number = {8}, publisher = {Elsevier}, address = {London}, issn = {1360-1385}, doi = {10.1016/j.tplants.2015.04.012}, pages = {490 -- 497}, year = {2015}, abstract = {In most plants, carbohydrates represent the major energy store as well as providing the building blocks for essential structural polymers. Although the major pathways for carbohydrate biosynthesis, degradation, and transport are well characterized, several key steps have only recently been discovered. In addition, several novel minor metabolic routes have been uncovered in the past few years. Here we review current studies of plant carbohydrate metabolism detailing the expanding compendium of functionally characterized transport proteins as well as our deeper comprehension of more minor and conditionally activated metabolic pathways. We additionally explore the pertinent questions that will allow us to enhance our understanding of the response of both major and minor carbohydrate fluxes to changing cellular circumstances.}, language = {en} } @article{KunzZamaniNourHaeusleretal.2014, author = {Kunz, Hans-Henning and Zamani-Nour, Shirin and Haeusler, Rainer E. and Ludewig, Katja and Schroeder, Julian I. and Malinova, Irina and Fettke, J{\"o}rg and Fluegge, Ulf-Ingo and Gierth, Markus}, title = {Loss of cytosolic phosphoglucose isomerase affects carbohydrate metabolism in leaves and is essential for fertility of arabidopsis}, series = {Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants}, volume = {166}, 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.114.241091}, pages = {753 -- U960}, year = {2014}, abstract = {Carbohydrate metabolism in plants is tightly linked to photosynthesis and is essential for energy and carbon skeleton supply of the entire organism. Thus, the hexose phosphate pools of the cytosol and the chloroplast represent important metabolic resources that are maintained through action of phosphoglucose isomerase (PGI) and phosphoglucose mutase interconverting glucose 6-phosphate, fructose 6-phosphate, and glucose 1-phosphate. Here, we investigated the impact of disrupted cytosolic PGI (cPGI) function on plant viability and metabolism. Overexpressing an artificial microRNA targeted against cPGI (amiR-cpgi) resulted in adult plants with vegetative tissue essentially free of cPGI activity. These plants displayed diminished growth compared with the wild type and accumulated excess starch in chloroplasts but maintained low sucrose content in leaves at the end of the night. Moreover, amiR-cpgi plants exhibited increased nonphotochemical chlorophyll a quenching during photosynthesis. In contrast to amiR-cpgi plants, viable transfer DNA insertion mutants disrupted in cPGI function could only be identified as heterozygous individuals. However, homozygous transfer DNA insertion mutants could be isolated among plants ectopically expressing cPGI. Intriguingly, these plants were only fertile when expression was driven by the ubiquitin10 promoter but sterile when the seed-specific unknown seed protein promoter or the Cauliflower mosaic virus 35S promoter were employed. These data show that metabolism is apparently able to compensate for missing cPGI activity in adult amiR-cpgi plants and indicate an essential function for cPGI in plant reproduction. Moreover, our data suggest a feedback regulation in amiR-cpgi plants that fine-tunes cytosolic sucrose metabolism with plastidic starch turnover.}, language = {en} } @article{BrustLehmannFettke2014, author = {Brust, Henrike and Lehmann, Tanja and Fettke, J{\"o}rg}, title = {Analysis of the functional interaction of arabidopsis starch synthase and branching enzyme isoforms reveals that the cooperative action of SSI and BEs results in glucans with polymodal chain length distribution similar to amylopectin}, series = {PLoS one}, volume = {9}, journal = {PLoS one}, number = {7}, publisher = {PLoS}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0102364}, pages = {14}, year = {2014}, abstract = {Starch synthase (SS) and branching enzyme (BE) establish the two glycosidic linkages existing in starch. Both enzymes exist as several isoforms. Enzymes derived from several species were studied extensively both in vivo and in vitro over the last years, however, analyses of a functional interaction of SS and BE isoforms are missing so far. Here, we present data from in vitro studies including both interaction of leaf derived and heterologously expressed SS and BE isoforms. We found that SSI activity in native PAGE without addition of glucans was dependent on at least one of the two BE isoforms active in Arabidopsis leaves. This interaction is most likely not based on a physical association of the enzymes, as demonstrated by immunodetection and native PAGE mobility analysis of SSI, BE2, and BE3. The glucans formed by the action of SSI/BEs were analysed using leaf protein extracts from wild type and be single mutants (Atbe2 and Atbe3 mutant lines) and by different combinations of recombinant proteins. Chain length distribution (CLD) patterns of the formed glucans were irrespective of SSI and BE isoforms origin and still independent of assay conditions. Furthermore, we show that all SS isoforms (SSI-SSIV) were able to interact with BEs and form branched glucans. However, only SSI/BEs generated a polymodal distribution of glucans which was similar to CLD pattern detected in amylopectin of Arabidopsis leaf starch. We discuss the impact of the SSI/BEs interplay for the CLD pattern of amylopectin.}, language = {en} } @article{MahlowHejaziKuhnertetal.2014, author = {Mahlow, Sebastian and Hejazi, Mahdi and Kuhnert, Franziska and Garz, Andreas and Brust, Henrike and Baumann, Otto and Fettke, J{\"o}rg}, title = {Phosphorylation of transitory starch by -glucan, water dikinase during starch turnover affects the surface properties and morphology of starch granules}, series = {New phytologist : international journal of plant science}, volume = {203}, journal = {New phytologist : international journal of plant science}, number = {2}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {0028-646X}, doi = {10.1111/nph.12801}, pages = {495 -- 507}, year = {2014}, abstract = {Glucan, water dikinase (GWD) is a key enzyme of starch metabolism but the physico-chemical properties of starches isolated from GWD-deficient plants and their implications for starch metabolism have so far not been described. Transgenic Arabidopsis thaliana plants with reduced or no GWD activity were used to investigate the properties of starch granules. In addition, using various in vitro assays, the action of recombinant GWD, -amylase, isoamylase and starch synthase 1 on the surface of native starch granules was analysed. The internal structure of granules isolated from GWD mutant plants is unaffected, as thermal stability, allomorph, chain length distribution and density of starch granules were similar to wild-type. However, short glucan chain residues located at the granule surface dominate in starches of transgenic plants and impede GWD activity. A similarly reduced rate of phosphorylation by GWD was also observed in potato tuber starch fractions that differ in the proportion of accessible glucan chain residues at the granule surface. A model is proposed to explain the characteristic morphology of starch granules observed in GWD transgenic plants. The model postulates that the occupancy rate of single glucan chains at the granule surface limits accessibility to starch-related enzymes.}, language = {en} } @article{MalinovaKunzAlseekhetal.2014, author = {Malinova, Irina and Kunz, Hans-Henning and Alseekh, Saleh and Herbst, Karoline and Fernie, Alisdair R. and Gierth, Markus and Fettke, J{\"o}rg}, title = {Reduction of the cytosolic phosphoglucomutase in arabidopsis reveals impact on plant growth, seed and root development, and carbohydrate partitioning}, series = {PLoS one}, volume = {9}, journal = {PLoS one}, number = {11}, publisher = {PLoS}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0112468}, pages = {11}, year = {2014}, abstract = {Phosphoglucomutase (PGM) catalyses the interconversion of glucose 1-phosphate (G1P) and glucose 6-phosphate (G6P) and exists as plastidial (pPGM) and cytosolic (cPGM) isoforms. The plastidial isoform is essential for transitory starch synthesis in chloroplasts of leaves, whereas the cytosolic counterpart is essential for glucose phosphate partitioning and, therefore, for syntheses of sucrose and cell wall components. In Arabidopsis two cytosolic isoforms (PGM2 and PGM3) exist. Both PGM2 and PGM3 are redundant in function as single mutants reveal only small or no alterations compared to wild type with respect to plant primary metabolism. So far, there are no reports of Arabidopsis plants lacking the entire cPGM or total PGM activity, respectively. Therefore, amiRNA transgenic plants were generated and used for analyses of various parameters such as growth, development, and starch metabolism. The lack of the entire cPGM activity resulted in a strongly reduced growth revealed by decreased rosette fresh weight, shorter roots, and reduced seed production compared to wild type. By contrast content of starch, sucrose, maltose and cell wall components were significantly increased. The lack of both cPGM and pPGM activities in Arabidopsis resulted in dwarf growth, prematurely die off, and inability to develop a functional inflorescence. The combined results are discussed in comparison to potato, the only described mutant with lack of total PGM activity.}, language = {en} } @article{MalinovaMahlowAlseekhetal.2014, author = {Malinova, Irina and Mahlow, Sebastian and Alseekh, Saleh and Orawetz, Tom and Fernie, Alisdair R. 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} } @article{ZhangSunFettkeetal.2014, author = {Zhang, Youjun and Sun, Feng and Fettke, J{\"o}rg and Schoettler, Mark Aurel and Ramsden, Lawrence and Fernie, Alisdair R. and Lim, Boon Leong}, title = {Heterologous expression of AtPAP2 in transgenic potato influences carbon metabolism and tuber development}, series = {FEBS letters : the journal for rapid publication of short reports in molecular biosciences}, volume = {588}, journal = {FEBS letters : the journal for rapid publication of short reports in molecular biosciences}, number = {20}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0014-5793}, doi = {10.1016/j.febslet.2014.08.019}, pages = {3726 -- 3731}, year = {2014}, abstract = {Changes in carbon flow and sink/source activities can affect floral, architectural, and reproductive traits of plants. In potato, overexpression (OE) of the purple acid phosphatase 2 of Arabidopsis (AtPAP2) resulted in earlier flowering, faster growth rate, increased tubers and tuber starch content, and higher photosynthesis rate. There was a significant change in sucrose, glucose and fructose levels in leaves, phloem and sink biomass of the OE lines, consistent with an increased expression of sucrose transporter 1 (StSUT1). Furthermore, the expression levels and enzyme activity of sucrose-phosphate synthase (SPS) were also significantly increased in the OE lines. These findings strongly suggest that higher carbon supply from the source and improved sink strength can improve potato tuber yield. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.}, 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{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{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{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{SchenkFettkeLenzetal.2012, author = {Schenk, J{\"o}rg A. and Fettke, J{\"o}rg and Lenz, Christine and Albers, Katharina and Mallwitz, Frank and Gajovic-Eichelmann, Nenad and Ehrentreich-F{\"o}rster, Eva and Kusch, Emely and Sellrie, Frank}, title = {Secretory leukocyte protease inhibitor (SLPI) might contaminate murine monoclonal antibodies after purification on protein G}, series = {Journal of biotechnology}, volume = {158}, journal = {Journal of biotechnology}, number = {1-2}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0168-1656}, doi = {10.1016/j.jbiotec.2011.12.025}, pages = {34 -- 35}, year = {2012}, abstract = {The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14 kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). This SLPI contamination lead to a failure of the fiber-optic based competitive fluorescence assay to detect progesterone in milk. Purification of the monoclonal antibody using protein A columns circumvented this problem.}, language = {en} } @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{FettkeMalinovaAlbrechtetal.2011, author = {Fettke, J{\"o}rg and Malinova, Irina and Albrecht, Tanja and Hejazi, Mahdi and Steup, Martin}, title = {Glucose-1-Phosphate transport into protoplasts and chloroplasts from leaves of arabidopsis}, series = {Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants}, volume = {155}, journal = {Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants}, number = {4}, publisher = {American Society of Plant Physiologists}, address = {Rockville}, issn = {0032-0889}, doi = {10.1104/pp.110.168716}, pages = {1723 -- 1734}, year = {2011}, abstract = {Almost all glucosyl transfer reactions rely on glucose-1-phosphate (Glc-1-P) that either immediately acts as glucosyl donor or as substrate for the synthesis of the more widely used Glc dinucleotides, ADPglucose or UDPglucose. In this communication, we have analyzed two Glc-1-P-related processes: the carbon flux from externally supplied Glc-1-P to starch by either mesophyll protoplasts or intact chloroplasts from Arabidopsis (Arabidopsis thaliana). When intact protoplasts or chloroplasts are incubated with [U-C-14]Glc-1-P, starch is rapidly labeled. Incorporation into starch is unaffected by the addition of unlabeled Glc-6-P or Glc, indicating a selective flux from Glc-1-P to starch. However, illuminated protoplasts incorporate less C-14 into starch when unlabeled bicarbonate is supplied in addition to the C-14-labeled Glc-1-P. Mesophyll protoplasts incubated with [U-C-14] Glc-1-P incorporate C-14 into the plastidial pool of adenosine diphosphoglucose. Protoplasts prepared from leaves of mutants of Arabidopsis that lack either the plastidial phosphorylase or the phosphoglucomutase isozyme incorporate C-14 derived from external Glc-1-P into starch, but incorporation into starch is insignificant when protoplasts from a mutant possessing a highly reduced ADPglucose pyrophosphorylase activity are studied. Thus, the path of assimilatory starch biosynthesis initiated by extraplastidial Glc-1-P leads to the plastidial pool of adenosine diphosphoglucose, and at this intermediate it is fused with the Calvin cycle-driven route. Mutants lacking the plastidial phosphoglucomutase contain a small yet significant amount of transitory starch.}, language = {en} } @phdthesis{Fettke2011, author = {Fettke, J{\"o}rg}, title = {Analysen St{\"a}rke-bezogener Kohlenstofffl{\"u}sse}, address = {Potsdam}, year = {2011}, language = {de} } @article{DortaySchmoeckelFettkeetal.2011, author = {Dortay, Hakan and Schm{\"o}ckel, Sandra M. and Fettke, J{\"o}rg and M{\"u}ller-R{\"o}ber, Bernd}, title = {Expression of human c-reactive protein in different systems and its purification from Leishmania tarentolae}, series = {Protein expression and purification}, volume = {78}, journal = {Protein expression and purification}, number = {1}, publisher = {Elsevier}, address = {San Diego}, issn = {1046-5928}, doi = {10.1016/j.pep.2011.03.010}, pages = {55 -- 60}, year = {2011}, abstract = {With its homo-pentameric structure and calcium-dependent specificity for phosphocholine (PCh), human c-reactive protein (CRP) is produced by the liver and secreted in elevated quantities in response to inflammation. CRP is widely accepted as a cardiac marker, e.g. in point-of-care diagnostics, however, its heterologous expression has proven difficult. Here, we demonstrate the expression of CRP in different Escherichia coli strains as well as by in vitro transcription/translation. Although expression in these systems was straightforward, most of the protein that accumulated was insoluble. We therefore expanded our study to include the expression of CRP in two eukaryotic hosts, namely the yeast Kluyveromyces lactis and the protozoon Leishmania tarentolae. Both expression systems are optimized for secretion of recombinant proteins and here allowed successful expression of soluble CRP. We also demonstrate the purification of recombinant CRP from Leishmania growth medium; the purification of protein expressed from K. lactis was not successful. Functional and intact CRP pentamer is known to interact with PCh in Ca(2+)-dependent manner. In this report we verify the binding specificity of recombinant CRP from L tarentolae (2 mu g/mL culture medium) for PCh.}, language = {en} } @article{FettkeNunesNesiFernieetal.2011, author = {Fettke, J{\"o}rg and Nunes-Nesi, Adriano and Fernie, Alisdair R. 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{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{FettkeAlbrechtHejazietal.2010, author = {Fettke, J{\"o}rg and Albrecht, Tanja and Hejazi, Mahdi and Mahlow, Sebastian and Nakamura, Yasunori and Steup, Martin}, title = {Glucose 1-phosphate is efficiently taken up by potato (Solanum tuberosum) tuber parenchyma cells and converted to reserve starch granules}, issn = {0028-646X}, doi = {10.1111/j.1469-8137.2009.03126.x}, year = {2010}, abstract = {Reserve starch is an important plant product but the actual biosynthetic process is not yet fully understood. Potato (Solanum tuberosum) tuber discs from various transgenic plants were used to analyse the conversion of external sugars or sugar derivatives to starch. By using in vitro assays, a direct glucosyl transfer from glucose 1-phosphate to native starch granules as mediated by recombinant plastidial phosphorylase was analysed. Compared with labelled glucose, glucose 6-phosphate or sucrose, tuber discs converted externally supplied [C-14] glucose 1-phosphate into starch at a much higher rate. Likewise, tuber discs from transgenic lines with a strongly reduced expression of cytosolic phosphoglucomutase, phosphorylase or transglucosidase converted glucose 1-phosphate to starch with the same or even an increased rate compared with the wild-type. Similar results were obtained with transgenic potato lines possessing a strongly reduced activity of both the cytosolic and the plastidial phosphoglucomutase. Starch labelling was, however, significantly diminished in transgenic lines, with a reduced concentration of the plastidial phosphorylase isozymes. Two distinct paths of reserve starch biosynthesis are proposed that explain, at a biochemical level, the phenotype of several transgenic plant lines.}, language = {en} } @article{FettkeHejaziSmirnovaetal.2009, author = {Fettke, J{\"o}rg and Hejazi, Mahdi and Smirnova, Julia and Hoechel, Erik and Stage, Marion and Steup, Martin}, title = {Eukaryotic starch degradation : integration of plastidial and cytosolic pathways}, issn = {0022-0957}, doi = {10.1093/Jxb/Erp054}, year = {2009}, abstract = {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.}, 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{FettkeMalinovaEckermannetal.2009, author = {Fettke, J{\"o}rg and Malinova, Irina and Eckermann, Nora and Steup, Martin}, title = {Cytosolic heteroglycans in photoautotrophic and in heterotrophic plant cells}, issn = {0031-9422}, doi = {10.1016/j.phytochem.2009.03.016}, year = {2009}, abstract = {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.}, 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{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{FettkeEckermannTiessenetal.2005, author = {Fettke, J{\"o}rg and Eckermann, Nora and Tiessen, Axel and Geigenberger, Peter Ludwig and Steup, Martin}, title = {Identification, subcellular localization and biochemical characterization of water-soluble heteroglycans (SHG) in leaves of Arabidopsis thaliana L. : distinct SHG reside in the cytosol and in the apoplast}, issn = {0960-7412}, year = {2005}, abstract = {Water-soluble heteroglycans (SHG) were isolated from leaves of wild-type Arabidopsis thaliana L. and from two starch-deficient mutants. Major constituents of the SHG are arabinose, galactose, rhamnose, and glucose. SHG was separated into low (< 10 kDa; SHG(S)) and high (> 10 kDa; SHG(L)) molecular weight compounds. SHG(S) was resolved into approximately 25 distinct oligoglycans by ion exchange chromatography. SHG(L) was further separated into two subfractions, designated as subfraction I and II, by field flow fractionation. For the intracellular localization of the various SHG compounds several approaches were chosen: first, leaf material was subjected to non-aqueous fractionation. The apolar gradient fractions were characterized by monitoring markers and were used as starting material for the SHG isolation. Subfraction I and SHG(S) exhibited a distribution similar to that of cytosolic markers whereas subfraction II cofractionated with crystalline cellulose. Secondly, intact organelles were isolated and used for SHG isolation. Preparations of intact organelles (mitochondria plus peroxisomes) contained no significant amount of any heteroglycan. In isolated intact microsomes a series of oligoglycans was recovered but neither subfraction I nor II. In in vitro assays using glucose 1-phosphate and recombinant cytosolic (Pho 2) phosphorylase both SHG(S) and subfraction I acted as glucosyl acceptor whereas subfraction II was essentially inactive. Rabbit muscle phosphorylase a did not utilize any of the plant glycans indicating a specific Pho 2-glycan interaction. As revealed by in vivo labeling experiments using (CO2)-C-14 carbon fluxes into subfraction I and II differed. Furthermore, in leaves the pool size of subfraction I varied during the light-dark regime}, language = {en} } @article{FettkePoesteEckermannetal.2005, author = {Fettke, J{\"o}rg and Poeste, Simon and Eckermann, Nora and Tiessen, Axel and Pauly, Markus and Geigenberger, Peter Ludwig and Steup, Martin}, title = {Analysis of cytosolic heteroglycans from leaves of transgenic potato (Solanum tuberosum L.) plants that under- or overexpress the Pho 2 phosphorylase isozyme}, year = {2005}, abstract = {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}, 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} } @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{EckermannFettkeSteup2002, author = {Eckermann, Nora and Fettke, J{\"o}rg and Steup, Martin}, title = {Identification of polysaccharide binding proteins by affinity electrophoresis in inhomogeneous polyacrylamide gels and subsequent SDS-PAGE/MALDI-TOF analysis}, year = {2002}, language = {en} }