Search

Phosphorylation and dephosphorylation of starch and glycogen are important for their physicochemical properties and also their physiological functions. It is therefore desirable to reliably determine the phosphorylation sites. Heteronuclear multidimensional NMR-spectroscopy is in principle a straightforward analytical approach even for complex carbohydrate molecules. With heterogeneous samples from natural sources, however, the task becomes more difficult because a full assignment of the resonances of the carbohydrates is impossible to obtain. Here, we show that the combination of heteronuclear (1) H,(13) C and (1) H,(13) C,(31) P techniques and information derived from spectra of a set of reference compounds can lead to an unambiguous determination of the phosphorylation sites even in heterogeneous samples.

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 α-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 α-glucans, the two mutated dikinase proteins are capable of degrading ATP. Thus, consumption of ATP and phosphorylation of α-glucans are not strictly coupled processes but, to some extent, occur as independent phosphotransfer reactions. As revealed by incubation of the GWDs with [γ-33P]ATP, the consumption of ATP includes the transfer of the γ-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, α-glucans, AMP and ADP. A model is presented describing the complex phosphotransfer reactions of GWDs as affected by the availability of the various acceptors. Structured digital abstract • StGWD autophosphorylates by protein kinase assay (View interaction) • AtGWD autophosphorylates by protein kinase assay (View interaction)

Biomarkers are used to predict phenotypical properties before these features become apparent and, therefore, are valuable tools for both fundamental and applied research. Diagnostic biomarkers have been discovered in medicine many decades ago and are now commonly applied. While this is routine in the field of medicine, it is of surprise that in agriculture this approach has never been investigated. Up to now, the prediction of phenotypes in plants was based on growing plants and assaying the organs of interest in a time intensive process. For the first time, we demonstrate in this study the application of metabolomics to predict agronomic important phenotypes of a crop plant that was grown in different environments. Our procedure consists of established techniques to screen untargeted for a large amount of metabolites in parallel, in combination with machine learning methods. By using this combination of metabolomics and biomathematical tools metabolites were identified that can be used as biomarkers to improve the prediction of traits. The predictive metabolites can be selected and used subsequently to develop fast, targeted and low-cost diagnostic biomarker assays that can be implemented in breeding programs or quality assessment analysis. The identified metabolic biomarkers allow for the prediction of crop product quality. Furthermore, marker-assisted selection can benefit from the discovery of metabolic biomarkers when other molecular markers come to its limitation. The described marker selection method was developed for potato tubers, but is generally applicable to any crop and trait as it functions independently of genomic information.

Arabidopsis thaliana mutants impaired in starch biosynthesis due to defects in either ADP glucose pyrophosphorylase (adg1-1), plastidic phosphoglucose mutase (pgm) or a new allele of plastidic phosphoglucose isomerase (pgi1-2) exhibit substantial activity of glucose-6-phosphate (Glc6P) transport in leaves that is mediated by a Glc6P/phosphate translocator (GPT) of the inner plastid envelope membrane. In contrast to the wild type, GPT2, one of two functional GPT genes of A. thaliana, is strongly induced in these mutants during the light period. The proposed function of the GPT in plastids of non-green tissues is the provision of Glc6P for starch biosynthesis and/or the oxidative pentose phosphate pathway. The function of GPT in photosynthetic tissues, however, remains obscure. The adg1-1 and pgi1-2 mutants were crossed with the gpt2-1 mutant defective in GPT2. Whereas adg1-1/gpt2-1 was starch-free, residual starch could be detected in pgi1-2/gpt2-1 and was confined to stomatal guard cells, bundle sheath cells and root tips, which parallels the reported spatial expression profile of AtGPT1. Glucose content in the cytosolic heteroglycan increased substantially in adg1-1 but decreased in pgi1-2, suggesting that the plastidic Glc6P pool contributes to its biosynthesis. The abundance of GPT2 mRNA correlates with increased levels of soluble sugars, in particular of glucose in leaves, suggesting induction by the sugar-sensing pathway. The possible function of GPT2 in starch-free mutants is discussed in the background of carbon requirement in leaves during the light-dark cycle.

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.

Among the three distinct starch phosphorylase activities detected in Chlamydomonas reinhardtii, two distinct plastidial enzymes (PhoA and PhoB) are documented while a single extraplastidial form (PhoC) displays a higher affinity for glycogen as in vascular plants. The two plastidial phosphorylases are shown to function as homodimers containing two 91-kDa (PhoA) subunits and two 110-kDa (PhoB) subunits. Both lack the typical 80-amino-acid insertion found in the higher plant plastidial forms. PhoB is exquisitely sensitive to inhibition by ADP-glucose and has a low affinity for malto-oligosaccharides. PhoA is more similar to the higher plant plastidial phosphorylases: it is moderately sensitive to ADP-glucose inhibition and has a high affinity for unbranched malto-oligosaccharides. Molecular analysis establishes that STA4 encodes PhoB. Chlamydomonas reinhardtii strains carrying mutations at the STA4 locus display a significant decrease in amounts of starch during storage that correlates with the accumulation of abnormally shaped granules containing a modified amylopectin structure and a high amylose content. The wild-type phenotype could be rescued by reintroduction of the cloned wild-type genomic DNA, thereby demonstrating the involvement of phosphorylase in storage starch synthesis.

Glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) are required for normal starch metabo- lism. We analysed starch phosphorylation in Arabidopsis wild- type plants and mutants lacking either GWD or PWD using 31 P NMR. Phosphorylation at both C6- and C3-positions of glu- cose moieties in starch was drastically decreased in GWD-defi- cient mutants. In starch from PWD-deficient plants C3-bound phosphate was reduced to levels close to the detection limit. The latter result contrasts with previous reports according to which GWD phosphorylates both C6- and C3-positions. In these studies, phosphorylation had been analysed by HPLC of acid- hydrolysed glucans. We now show that maltose-6-phosphate, a product of incomplete starch hydrolysis, co-eluted with glu- cose-3-phosphate under the chromatographic conditions applied. Re-examination of the specificity of the dikinases using an im- proved method demonstrates that C6- and C3-phosphorylation is selectively catalysed by GWD and PWD, respectively. 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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(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

Saccharomyces cerevisiae possesses two glycogenin isoforms (designated as Glg1p and Glg2p) that both contain a conserved tyrosine residue, Tyr232. However, Glg2p possesses an additional tyrosine residue, Tyr230 and therefore two potential autoglucosylation sites. Glucosylation of Glg2p was studied using both matrix-assisted laser desorption ionization and electrospray quadrupole time of flight mass spectrometry. Glg2p, carrying a C-terminal (His6) tag, was produced in Escherichia coli and purified. By tryptic digestion and reversed phase chromatography a peptide (residues 219–246 of the complete Glg2p sequence) was isolated that contained 4–25 glucosyl residues. Following incubation of Glg2p with UDPglucose, more than 36 glucosyl residues were covalently bound to this peptide. Using a combination of cyanogen bromide cleavage of the protein backbone, enzymatic hydrolysis of glycosidic bonds and reversed phase chromatography, mono- and diglucosylated peptides having the sequence PNYGYQSSPAM were generated. MS/MS spectra revealed that glucosyl residues were attached to both Tyr232 and Tyr230 within the same peptide. The formation of the highly glucosylated eukaryotic Glg2p did not favour the bacterial glycogen accumulation. Under various experimental conditions Glg2p-producing cells accumulated approximately 30% less glycogen than a control transformed with a Glg2p lacking plasmid. The size distribution of the glycogen and extractable activities of several glycogen-related enzymes were essentially unchanged. As revealed by high performance anion exchange chromatography, the intracellular maltooligosaccharide pattern of the bacterial cells expressing the functional eukaryotic transgene was significantly altered. Thus, the eukaryotic glycogenin appears to be incompatible with the bacterial initiation of glycogen biosynthesis.

Two monoclonal anti-diuron antibodies were generated that bind to diuron with an extremely low equilibrium dissociation constant. The antibodies prevented and restored in vitro and in vivo the diuron-dependent inhibition of photosynthesis. In isolated thylakoids prepared from spinach leaves (Spinacia oleracea L.) the diuron-inhibited Hill reaction was reconstituted immediately after the addition of the monoclonal antibodies. The antibodies also restored the diuron-dependent inhibition of the photosynthetic oxygen evolution of the cell wall-deficient mutant cw15 of the green alga Chlamydomonas reinhardtii Dangeard.

Second harmonic generation (SHG) is a nonlinear optical process that inherently generates signal in non-centrosymmetric materials, such as starch granules, and therefore can be used for label-free imaging. Both intensity and polarization of SHG are determined by material properties that are characterized by the nonlinear susceptibility tensor, χ(2). Examination of the tensor is performed for each focal volume (voxel) of the image by measuring the outgoing polarization state of the SHG signal for a set of incoming laser beam polarizations. Mapping of nonlinear properties expressed as the susceptibility ratio reveals structural features including the organization of crystalline material within a single starch granule, and the distribution of structural properties in a population of granules. Isolated granules, as well as in situ starch, can be analyzed using polarimetric SHG microscopy. Due to the fast sample preparation and short imaging times, polarimetric SHG microscopy allows for a quick assessment of starch structure and permits rapid feedback for bioengineering applications. This article presents the basics of SHG theory and microscopy applications for starch-containing materials. Quantification of ultrastructural features within individual starch granules is described. New results obtained by polarization resolved SHG microscopy of starch granules are presented for various maize genotypes revealing heterogeneity within a single starch particle and between various granules.

Starch phosphorylation by glucan, water dikinase (GWD; EC 2.7.9.4) is an essential step in the breakdown of native starch particles, but the underlying mechanisms have remained obscure. In this paper, the initial reactions of starch degradation were analyzed using crystallized maltodextrins as model carbohydrates. As revealed by X-ray diffraction analysis, the crystallized maltodextrins represent the B-type starch allomorph. Recombinant GWD phosphorylated crystalline maltodextrins with a high specific activity (55-60 nmol mg-1 protein min-1), but exhibited very little activity with the same maltodextrins that had been solubilized by heat treatment. Recombinant phosphoglucan, water dikinase (PWD; EC 2.7.9.5) utilized the crystalline maltodextrins only when pre-phosphorylated by GWD. Phosphorylation of crystalline maltodextrins, as catalyzed by GWD, initiated solubilization of neutral as well as phosphorylated glucans. In both the insoluble and the soluble state, mono-, di- and triphosphorylated alpha-glucans were observed, with wide and overlapping ranges of degree of polymerization. Thus, the substrate specificity of the GWD is defined by the physical arrangement of alpha-glucans rather than by structural parameters, such as the distribution of branching points or degree of polymerization. Unlike GWD and PWD, recombinant beta-amylase isozyme 3 (BAM3), which has been shown to be essential for plastidial starch degradation, preferentially degraded soluble maltodextrins rather than crystallized glucans. In summary, two conclusions were reached. Firstly, carbohydrate targets of GWD are primarily defined by the molecular order of glucan helices. Secondly, GWD-catalyzed phosphorylation mediates the phase transition of glucans from a highly ordered to a less ordered and hydrated state.

Extracts of Synechococcus leopoliensis (Anacystis nidulans) contain two forms of D-fructose-1,6-bisphosphatase (EC 3.1.3.11) previously designated as forms A and B [Gerbling, K.-P., Steup, M., and Latzko, E. (1984) Arch. Microbiol. 137, 109-114]. Form B, which probably represents the major part of the total extractable fructose-1,6-bisphosphatase activity, has been purified to apparent homogeneity. Gel filtration, non-denaturing polyacrylamide gel electrophoresis, and cross-linking with bis(sulfosuccinimidyl)suberate revealed that the fructose-1,6-bisphosphatase B exists in either a dimeric or in a tetrameric subform, depending upon the absence or presence of fructose-1,6-bisphosphate and Mg2+. The dimer--tetramer interconversion was readily reversible. The results provide evidence for a two-step activation of fructose-1,6-bisphosphatase B involving the reduction of the dimeric subform and the subsequent substrate-dependent conversion of the reduced dimer to a reduced tetramer, which is the only catalytically active state. In contrast to form B, no substrate-dependent interconversion was detected with form A from S. leopoliensis.

α-1,4-Glucan phosphorylase (EC 2.4.1.1) forms from light or dark grown shoots of Pisum sativum L. cv. ‘Kleine Rheinlanderin’ have been studied using various electrophoretic techniques. The phosphorylase patterns of green and etiolated shoots differed. Etiolated shoots contained two enzyme forms, one residing inside and the other outside the etioplast; this was shown by electrophoresis of extracts of isolated etioplasts. Purity and intactness of the organelle preparation were ascertained by electron microscopy. Light-grown shoots contained, in addition to these two enzyme forms, a third phosphorylase which appears to be chloroplast-specific. The two plastidic phosphorylase forms differed slightly in their apparent molecular masses (as determined by non-denaturing polyacrylamide gel electrophoresis) and in their affinities towards branched polyglucans (as revealed by affinity electrophoresis). The apparent affinity of the extrachloroplastic phosphorylase form to these polyglucans was orders of magnitude higher than that of the two plastidic enzyme forms. The development of the chloroplast-specific phosphorylase pattern is under photocontrol. Investigations performed with red or far-red illuminated wild-type plants and with a pale mutant which has a highly reduced pigment and thylakoid content suggest that this photocontrol is mediated by phytochrome.

In extracts of the unicellular green alga Eremosphaera viridis DeBary (Chlorococcales, Chlorophyceae) the average specific activity of α-1,4-glucan phosphorylase (E.C. 2.4.1.1) was 200 nmol glucose 1-phosphate formed per min and mg protein. Using continuous and discontinuous electrophoresis on polyacrylamide gels, three phosphorylase forms were found. When the log of the relative mobility of the three enzyme forms was plotted versus the acrylamide gel concentration (Ferguson plot) parallel lines were obtained, indicating that the three enzymes were indiscernible with respect to molecular weight. Electrophoresis on density gradient gels resulted in three activity zones lying close to each other. The relative molecular mass (Mr) of the three enzymes was estimated to be around 180,000 with a difference of less than 7,000 between the small and the large forms.