Simultaneous antisense inhibition of two starch-synthase isoforms in potato tubers leads to accumulation of grossly modified amylopectin.
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A chimaeric antisense construct was used to reduce the activities of the two major starch-synthase isoforms in potato tubers simultaneously. A range of reductions in total starch-synthase activities were found in the resulting transgenic plants, up to a maximum of 90% inhibition. The reduction in starch-synthase activity had a profound effect on the starch granules, which became extremely distorted in appearance compared with the control lines. Analysis of the starch indicated that the amounts produced in the tubers, and the amylose content of the starch, were not affected by the reduction in activity. In order to understand why the starch granules were distorted, amylopectin was isolated and the constituent chain lengths analysed. This indicated that the amylopectin was very different to that of the control. It contained more chains of fewer than 15 glucose units in length, and fewer of between 15 and 80 glucose units. In addition, the amylopectin contained more very long chains. Amylopectin from plants repressed in just one of the activities of the two starch-synthase isoforms, which we have reported upon previously, were also analysed. Using a technique different to that used previously we show that both isoforms also affect the amylopectin, but in a way that is different to when both isoforms are repressed together. (+info)
Amylopectinosis in fetal and neonatal Quarter Horses.
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Three Quarter Horses, a stillborn filly (horse No. 1), a female fetus aborted at approximately 6 months of gestation (horse No. 2), and a 1-month-old colt that had been weak at birth (horse No. 3), had myopathy characterized histologically by large spherical or ovoid inclusions in skeletal and cardiac myofibers. Smaller inclusions were also found in brain and spinal cord and in some cells of all other tissues examined. These inclusions were basophilic, red-purple after staining with periodic acid-Schiff (both before and after digestion with diastase), and moderately dark blue after staining with toluidine blue. The inclusions did not react when stained with Congo red. Staining with iodine ranged from pale blue to black. Their ultrastructural appearance varied from amorphous to somewhat filamentous. On the basis of staining characteristics and diastase resistance, we concluded that these inclusions contained amylopectin. A distinctly different kind of inclusion material was also present in skeletal muscle and tongue of horse Nos. 1 and 3. These inclusions were crystalline with a sharply defined ultrastructural periodicity. The crystals were eosinophilic and very dark blue when stained with toluidine blue but did not stain with iodine. Crystals sometimes occurred freely within the myofibers but more often were encased by deposits of amylopectin. This combination of histologic and ultrastructural features characterizes a previously unreported storage disease in fetal and neonatal Quarter Horses, with findings similar to those of glycogen storage disease type IV. We speculate that a severe inherited loss of glycogen brancher enzyme activity may be responsible for these findings. The relation of amylopectinosis to the death of the foals is unknown. (+info)
Essential arginine residues in maize starch synthase IIa are involved in both ADP-glucose and primer binding.
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The arginine-specific reagent phenylglyoxal inactivated the activity of maize starch synthase IIa (SSIIa), due to the modification of at least one arginine residue out of a possible 42. The addition of ADPGlc completely protected SSIIa from the inactivation, indicating that arginine may be involved in the interaction of this anionic substrate with SSIIa. However, site-directed mutagenesis of the conserved Arg-214 in SSIIa showed that this amino acid is important for apparent affinity of SSIIa for its primer (amylopectin and glycogen), as evidenced by a marked increase in the K(m) for primer upon substitution of this amino acid with no concomitant change in V(max), K(m) for ADPGlc, or secondary structure. Therefore, Arg-214 of SSIIa appears to play a role in its primer binding. (+info)
Interaction with amylopectin influences the ability of granule-bound starch synthase I to elongate malto-oligosaccharides.
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This paper examines the properties in soluble form of two isoforms of starch synthase. One of these, granule-bound starch synthase I (GBSSI), is responsible for the synthesis of amylose inside the amylopectin matrix of the starch granule in vivo. The other, starch synthase II (SSII), is involved in amylopectin synthesis. Both isoforms can use amylopectin and malto-oligosaccharide as substrates in vitro. As well as acting as a substrate for GBSSI, amylopectin acts as an effector of this isoform, increasing the rate at which it elongates malto-oligosaccharides and promoting a processive rather than distributive mode of elongation of these compounds. The affinity of GBSSI for amylopectin as an effector is greater than its affinity for amylopectin as a substrate. The rate and mode of elongation of malto-oligosaccharides by SSII are not influenced by amylopectin. These results suggest that specific interaction with amylopectin in the matrix of the starch granule is a unique property of GBSSI and is critical in determining the nature of its products. (+info)
In vitro utilization of amylopectin and high-amylose maize (Amylomaize) starch granules by human colonic bacteria.
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It has been well established that a certain amount of ingested starch can escape digestion in the human small intestine and consequently enters the large intestine, where it may serve as a carbon source for bacterial fermentation. Thirty-eight types of human colonic bacteria were screened for their capacity to utilize soluble starch, gelatinized amylopectin maize starch, and high-amylose maize starch granules by measuring the clear zones on starch agar plates. The six cultures which produced clear zones on amylopectin maize starch- containing plates were selected for further studies for utilization of amylopectin maize starch and high-amylose maize starch granules A (amylose; Sigma) and B (Culture Pro 958N). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect bacterial starch-degrading enzymes. It was demonstrated that Bifidobacterium spp., Bacteroides spp., Fusobacterium spp., and strains of Eubacterium, Clostridium, Streptococcus, and Propionibacterium could hydrolyze the gelatinized amylopectin maize starch, while only Bifidobacterium spp. and Clostridium butyricum could efficiently utilize high-amylose maize starch granules. In fact, C. butyricum and Bifidobacterium spp. had higher specific growth rates in the autoclaved medium containing high-amylose maize starch granules and hydrolyzed 80 and 40% of the amylose, respectively. Starch-degrading enzymes were cell bound on Bifidobacterium and Bacteroides cells and were extracellular for C. butyricum. Active staining for starch-degrading enzymes on SDS-PAGE gels showed that the Bifidobacterium cells produced several starch-degrading enzymes with high relative molecular (M(r)) weights (>160,000), medium-sized relative molecular weights (>66,000), and low relative molecular weights (<66,000). It was concluded that Bifidobacterium spp. and C. butyricum degraded and utilized granules of amylomaize starch. (+info)
Specificity of starch synthase isoforms from potato.
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In higher plants several isoforms of starch synthase contribute to the extension of glucan chains in the synthesis of starch. Different isoforms are responsible for the synthesis of essentially linear amylose chains and branched, amylopectin chains. The activity of granule-bound starch synthase I from potato has been compared with that of starch synthase II from potato following expression of both isoforms in Escherichia coli. Significant differences in their activities are apparent which may be important in determining their specificities in vivo. These differences include affinities for ADPglucose and glucan substrates, activation by amylopectin, response to citrate, thermosensitivity and the processivity of glucan chain extension. To define regions of the isoforms determining these characteristic traits, chimeric proteins have been produced by expression in E. coli. These experiments reveal that the C-terminal region of granule-bound starch synthase I confers most of the specific properties of this isoform, except its processive elongation of glucan chains. This region of granule-bound starch synthase I is distinct from the C-terminal region of other starch synthases. The specific properties it confers may be important in defining the specificity of granule-bound starch synthase I in producing amylose in vivo. (+info)
Identification of the maize amyloplast stromal 112-kD protein as a plastidic starch phosphorylase.
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Amyloplast is the site of starch synthesis in the storage tissue of maize (Zea mays). The amyloplast stroma contains an enriched group of proteins when compared with the whole endosperm. Proteins with molecular masses of 76 and 85 kD have been identified as starch synthase I and starch branching enzyme IIb, respectively. A 112-kD protein was isolated from the stromal fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to tryptic digestion and amino acid sequence analysis. Three peptide sequences showed high identity to plastidic forms of starch phosphorylase (SP) from sweet potato, potato, and spinach. SP activity was identified in the amyloplast stromal fraction and was enriched 4-fold when compared with the activity in the whole endosperm fraction. Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that SP activity was associated with the amyloplast stromal 112-kD protein. In addition, antibodies raised against the potato plastidic SP recognized the amyloplast stromal 112-kD protein. The amyloplast stromal 112-kD SP was expressed in whole endosperm isolated from maize harvested 9 to 24 d after pollination. Results of affinity electrophoresis and enzyme kinetic analyses showed that the amyloplast stromal 112-kD SP preferred amylopectin over glycogen as a substrate in the synthetic reaction. The maize shrunken-4 mutant had reduced SP activity due to a decrease of the amyloplast stromal 112-kD enzyme. (+info)
Two loci control phytoglycogen production in the monocellular green alga Chlamydomonas reinhardtii.
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The STA8 locus of Chlamydomonas reinhardtii was identified in a genetic screen as a factor that controls starch biosynthesis. Mutations of STA8 cause a significant reduction in the amount of granular starch produced during nutrient limitation and accumulate phytoglycogen. The granules remaining in sta8 mutants are misshapen, and the abundance of amylose and long chains in amylopectin is altered. Mutations of the STA7 locus, which completely lack isoamylase activity, also cause accumulation of phytoglycogen, although sta8 and sta7 mutants differ in that there is a complete loss of granular starch in the latter. This is the first instance in which mutations of two different genetic elements in one plant species have been shown to cause phytoglycogen accumulation. An analytical procedure that allows assay of isoamylase in total extracts was developed and used to show that sta8 mutations cause a 65% reduction in the level of this activity. All other enzymes known to be involved in starch biosynthesis were shown to be unaffected in sta8 mutants. The same amount of total isoamylase activity (approximately) as that present in sta8 mutants was observed in heterozygous triploids containing two sta7 mutant alleles and one wild-type allele. This strain, however, accumulates normal levels of starch granules and lacks phytoglycogen. The total level of isoamylase activity, therefore, is not the major determinant of whether granule production is reduced and phytoglycogen accumulates. Instead, a qualitative property of the isoamylase that is affected by the sta8 mutation is likely to be the critical factor in phytoglycogen production. (+info)