TY - JOUR A1 - Garz, Andreas A1 - Sandmann, Michael A1 - Rading, Michael A1 - Ramm, Sascha A1 - Menzel, Ralf A1 - Steup, Martin T1 - Cell-to-cell diversity in a synchronized chlamydomonas culture as revealed by single-cell analyses JF - Biophysical journal N2 - In a synchronized photoautotrophic culture of Chlamydomonas reinhardtii, cell size, cell number, and the averaged starch content were determined throughout the light-dark cycle. For single-cell analyses, the relative cellular starch was quantified by measuring the second harmonic generation (SHG). In destained cells, amylopectin essentially represents the only biophotonic structure. As revealed by various validation procedures, SHG signal intensities are a reliable relative measure of the cellular starch content. During photosynthesis-driven starch biosynthesis, synchronized Chlamydomonas cells possess an unexpected cell-to-cell diversity both in size and starch content, but the starch-related heterogeneity largely exceeds that of size. The cellular volume, starch content, and amount of starch/cell volume obey lognormal distributions. Starch degradation was initiated by inhibiting the photosynthetic electron transport in illuminated cells or by darkening. Under both conditions, the averaged rate of starch degradation is almost constant, but it is higher in illuminated than in darkened cells. At the single-cell level, rates of starch degradation largely differ but are unrelated to the initial cellular starch content. A rate equation describing the cellular starch degradation Y1 - 2012 U6 - https://doi.org/10.1016/j.bpj.2012.07.026 SN - 0006-3495 VL - 103 IS - 5 SP - 1078 EP - 1086 PB - Cell Press CY - Cambridge ER - TY - JOUR A1 - Nakamura, Yasunori A1 - Ono, Masami A1 - Utsumi, Chikako A1 - Steup, Martin T1 - Functional interaction between plastidial starch phosphorylase and starch branching enzymes from rice during the synthesis of branched maltodextrins JF - Plant & cell physiology N2 - The present study established the way in which plastidial alpha-glucan phosphorylase (Pho1) synthesizes maltodextrin (MD) which can be the primer for starch biosynthesis in rice endosperm. The synthesis of MD by Pho1 was markedly accelerated by branching enzyme (BE) isozymes, although the greatest effect was exhibited by the presence of branching isozyme I (BEI) rather than by isozyme IIa (BEIIa) or isozyme IIb (BEIIb). The enhancement of the activity of Pho1 by BE was not merely due to the supply of a non-reducing ends. At the same time, Pho1 greatly enhanced the BE activity, possibly by generating a branched carbohydrate substrate which is used by BE with a higher affinity. The addition of isoamylase to the reaction mixture did not prevent the concerted action of Pho1 and BEI. Furthermore, in the product, the branched structure was, at least to some extent, maintained. Based on these results we propose that the interaction between Pho1 and BE is not merely due to chain-elongating and chain-branching reactions, but occurs in a physically and catalytically synergistic manner by each activating the mutual capacity of the other, presumably forming a physical association of Pho1, BEI and branched MDs. This close interaction might play a crucial role in the synthesis of branched MDs and the branched MDs can act as a primer for the biosynthesis of amylopectin molecules. KW - Amylopectin KW - Glucan KW - Phosphorylase KW - Rice KW - Starch KW - Starch branching enzyme Y1 - 2012 U6 - https://doi.org/10.1093/pcp/pcs030 SN - 0032-0781 VL - 53 IS - 5 SP - 869 EP - 878 PB - Oxford Univ. Press CY - Oxford 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 - Hejazi, Mahdi A1 - Steup, Martin A1 - Fettke, Jörg T1 - The plastidial glucan, water dikinase (GWD) catalyses multiple phosphotransfer reactions JF - The FEBS journal N2 - 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. KW - glucan phosphorylation KW - glucan KW - water dikinase KW - protein autophosphorylation KW - starch metabolism KW - starch phosphorylation Y1 - 2012 U6 - https://doi.org/10.1111/j.1742-4658.2012.08576.x SN - 1742-464X VL - 279 IS - 11 SP - 1953 EP - 1966 PB - Wiley-Blackwell CY - Malden 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 -