Integrated functions among multiple starch synthases determine both amylopectin chain length and branch linkage location in Arabidopsis leaf starch

This study assessed the impact on starch metabolism in Arabidopsis leaves of simultaneously eliminating multiple soluble starch synthases (SS) from among SS1, SS2, and SS3. Double mutant ss1- ss2- or ss1- ss3- lines were generated using confirmed null mutations. These were compared to the wild type, each single mutant, and ss1- ss2- ss3- triple mutant lines grown in standardized environments. Double mutant plants developed similarly to the wild type, although they accumulated less leaf starch in both short-day and long-day diurnal cycles. Despite the reduced levels in the double mutants, lines containing only SS2 and SS4, or SS3 and SS4, are able to produce substantial amounts of starch granules. In both double mutants the residual starch was structurally modified including higher ratios of amylose:amylopectin, altered glucan chain length distribution within amylopectin, abnormal granule morphology, and altered placement of α(1→6) branch linkages relative to the reducing end of each linear chain. The data demonstrate that SS activity affects not only chain elongation but also the net result of branch placement accomplished by the balanced activities of starch branching enzymes and starch debranching enzymes. SS3 was shown partially to overlap in function with SS1 for the generation of short glucan chains within amylopectin. Compensatory functions that, in some instances, allow continued residual starch production in the absence of specific SS classes were identified, probaby accomplished by the granule bound starch synthase GBSS1.ANR Génoplante GPLA0611GEuropean Union-FEDER, Région Nord Pas de Calais ARCir PlantTEQ5National Science Foundation DBI-0209789Comisión Interministerial de Ciencia y Tecnología BIO2009-07040Junta de Andalucía P09-CVI-470

Overlapping functions of the starch synthases SSII and SSIII in amylopectin biosynthesis in Arabidopsis

Background: The biochemical mechanisms that determine the molecular architecture of amylopectin are central in plant biology because they allow long-term storage of reduced carbon. Amylopectin structure imparts the ability to form semi-crystalline starch granules, which in turn provides its glucose storage function. The enzymatic steps of amylopectin biosynthesis resemble those of the soluble polymer glycogen, however, the reasons for amylopectin's architectural distinctions are not clearly understood. The multiplicity of starch biosynthetic enzymes conserved in plants likely is involved. For example, amylopectin chain elongation in plants involves five conserved classes of starch synthase (SS), whereas glycogen biosynthesis typically requires only one class of glycogen synthase. Results: Null mutations were characterized in AtSS2, which codes for SSII, and mutant lines were compared to lines lacking SSIII and to an Atss2, Atss3 double mutant. Loss of SSII did not affect growth rate or starch quantity, but caused increased amylose/amylopectin ratio, increased total amylose, and deficiency in amylopectin chains with degree of polymerization (DP) 12 to DP28. In contrast, loss of both SSII and SSIII caused slower plant growth and dramatically reduced starch content. Extreme deficiency in DP12 to DP28 chains occurred in the double mutant, far more severe than the summed changes in SSII- or SSIII-deficient plants lacking only one of the two enzymes. Conclusion: SSII and SSIII have partially redundant functions in determination of amylopectin structure, and these roles cannot be substituted by any other conserved SS, specifically SSI, GBSSI, or SSIV. Even though SSIII is not required for the normal abundance of glucan chains of DP12 to DP18, the enzyme clearly is capable of functioning in production such chains. The role of SSIII in producing these chains cannot be detected simply by analysis of an individual mutation. Competition between different SSs for binding to substrate could in part explain the specific distribution of glucan chains within amylopectin

The priming of storage glucan synthesis from bacteria to plants: current knowledge and new developments.

International audienceSummary Starch is the main polymer in which carbon and energy are stored in land plants, algae and some cyanobacteria. It plays a crucial role in the physiology of these organisms and also represents an important polymer for humans, in terms of both diet and nonfood industry uses. Recent efforts have elucidated most of the steps involved in the synthesis of starch. However, the process that initiates the synthesis of the starch granule remains unclear. Here, we outline the similarities between the synthesis of starch and the synthesis of glycogen, the other widespread and abundant glucose-based polymer in living cells. We place special emphasis on the mechanisms of initiation of the glycogen granule and current knowledge concerning the initiation of the starch granule. We also discuss recent discoveries regarding the function of starch synthases in the priming of the starch granule and possible interactions with other elements of the starch synthesis machinery

18F-FDG PET/CT and MRI of a Mediastinal Malignant Granular Cell Tumor With Associated Recurrent Pericarditis.

We report a rare case of a mediastinal malignant granular cell tumor in a 41-year-old man presenting with dyspnea, retrosternal chest pain, and recurrent pericardial effusion under treatment with corticosteroids. Because of recurrent episodes, further investigations with chest MRI and F-FDG PET/CT revealed a large infiltrating and strongly hypermetabolic mass in the mediastinum with pericarditis. Histopathology and immunohistochemical analysis showed large cells with greater pleomorphism and eosinophilic granular cytoplasm with positive staining for S100 protein and CD68 as well as high Ki67 proliferative index (15%) confirming the diagnosis of a malignant granular cell tumor.status: publishe

The virtual shop floor: a shop floor control environment

The Virtual Shop Floor is an integrated software system for monitoring, management, and control of manufacturing shop floors. It is being developed as a long-term objective of IMTI's Distributed Control project. This report presents the current status of the VSF and outlines a plan for its further development.NRC publication: Ye

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

<div><p>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 <i>in vivo</i> and <i>in vitro</i> over the last years, however, analyses of a functional interaction of SS and BE isoforms are missing so far. Here, we present data from <i>in vitro</i> 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 <i>be</i> single mutants (<i>Atbe2</i> and <i>Atbe3</i> 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.</p></div

The 88 KD DEBRANCHING ENZYME MISSING IN GLYCOGEN ACCUMULATING MUTANTS OF CLAMYDOMONAS REINHARDTII DISPLAYS AN ISOAMYLASE-TYPE SPECIFICITY

International audienceTo investigate the functions of debranching enzymes in starch biosynthesis, we have partially purified and characterized these activities from wild-type and mutant sta7 Clamydomonas Reinhardtii. Two distinct debranching enzymes of 95 and 88 kD were detected. The 88 kD enzyme behaved as a part of a very large homo and heteromultimeric complex containing a minimum of 4 subunits. The 95 kD debranching enzyme cleaved -1,6 linkages separated by as few as 3 glucose residues while the multimeric complex containing the 88 kD hydrolase was unable to do so. Both enzymes were able to debranch amylopectin efficiently while the -1,6 linkages of glycogen were completely debranched by the 88 kD hydrolase only. Therefore the 95 and 88 kD debranching enzymes display respectively the limit-dextrinase (pullulanase) and isoamylase-type specificities. Various mutations in the STA7 locus caused the loss of the 88 kD isoamylase. At variance with the results obtained from maize and rice, however, the isoamylase deficiency did not result in any qualitative or quantitative difference in pullulanase activity. Morever, because the isoamylase activity accounted for over 95% of the total debranching enzyme activity we believe that the relative abundance of both types of debranching enzymes differs markedly from that found in vascular plants. The consequences of these findings with respect to the recently proposed mechanism for plant amylopectin synthesis are discussed

SS activities from recombinant SS isoforms.

<p>0.8 µg of rSSs (rSSI, rSSII, rSSIII and rSSIV) were electrophoretically separated under non-denaturing conditions. (A) Separation gel containing oyster glycogen was incubated with 1 mM ADPglucose. (B) Glucan free gel was incubated with 1 mM ADPglucose in presence of citrate. After incubation overnight at room temperature gels were washed with water and stained with iodine solution.</p

SS activities from different <i>be</i> knock-out plants.

<p>Leaf protein extracts from <i>Atbe3</i>, <i>Atbe2</i>, <i>Atbe3/2</i> and additionally from wild type (Ws) and <i>AtssI</i> were electrophoretically separated under non-denaturing conditions (65 µg protein per lane). (A) Glucan free gel was incubated with 1 mM ADPglucose in presence of citrate (buffer b). (B) Separation gel containing oyster glycogen was incubated with 1 mM ADPglucose (buffer a). After Incubation overnight at room temperature gels were washed with water and stained with iodine solution. Open triangle indicates the position of rubisco.</p