TY - JOUR A1 - Schwarte, Sandra A1 - Wegner, Fanny A1 - Havenstein, Katja A1 - Groth, Detlef A1 - Steup, Martin A1 - Tiedemann, Ralph T1 - Sequence variation, differential expression, and divergent evolution in starch-related genes among accessions of Arabidopsis thaliana JF - Plant molecular biology : an international journal of fundamental research and genetic engineering N2 - Transitory starch metabolism is a nonlinear and highly regulated process. It originated very early in the evolution of chloroplast-containing cells and is largely based on a mosaic of genes derived from either the eukaryotic host cell or the prokaryotic endosymbiont. Initially located in the cytoplasm, starch metabolism was rewired into plastids in Chloroplastida. Relocation was accompanied by gene duplications that occurred in most starch-related gene families and resulted in subfunctionalization of the respective gene products. Starch-related isozymes were then evolutionary conserved by constraints such as internal starch structure, posttranslational protein import into plastids and interactions with other starch-related proteins. 25 starch-related genes in 26 accessions of Arabidopsis thaliana were sequenced to assess intraspecific diversity, phylogenetic relationships, and modes of selection. Furthermore, sequences derived from additional 80 accessions that are publicly available were analyzed. Diversity varies significantly among the starch-related genes. Starch synthases and phosphorylases exhibit highest nucleotide diversities, while pyrophosphatases and debranching enzymes are most conserved. The gene trees are most compatible with a scenario of extensive recombination, perhaps in a Pleistocene refugium. Most genes are under purifying selection, but disruptive selection was inferred for a few genes/substitutiones. To study transcript levels, leaves were harvested throughout the light period. By quantifying the transcript levels and by analyzing the sequence of the respective accessions, we were able to estimate whether transcript levels are mainly determined by genetic (i.e., accession dependent) or physiological (i.e., time dependent) parameters. We also identified polymorphic sites that putatively affect pattern or the level of transcripts. KW - Arabidopsis thaliana KW - Divergent evolution KW - Intraspecific genetic variation KW - Positive selection KW - Starch metabolizing enzymes KW - Transcript levels Y1 - 2015 U6 - https://doi.org/10.1007/s11103-015-0293-2 SN - 0167-4412 SN - 1573-5028 VL - 87 IS - 4-5 SP - 489 EP - 519 PB - Springer CY - Dordrecht ER - TY - JOUR A1 - Cisek, Richard A1 - Tokarz, Danielle A1 - Steup, Martin A1 - Tetlow, Ian J. A1 - Emes, Michael J. A1 - Hebelstrup, Kim H. A1 - Blennow, Andreas A1 - Barzda, Virginijus T1 - Second harmonic generation microscopy investigation of the crystalline ultrastructure of three barley starch lines affected by hydration JF - Biomedical optics express N2 - Second harmonic generation (SHG) microscopy is employed to study changes in crystalline organization due to altered gene expression and hydration in barley starch granules. SHG intensity and susceptibility ratio values (R'(SHG)) are obtained using reduced Stokes-Mueller polarimetric microscopy. The maximum R'(SHG) values occur at moderate moisture indicating the narrowest orientation distribution of nonlinear dipoles from the cylindrical axis of glucan helices. The maximum SHG intensity occurs at the highest moisture and amylopectin content. These results support the hypothesis that SHG is caused by ordered hydrogen and hydroxyl bond networks which increase with hydration of starch granules. (C) 2015 Optical Society of America Y1 - 2015 U6 - https://doi.org/10.1364/BOE.6.003694 SN - 2156-7085 VL - 6 IS - 10 SP - 3694 EP - 3700 PB - Optical Society of America CY - Washington ER - TY - JOUR A1 - Cencil, Ugo A1 - Nitschke, Felix A1 - Steup, Martin A1 - Minassian, Berge A. A1 - Colleoni, Christophe A1 - Ball, Steven G. T1 - Transition from glycogen to starch metabolism in Archaeplastida JF - Trends in plant science N2 - In this opinion article we propose a scenario detailing how two crucial components have evolved simultaneously to ensure the transition of glycogen to starch in the cytosol of the Archaeplastida last common ancestor: (i) the recruitment of an enzyme from intracellular Chlamydiae pathogens to facilitate crystallization of alpha-glucan chains; and (ii) the evolution of novel types of polysaccharide (de)phosphorylating enzymes from preexisting glycogen (de)phosphorylation host pathways to allow the turnover of such crystals. We speculate that the transition to starch benefitted Archaeplastida in three ways: more carbon could be packed into osmotically inert material; the host could resume control of carbon assimilation from the chlamydial pathogen that triggered plastid endosymbiosis; and cyanobacterial photosynthate export could be integrated in the emerging Archaeplastida. KW - evolution of plastids KW - starch and glycogen metabolism KW - polyglucan debranching reactions KW - starch and glycogen (de)phosphorylation KW - Chlamydia-like bacteria KW - Lafora disease Y1 - 2014 U6 - https://doi.org/10.1016/j.tplants.2013.08.004 SN - 1360-1385 VL - 19 IS - 1 SP - 18 EP - 28 PB - Elsevier CY - London ER - TY - JOUR A1 - Malinova, Irina A1 - Mahlow, Sebastian A1 - Alseekh, Saleh A1 - Orawetz, Tom A1 - Fernie, Alisdair R. A1 - Baumann, Otto A1 - Steup, Martin A1 - Fettke, Jörg T1 - Double knockout mutants of arabidopsis grown under normal conditions reveal that the plastidial phosphorylase isozyme participates in transitory starch metabolism JF - Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants N2 - 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. Y1 - 2014 U6 - https://doi.org/10.1104/pp.113.227843 SN - 0032-0889 SN - 1532-2548 VL - 164 IS - 2 SP - 907 EP - 921 PB - American Society of Plant Physiologists CY - Rockville ER - TY - JOUR A1 - Tenenboim, Hezi A1 - Smirnova, Julia A1 - Willmitzer, Lothar A1 - Steup, Martin A1 - Brotman, Yariv T1 - VMP1-deficient Chlamydomonas exhibits severely aberrant cell morphology and disrupted cytokinesies JF - BMC plant biology N2 - Background: The versatile Vacuole Membrane Protein 1 (VMP1) has been previously investigated in six species. It has been shown to be essential in macroautophagy, where it takes part in autophagy initiation. In addition, VMP1 has been implicated in organellar biogenesis; endo-, exo- and phagocytosis, and protein secretion; apoptosis; and cell adhesion. These roles underly its proven involvement in pancreatitis, diabetes and cancer in humans. Results: In this study we analyzed a VMP1 homologue from the green alga Chlamydomonas reinhardtii. CrVMP1 knockdown lines showed severe phenotypes, mainly affecting cell division as well as the morphology of cells and organelles. We also provide several pieces of evidence for its involvement in macroautophagy. KW - VMP1 KW - Autophagy KW - Cytokinesis Y1 - 2014 U6 - https://doi.org/10.1186/1471-2229-14-121 SN - 1471-2229 VL - 14 PB - BioMed Central CY - London ER - TY - JOUR A1 - Hemme, Dorothea A1 - Veyel, Daniel A1 - Muehlhaus, Timo A1 - Sommer, Frederik A1 - Jueppner, Jessica A1 - Unger, Ann-Katrin A1 - Sandmann, Michael A1 - Fehrle, Ines A1 - Schoenfelder, Stephanie A1 - Steup, Martin A1 - Geimer, Stefan A1 - Kopka, Joachim A1 - Giavalisco, Patrick A1 - Schroda, Michael T1 - Systems-wide analysis of acclimation responses to long-term heat stress and recovery in the photosynthetic model organism Chlamydomonas reinhardtii JF - The plant cell N2 - We applied a top-down systems biology approach to understand how Chlamydomonas reinhardtii acclimates to long-term heat stress (HS) and recovers from it. For this, we shifted cells from 25 to 42 degrees C for 24 h and back to 25 degrees C for >= 8 h and monitored abundances of 1856 proteins/protein groups, 99 polar and 185 lipophilic metabolites, and cytological and photosynthesis parameters. Our data indicate that acclimation of Chlamydomonas to long-term HS consists of a temporally ordered, orchestrated implementation of response elements at various system levels. These comprise (1) cell cycle arrest; (2) catabolism of larger molecules to generate compounds with roles in stress protection; (3) accumulation of molecular chaperones to restore protein homeostasis together with compatible solutes; (4) redirection of photosynthetic energy and reducing power from the Calvin cycle to the de novo synthesis of saturated fatty acids to replace polyunsaturated ones in membrane lipids, which are deposited in lipid bodies; and (5) when sinks for photosynthetic energy and reducing power are depleted, resumption of Calvin cycle activity associated with increased photorespiration, accumulation of reactive oxygen species scavengers, and throttling of linear electron flow by antenna uncoupling. During recovery from HS, cells appear to focus on processes allowing rapid resumption of growth rather than restoring pre-HS conditions. Y1 - 2014 U6 - https://doi.org/10.1105/tpc.114.130997 SN - 1040-4651 SN - 1532-298X VL - 26 IS - 11 SP - 4270 EP - 4297 PB - American Society of Plant Physiologists CY - Rockville ER - TY - JOUR A1 - Cisek, Richard A1 - Tokarz, Danielle A1 - Krouglov, Serguei A1 - Steup, Martin A1 - Emes, Michael J. A1 - Tetlow, Ian J. A1 - Barzda, Virginijus T1 - Second harmonic generation mediated by aligned water in starch granules JF - The journal of physical chemistry : B, Condensed matter, materials, surfaces, interfaces & biophysical chemistry N2 - The origin of second harmonic generation (SHG) in starch granules was investigated using ab initio quantum mechanical modeling and experimentally examined using polarization-in, polarization-out (PIPO) second harmonic generation microscopy. Ab initio calculations revealed that the largest contribution to the SHG signal from A- and B-type allomorphs of starch originates from the anisotropic organization of hydroxide and hydrogen bonds mediated by aligned water found in the polymers. The hypothesis was experimentally tested by imaging maize starch granules under various hydration and heat treatment conditions that alter the hydrogen bond network. The highest SHG intensity was found in fully hydrated starch granules, and heat treatment diminished the SHG intensity. The PIPO SHG imaging showed that dried starch granules have a much higher nonlinear optical susceptibility component ratio than fully hydrated granules. In contrast, deuterated starch granules showed a smaller susceptibility component ratio demonstrating that SHG is highly sensitive to the organization of the hydroxyl and hydrogen bond network. The polarization SHG imaging results of potato starch granules, representing starch allomorph B, were compared to those of maize starch granules representing allomorph A. The results showed that the amount of aligned water was higher in the maize granules. Nonlinear microscopy of starch granules provides evidence that varying hydration conditions leads to significant changes in the nonlinear susceptibility ratio as well as the SHG intensity, supporting the hypothesis from ab initio calculations that the dominant contribution to SHG is due to the ordered hydroxide and hydrogen bond network. Y1 - 2014 U6 - https://doi.org/10.1021/jp508751s SN - 1520-6106 VL - 118 IS - 51 SP - 14785 EP - 14794 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Fettke, Jörg A1 - Malinova, Irina A1 - Albrecht, Tanja A1 - Hejazi, Mahdi A1 - Steup, Martin T1 - Glucose-1-Phosphate transport into protoplasts and chloroplasts from leaves of arabidopsis JF - Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants N2 - 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. Y1 - 2011 U6 - https://doi.org/10.1104/pp.110.168716 SN - 0032-0889 VL - 155 IS - 4 SP - 1723 EP - 1734 PB - American Society of Plant Physiologists CY - Rockville ER - TY - JOUR A1 - Fettke, Jörg A1 - Nunes-Nesi, Adriano A1 - Fernie, Alisdair R. A1 - Steup, Martin T1 - Identification of a novel heteroglycan-interacting protein, HIP 1.3, from Arabidopsis thaliana JF - Journal of plant physiology : biochemistry, physiology, molecular biology and biotechnology of plants N2 - 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. KW - Arabidopsis thaliana KW - Carbohydrate binding proteins KW - Cytosolic heteroglycans KW - Maltose metabolism KW - Starch metabolism Y1 - 2011 U6 - https://doi.org/10.1016/j.jplph.2010.09.008 SN - 0176-1617 VL - 168 IS - 12 SP - 1415 EP - 1425 PB - Elsevier CY - Jena ER - TY - JOUR A1 - Malinova, Irina A1 - Steup, Martin A1 - Fettke, Jörg T1 - Starch-related cytosolic heteroglycans in roots from Arabidopsis thaliana JF - Journal of plant physiology : biochemistry, physiology, molecular biology and biotechnology of plants N2 - 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. KW - Cytosolic heteroglycans KW - Cytosolic glucosyl transferases KW - Photoautotrophic tissues KW - Heterotrophic tissues KW - Starch metabolism Y1 - 2011 U6 - https://doi.org/10.1016/j.jplph.2010.12.008 SN - 0176-1617 VL - 168 IS - 12 SP - 1406 EP - 1414 PB - Elsevier CY - Jena ER -