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Abstract Background Starch is stored in higher plants as granules composed of semi-crystalline amylopectin and amorphous amylose. Starch granules provide energy for the plant during dark periods and for germination of seeds and tubers. Dietary starch is also a highly glycemic carbohydrate being degraded to glucose and rapidly absorbed in the small intestine. But a portion of dietary starch, termed “resistant starch” (RS) escapes digestion and reaches the large intestine, where it is fermented by colonic bacteria producing short chain fatty acids (SCFA) which are linked to several health benefits. The RS is preferentially derived from amylose, which can be increased by suppressing amylopectin synthesis by silencing of starch branching enzymes (SBEs). However all the previous works attempting the production of high RS crops resulted in only partly increased amylose-content and/or significant yield loss. Results In this study we invented a new method for silencing of multiple genes. Using a chimeric RNAi hairpin we simultaneously suppressed all genes coding for starch branching enzymes (SBE I, SBE IIa, SBE IIb) in barley (Hordeum vulgare L.), resulting in production of amylose-only starch granules in the endosperm. This trait was segregating 3:1. Amylose-only starch granules were irregularly shaped and showed peculiar thermal properties and crystallinity. Transgenic lines retained high-yield possibly due to a pleiotropic upregualtion of other starch biosynthetic genes compensating the SBEs loss. For gelatinized starch, a very high content of RS (65 %) was observed, which is 2.2-fold higher than control (29%). The amylose-only grains germinated with same frequency as control grains. However, initial growth was delayed in young plants. Conclusions This is the first time that pure amylose has been generated with high yield in a living organism. This was achieved by a new method of simultaneous suppression of the entire complement of genes encoding starch branching enzymes. We demonstrate that amylopectin is not essential for starch granule crystallinity and integrity. However the slower initial growth of shoots from amylose-only grains may be due to an important physiological role played by amylopectin ordered crystallinity for rapid starch remobilization explaining the broad conservation in the plant kingdom of the amylopectin structure.

Lignocellulosic materials from municipal solid waste emerge as attractive resources for anaerobic digestion biorefinery. To increase the knowledge required for establishing efficient bioprocesses, dynamics of batch fermentation by the cellulolytic bacterium Ruminiclostridium cellulolyticum were compared using three cellulosic materials, paper handkerchief, cotton discs and Whatman filter paper. Fermentation of paper handkerchief occurred the fastest and resulted in a specific metabolic profile: it resulted in the lowest acetate-to-lactate and acetate-to-ethanol ratios. By shotgun proteomic analyses of paper handkerchief and Whatman paper incubations, 151 proteins with significantly different levels were detected, including 20 of the 65 cellulosomal components, 8 non-cellulosomal CAZymes and 44 distinct extracytoplasmic proteins. Consistent with the specific metabolic profile observed, many enzymes from the central carbon catabolic pathways had higher levels in paper handkerchief incubations. Among the quantified CAZymes and cellulosomal components, 10 endoglucanases mainly from the GH9 families and 7 other cellulosomal subunits had lower levels in paper handkerchief incubations. An in-depth characterization of the materials used showed that the lower levels of endoglucanases in paper handkerchief incubations could hypothetically result from its lower crystallinity index (50%) and degree of polymerization (970). By contrast, the higher hemicellulose rate in paper handkerchief (13.87%) did not result in the enhanced expression of enzyme with xylanase as primary activity, including enzymes from the “xyl-doc” cluster. It suggests the absence, in this material, of molecular structures that specifically lead to xylanase induction. The integrated approach developed in this work shows that subtle differences among cellulosic materials regarding chemical and structural characteristics have significant effects on expressed bacterial functions, in particular the cellulolysis machinery, resulting in different metabolic patterns and degradation dynamics. [ABSTRACT FROM AUTHOR]

The many studies about amylolysis have collected considerable information regarding the contribution of the starch physico-chemical properties. But the inherent elaborate and variable structure of granular starch and, consequently, the multifactorial condition of the system hinders the interpretation of the experimental results. The immediate benefit of multivariate statistical analysis approaches with that regard is twofold: considering the factors, possibly interrelated, all together and not independently, providing a first estimation of the magnitude and confidence level of the relations between factors and amylolysis kinetic parameters. Based on data of amylolysis of 13 starch samples from wild type, single and double mutants of maize by porcine pancreatic α-amylase (PPA), a multivariate analysis is proposed. Amylolysis progress-curves were fitted by a Weibull function, as proposed in a previous work, to extract three kinetic parameters: the reaction rate coefficient during the first time-unit, k , the reaction rate retardation over time, h , and the final hydrolysis extent, X ∞ . Multivariate models relate the macromolecular composition and the fractions of crystalline polymorphic types to the kinetic parameters. h and X ∞ are found to be highly related to the measured properties. Thus the amylose content appears to be significantly correlated to the hydrolysis rate retardation, which sheds some light on the probable contribution of the amylose molecules contained in the granules. The multivariate models give correct prediction performances except for k whose a part of variability remains unexplained. A further analysis points out the extent of the characterisation effort of the granule structure needed to extend the fraction of explained variability. [ABSTRACT FROM AUTHOR]

ABSTRACT Morphology, molecular structure, and thermal properties of potato starch granules with low to high phosphate content were studied as an effect of mild acid hydrolysis (lintnerization) to 80% solubilization at two temperatures (25 and 45°C). Light microscopy showed that the lintners contained apparently intact granules, which disintegrated into fragments upon dehydration. Transmission electron microscopy of rehydrated lintners revealed lacy networks of smaller subunits. The molecular composition of the lintners suggested that they largely consisted of remnants of crystalline lamellae. When lintnerization was performed at 45°C, the lintners contained more of branched dextrins compared to 25°C in both low and intermediate phosphate-containing samples. High-phosphate-containing starch was, however, unaffected by temperature and this was probably due to an altered amylopectin structure rather than the phosphate content. After lintnerization, the melting endotherms were broad with decreased onset and increased peak melting temperatures. The relative crystallinity was lower in lintners prepared at 45°C. A hypothesis that combines the kinetics of lintnerization with the molecular and thermal characteristics of the lintners is presented. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 257-271, 2014. [ABSTRACT FROM AUTHOR]

Abstract: Background: Native starch accumulates as granules containing two glucose polymers: amylose and amylopectin. Phosphate (0.2–0.5%) and proteins (0.1–0.7%) are also present in some starches. Phosphate groups play a major role in starch metabolism while granule-bound starch synthase 1 (GBSS1) which represents up to 95% of the proteins bound to the granule is responsible for amylose biosynthesis. Methods: Synchrotron micro-X-ray fluorescence (μXRF) was used for the first time for high-resolution mapping of GBSS1 and phosphate groups based on the XRF signal of sulfur (S) and phosphorus (P), respectively. Wild-type starches were studied as well as their related mutants lacking GBSS1 or starch-phosphorylating enzyme. Results: Wild-type potato and maize starch exhibited high level of phosphorylation and high content of sulfur respectively when compared to mutant potato starch lacking glucan water dikinase (GWD) and mutant maize starch lacking GBSS1. Phosphate groups are mostly present at the periphery of wild-type potato starch granules, and spread all over the granule in the amylose-free mutant. P and S XRF were also measured within single small starch granules from Arabidopsis or Chlamydomonas not exceeding 3–5μm in diameter. Conclusions: Imaging GBSS1 (by S mapping) in potato starch sections showed that the antisense technique suppresses the expression of GBSS1 during biosynthesis. P mapping confirmed that amylose is mostly present in the center of the granule, which had been suggested before. General significance: μXRF is a potentially powerful technique to analyze the minor constituents of starch and understand starch structure/properties or biosynthesis by the use of selected genetic backgrounds. [Copyright &y& Elsevier]

Single-celled apicomplexan parasites are known to cause major diseases in humans and animals including malaria, toxoplasmosis, and coccidiosis. The presence of apicoplasts with the remnant of a plastid-like DNA argues that these parasites evolved from photosynthetic ancestors possibly related to the dinoflagellates.Toxoplasma gondiidisplays amylopectin-like polymers within the cytoplasm of the dormant brain cysts. Here we report a detailed structural and comparative analysis of theToxoplasma gondii, green algaChlamydomonas reinhardtii, and dinoflagellateCrypthecodinium cohniistorage polysaccharides. We showToxoplasma gondiiamylopectin to be similar to the semicrystalline floridean starch accumulated by red algae. Unlike green plants or algae, the nuclear DNA sequences as well as biochemical and phylogenetic analysis argue that theToxoplasma gondiiamylopectin pathway has evolved from a totally different UDP-glucose-based metabolism similar to that of the floridean starch accumulating red algaCyanidioschyzon merolaeand, to a lesser extent, to those of glycogen storing animals or fungi. In both red algae and apicomplexan parasites, isoamylase and glucan-water dikinase sequences are proposed to explain the appearance of semicrystalline starch-like polymers. Our results have built a case for the separate evolution of semicrystalline storage polysaccharides upon acquisition of photosynthesis in eukaryotes. [ABSTRACT FROM AUTHOR]

Whereas Glc is stored in small-sized hydrosoluble glycogen particles in archaea, eubacteria, fungi, and animal cells, photosynthetic eukaryotes have resorted to building starch, which is composed of several distinct polysaccharide fractions packed into a highly organized semicrystalline granule. In plants, both the initiation of polysaccharide synthesis and the nucleation mechanism leading to formation of new starch granules are currently not understood. Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, defines the tiniest eukaryote with one of the smallest genomes. We show that it accumulates a single starch granule at the chloroplast center by using the same pathway as higher plants. At the time of plastid division, we observe elongation of the starch and division into two daughter structures that are partitioned in each newly formed chloroplast. These observations suggest that in this system the information required to initiate crystalline polysaccharide growth of a new granule is contained within the preexisting polysaccharide structure and the design of the plastid division machinery. [ABSTRACT FROM AUTHOR]

Two new polysaccharides were isolated from the cell walls of the carrageenan producing red seaweed Kappaphycus alvarezii (Doty) Doty. They were characterized by chemical analyses, enzymatic degradations, and nuclear magnetic resonance spectroscopy. One was a 4.0 M NaOH soluble β-(1,4)-d-glucomannan that mostly precipitated upon neutralization and dialysis. It was composed of about 82 residues, and 70% of its glucose and mannose were released by a commercial cellulase enzyme complex. The disaccharide β-d-Man (1→4) d-Glc was recovered from the hydrolysate during the first hours of degradation and confirmed the chemical structure of the polysaccharide. The other polysaccharide was extracted with 1.5 M NaOH and was identified as a sulfated glucan of degree of polymerization of about 180 1,4-linked β-glucose containing 10% 1,3-linkages. The sulfate was located on C-6 of 64% of the 4-linked glucose residues. A third alkali-soluble polysaccharide rich in galactose was also detected. The distribution of the glucomannan and galactose containing polysaccharides was inversely related to the algal cell size. Potential functions of these alkali-soluble polymers are discussed in the context of cell wall polysaccharide assembly. [ABSTRACT FROM AUTHOR]