TY - JOUR A1 - Brust, Henrike A1 - Orzechowski, Slawomir A1 - Fettke, Jörg T1 - Starch and Glycogen Analyses BT - Methods and Techniques JF - Biomolecules N2 - For complex carbohydrates, such as glycogen and starch, various analytical methods and techniques exist allowing the detailed characterization of these storage carbohydrates. In this article, we give a brief overview of the most frequently used methods, techniques, and results. Furthermore, we give insights in the isolation, purification, and fragmentation of both starch and glycogen. An overview of the different structural levels of the glucans is given and the corresponding analytical techniques are discussed. Moreover, future perspectives of the analytical needs and the challenges of the currently developing scientific questions are included KW - starch KW - glycogen KW - analytics Y1 - 2020 U6 - https://doi.org/10.3390/biom10071020 SN - 2218-273X VL - 10 IS - 7 PB - MDPI CY - Basel ER - TY - JOUR A1 - Brust, Henrike A1 - Lehmann, Tanja A1 - Fettke, Jörg T1 - 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 JF - PLoS one N2 - 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. Y1 - 2014 U6 - https://doi.org/10.1371/journal.pone.0102364 SN - 1932-6203 VL - 9 IS - 7 PB - PLoS CY - San Fransisco ER - TY - JOUR A1 - Mahlow, Sebastian A1 - Hejazi, Mahdi A1 - Kuhnert, Franziska A1 - Garz, Andreas A1 - Brust, Henrike A1 - Baumann, Otto A1 - Fettke, Jörg T1 - Phosphorylation of transitory starch by -glucan, water dikinase during starch turnover affects the surface properties and morphology of starch granules JF - New phytologist : international journal of plant science N2 - 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. KW - Arabidopsis thaliana KW - glucan KW - water dikinase (GWD) KW - sex1-8 KW - starch granule surface KW - starch phosphorylation Y1 - 2014 U6 - https://doi.org/10.1111/nph.12801 SN - 0028-646X SN - 1469-8137 VL - 203 IS - 2 SP - 495 EP - 507 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Malinova, Irina A1 - Qasim, Hadeel M. A1 - Brust, Henrike A1 - Fettke, Jörg T1 - Parameters of Starch Granule Genesis in Chloroplasts of Arabidopsis thaliana JF - Frontiers in Plant Science N2 - Starch is the primary storage carbohydrate in most photosynthetic organisms and allows the accumulation of carbon and energy in form of an insoluble and semi-crystalline particle. In the last decades large progress, especially in the model plant Arabidopsis thaliana, was made in understanding the structure and metabolism of starch and its conjunction. The process underlying the initiation of starch granules remains obscure, although this is a fundamental process and seems to be strongly regulated, as in Arabidopsis leaves the starch granule number per chloroplast is fixed with 5-7. Several single, double, and triple mutants were reported in the last years that showed massively alterations in the starch granule number per chloroplast and allowed further insights in this important process. This mini review provides an overview of the current knowledge of processes involved in the initiation and formation of starch granules. We discuss the central role of starch synthase 4 and further proteins for starch genesis and affecting metabolic factors. KW - starch biosynthesis KW - starch granule biogenesis KW - starch synthase KW - plastidial phosphorylase KW - maltooligosaccharides Y1 - 2018 U6 - https://doi.org/10.3389/fpls.2018.00761 SN - 1664-462X VL - 9 SP - 1 EP - 7 PB - Frontiers Media CY - Lausanne ER - TY - JOUR A1 - Schwarte, Sandra A1 - Brust, Henrike A1 - Steup, Martin A1 - Tiedemann, Ralph T1 - Intraspecific sequence variation and differential expression in starch synthase genes of Arabidopsis thaliana Y1 - 2013 UR - http://www.biomedcentral.com/content/pdf/1756-0500-6-84.pdf U6 - https://doi.org/10.1186/1756-0500-6-84 ER - TY - JOUR A1 - Fettke, Jörg A1 - Leifels, Lydia A1 - Brust, Henrike A1 - Herbst, Karoline A1 - Steup, Martin T1 - Two carbon fluxes to reserve starch in potato (Solanum tuberosum L.) tuber cells are closely interconnected but differently modulated by temperature JF - Journal of experimental botany N2 - 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. KW - glucose 1-phosphate KW - phosphorylase KW - potato tubers KW - starch KW - starch synthase Y1 - 2012 U6 - https://doi.org/10.1093/jxb/ers014 SN - 0022-0957 VL - 63 IS - 8 SP - 3011 EP - 3029 PB - Oxford Univ. Press CY - Oxford ER - TY - JOUR A1 - Jüppner, Jessica A1 - Mubeen, Umarah A1 - Leisse, Andrea A1 - Caldana, Camila A1 - Brust, Henrike A1 - Steup, Martin A1 - Herrmann, Marion A1 - Steinhauser, Dirk A1 - Giavalisco, Patrick T1 - Dynamics of lipids and metabolites during the cell cycle of Chlamydomonas reinhardtii JF - The plant journal N2 - Metabolites and lipids are the final products of enzymatic processes, distinguishing the different cellular functions and activities of single cells or whole tissues. Understanding these cellular functions within a well-established model system requires a systemic collection of molecular and physiological information. In the current report, the green alga Chlamydomonas reinhardtii was selected to establish a comprehensive workflow for the detailed multi-omics analysis of a synchronously growing cell culture system. After implementation and benchmarking of the synchronous cell culture, a two-phase extraction method was adopted for the analysis of proteins, lipids, metabolites and starch from a single sample aliquot of as little as 10-15million Chlamydomonas cells. In a proof of concept study, primary metabolites and lipids were sampled throughout the diurnal cell cycle. The results of these time-resolved measurements showed that single compounds were not only coordinated with each other in different pathways, but that these complex metabolic signatures have the potential to be used as biomarkers of various cellular processes. Taken together, the developed workflow, including the synchronized growth of the photoautotrophic cell culture, in combination with comprehensive extraction methods and detailed metabolic phenotyping has the potential for use in in-depth analysis of complex cellular processes, providing essential information for the understanding of complex biological systems. KW - Chlamydomonas reinhardtii KW - synchronized cell cultures KW - photoautotrophic growth KW - cell cycle KW - metabolomics KW - lipidomics KW - systems biology KW - two-phase extraction KW - diurnal cycle KW - technical advance Y1 - 2017 U6 - https://doi.org/10.1111/tpj.13642 SN - 0960-7412 SN - 1365-313X VL - 92 SP - 331 EP - 343 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Paparelli, Eleonora A1 - Gonzali, Silvia A1 - Parlanti, Sandro A1 - Novi, Giacomo A1 - Giorgi, Federico M. A1 - Licausi, Francesco A1 - Kosmacz, Monika A1 - Feil, Regina A1 - Lunn, John Edward A1 - Brust, Henrike A1 - van Dongen, Joost T. A1 - Steup, Martin A1 - Perata, Pierdomenico T1 - Misexpression of a chloroplast aspartyl protease leads to severe growth defects and alters carbohydrate metabolism in arabidopsis JF - Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants N2 - The crucial role of carbohydrate in plant growth and morphogenesis is widely recognized. In this study, we describe the characterization of nana, a dwarf Arabidopsis (Arabidopsis thaliana) mutant impaired in carbohydrate metabolism. We show that the nana dwarf phenotype was accompanied by altered leaf morphology and a delayed flowering time. Our genetic and molecular data indicate that the mutation in nana is due to a transfer DNA insertion in the promoter region of a gene encoding a chloroplast-located aspartyl protease that alters its pattern of expression. Overexpression of the gene (oxNANA) phenocopies the mutation. Both nana and oxNANA display alterations in carbohydrate content, and the extent of these changes varies depending on growth light intensity. In particular, in low light, soluble sugar levels are lower and do not show the daily fluctuations observed in wild-type plants. Moreover, nana and oxNANA are defective in the expression of some genes implicated in sugar metabolism and photosynthetic light harvesting. Interestingly, some chloroplast-encoded genes as well as genes whose products seem to be involved in retrograde signaling appear to be down-regulated. These findings suggest that the NANA aspartic protease has an important regulatory function in chloroplasts that not only influences photosynthetic carbon metabolism but also plastid and nuclear gene expression. Y1 - 2012 U6 - https://doi.org/10.1104/pp.112.204016 SN - 0032-0889 VL - 160 IS - 3 SP - 1237 EP - 1250 PB - American Society of Plant Physiologists CY - Rockville ER - TY - JOUR A1 - Malinova, Irina A1 - Mahto, Harendra A1 - Brandt, Felix A1 - AL-Rawi, Shadha A1 - Qasim, Hadeel A1 - Brust, Henrike A1 - Hejazi, Mahdi A1 - Fettke, Jörg T1 - EARLY STARVATION1 specifically affects the phosphorylation action of starch-related dikinases JF - The plant journal N2 - Starch phosphorylation by starch-related dikinases glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) is a key step in starch degradation. Little information is known about the precise structure of the glucan substrate utilized by the dikinases and about the mechanisms by which these structures may be influenced. A 50-kDa starch-binding protein named EARLY STARVATION1 (ESV1) was analyzed regarding its impact on starch phosphorylation. In various invitro assays, the influences of the recombinant protein ESV1 on the actions of GWD and PWD on the surfaces of native starch granules were analyzed. In addition, we included starches from various sources as well as truncated forms of GWD. ESV1 preferentially binds to highly ordered, -glucans, such as starch and crystalline maltodextrins. Furthermore, ESV1 specifically influences the action of GWD and PWD at the starch granule surface. Starch phosphorylation by GWD is decreased in the presence of ESV1, whereas the action of PWD increases in the presence of ESV1. The unique alterations observed in starch phosphorylation by the two dikinases are discussed in regard to altered glucan structures at the starch granule surface. KW - Arabidopsis thaliana KW - EARLY STARVATION1 KW - glucan KW - phosphoglucan KW - starch granule surface KW - starch phosphorylation KW - water dikinase Y1 - 2018 U6 - https://doi.org/10.1111/tpj.13937 SN - 0960-7412 SN - 1365-313X VL - 95 IS - 1 SP - 126 EP - 137 PB - Wiley CY - Hoboken ER -