SummaryGlucan, 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, b-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.

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 10-15 million 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.

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-14C]glucose 1-phosphate, [U-14C]sucrose, [U-14C]glucose 1-phosphate plus unlabelled equimolar sucrose or [U-14C]sucrose plus unlabelled equimolar glucose 1-phosphate. 14C-incorporation into starch was monitored. In slices from freshly harvested tubers each unlabelled compound strongly enhanced 14C 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 °C but the flux of the sucrose-dependent route strongly increases above 20 °C. Results are confirmed by in vitro experiments using [U-14C]glucose 1-phosphate or adenosine-[U-14C]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 14C-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.

Essentially all living systems are capable of synthesizing large glycans, such as arabinogalactan, xylan, cellulose, fructan, laminarin, glycogen or the two starch-related polyglucans, amylose and amylopectin. Each of these glycans exists as a large group of chemically closely related but polydisperse biopolymers rather than as a distinct macromolecule. The biological functions exerted by glycans are highly heterogeneous and range from shaping and/or protecting cells or tissues to intra-cellular storage of reduced carbon including the realm of molecular interactions between plant cells or between a plant and a non-plant organism. Glycans have an enormous impact on human diet.1,2) Furthermore, polysaccharides are gaining increasing importance for several biotechnological applications and energy-converting processes that all are based on photosynthesis-driven biosynthesis of carbohydrates. Naturally occurring polyglucans are usually composed of either of two cyclic hemiacetals, designated as α-or β-glucopyranosyl residues. Both six-membered rings are interconnected by only one or two type(s) of precisely defined glucosidic linkages. The anomeric form of the pyranosyl residues and the type(s) of interglucose bonds largely determine the structure of the entire polysaccharide.As an extracellular product of plant cells, cellulose is composed of β-D-glucopyranosyl residues that are interconnected by β-1,4-bonds. By contrast, starch and glycogen are typical intracellular storage carbohydrate of plants and animals, respective. Both starch and glycogen are exclusively built of α-D-glucopyranosyl residues that are linked by two types of interglucose bonds, α-1,4-and α-1,6-linkages. Despite the chemical similarity, important physicochemical differences exist: Native starch is deposited as water-insoluble particles designated as granules. Native starch particles are capable of an essentially unlimited growth. Glycogen is a hydro-soluble polydispers molecule which appears to possess a strict upper size limit (for review see Ref. 9 11)).In photosynthesis-competent eukaryots and their heterotrophic derivatives starch granules act as the almost ubiquitously occurring storage carbohydrate that allows central carbon metabolism and growth to continue when photosynthesis is not functional and, at the same time, to prevent carbon starvation. Starch may, however, share these functions with other reduced carbon compounds, such as sucrose, fructans or even soluble non-carbohydrates and both accumulation and utilization of the various compounds are remarkably flexible. In green algae, mosses, ferns and higher plants, starch granules are synthesized inside the plastidial compartment but in many other eukaryotic algae the cytosol is the princi- We then give a schematic presentation of the structure of starch synthases from Arabidopsis thaliana and discuss the action of the different isoforms. The two types of phosphorylases that are common in plants are described and their in vivo actions are presented. We describe functional properties of...

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.