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At least 19 recordsLinked to original sources
PubMed1982

Soluble starch synthases and starch branching enzymes from cotyledons of smooth- and wrinkled-seeded lines of Pisum sativum L.

Soluble starch synthase and branching enzyme were purified from 18-day-old cotyledons of the smooth-seeded pea cultivar Alaska (RR) and wrinkled-seeded pea cultivar Progress #9 (rr) by DEAE-cellulose chromatography. Two coeluting peaks of primed and citrate-stimulated starch synthase activity and a major and minor peak of branching enzyme activity were observed in Alaska. However, in Progress #9, only one peak of synthase activity was found. When crude extracts of Progress #9 were centrifuged, over 70% of the starch synthase activity was recovered in the pelleted fraction, and additional washings of the pellet released no further activity. The addition of purified starch granules to Alaska crude extracts also resulted in the recovery of a greater proportion of synthase activity in pelleted fractions. The two peaks of branching enzyme activity in Alaska differed in their stimulation of phosphorylase, amylose branching activity, and activity in various buffers. The DEAE-cellulose profile of Progress #9 showed no distinct peak of branching enzyme and less than 10% of the total activity found in Alaska. The association of one form of soluble starch synthase with the pelleted fraction and the greatly reduced levels of branching enzyme provide a partial explanation for the appearance of high-amylose starch in Progress #9 cotyledons.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1993

Soluble isoforms of starch synthase and starch-branching enzyme also occur within starch granules in developing pea embryos.

Developing wild-type pea embryos contain two major isoforms of starch synthase and two isoforms of starch-branching enzyme. One of the starch synthases and both starch-branching enzymes occur both in the soluble fraction and tightly bound to starch granules. The other starch synthase, which is very similar to the waxy proteins of other species, is exclusively granule-bound., It is inactive when solubilized in a native form from starch granules, but activity is recovered when the SDS-denatured protein is reconstituted from polyacrylamide gels. Evidence is presented which indicates that all of these proteins become incorporated within the structure of the granule as it grows. It is proposed that the granule-bound waxy protein is active in vivo at the granule surface, whereas the remaining proteins are active in the soluble fraction of the amyloplast. The proteins become trapped within the granule matrix as the polymers they synthesize crystallize around them, and they probably play no further part in polymer synthesis.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1996

Physical association of starch biosynthetic enzymes with starch granules of maize endosperm. Granule-associated forms of starch synthase I and starch branching enzyme II.

Antibodies were used to probe the degree of association of starch biosynthetic enzymes with starch granules isolated from maize (Zea mays) endosperm. Graded washings of the starch granule, followed by release of polypeptides by gelatinization in 2% sodium dodecyl sulfate, enables distinction between strongly and loosely adherent proteins. Mild aqueous washing of granules resulted in near-complete solubilization of ADP-glucose pyrophosphorylase, indicating that little, if any, ADP-glucose pyrophosphorylase is granule associated. In contrast, all of the waxy protein plus significant levels of starch synthase I and starch branching enzyme II (BEII) remained granule associated. Stringent washings using protease and detergent demonstrated that the waxy protein, more than 85% total endosperm starch synthase I protein, and more than 45% of BEII protein were strongly associated with starch granules. Rates of polypeptide accumulation within starch granules remained constant during endosperm development. Soluble and granule-derived forms of BEII yielded identical peptide maps and overlapping tryptic fragments closely aligned with deduced amino acid sequences from BEII cDNA clones. These observations provide direct evidence that BEII exits as both soluble and granule-associated entities. We conclude that each of the known starch biosynthetic enzymes in maize endosperm exhibits a differential propensity to associate with, or to become irreversibly entrapped within, the starch granule.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1991

Molecular identification and comparison of the starch synthase bound to starch granules between endosperm and leaf blades in rice plants.

Normal (nonglutinous) rice plants (Oryza sativa and O. glaberrima) contain more than 18% amylose in endosperm starch, while waxy (glutinous) plants lack it in this starch. In contrast, leaf starch contained more than 3.6% amylose even in waxy plants. SDS-PAGE analysis of proteins bound to endosperm starch granules in the normal plants revealed a single band with a Mr of 60 kd, whereas waxy plants did not exhibit a similar band. The activity of starch synthase (NDP-glucose-starch glucosyltransferase) was completely inhibited by antibody against the 60-kd protein. Thus, we conclude that the 60-kd protein is the waxy protein encoded by the Wx allele, which also plays a role in the synthesis of nonglutinous starch in endosperm tissue. In leaf blades, the proteins bound to starch granules separated into five bands with Mr's of 53.6 to 64.9 kd on SDS-PAGE. Analysis of these proteins by immunoblotting using antiserum against Wx protein and inhibition of starch synthase activity by the synthase antibody revealed that none of these proteins was homologous to Wx protein. We suggest that the synthesis of amylose in leaf blades is brought about by a protein encoded by a gene(s) different from the Wx gene expressed in the endosperm.

Amylose↗
PubMed1999

Granule-bound starch synthase I in isolated starch granules elongates malto-oligosaccharides processively.

Isoforms of starch synthase belonging to the granule-bound starch synthase I (GBSSI) class synthesize the amylose component of starch in plants. Other granule-bound isoforms of starch synthase, such as starch synthase II (SSII), are unable to synthesize amylose. The kinetic properties of GBSSI and SSII that are responsible for these functional differences have been investigated using starch granules from embryos of wild-type peas and rug5 and lam mutant peas, which contain, respectively, both GBSSI and SSII, GBSSI but not SSII and SSII but not GBSSI. We show that GBSSI in isolated granules elongates malto-oligosaccharides processively, adding more than one glucose molecule for each enzyme-glucan encounter. Granule-bound SSII can elongate malto-oligosaccharides, but has a lower affinity for these than GBSSI and does not elongate processively. As a result of these properties GBSSI synthesizes longer malto-oligosaccharides than SSII. The significance of these results with respect to the roles of GBSSI and SSII in vivo is discussed.

Adenosine Diphosphate Glucose↗
PubMed2001

Multiple allelism as a control mechanism in metabolic pathways: GBSSI allelic composition affects the activity of granule-bound starch synthase I and starch composition in potato.

Multiple allelism in heterozygous autopolyploid species like potato not only occurs for genes that affect morphological characteristics but also for genes involved in metabolic pathways. Based on a combination of Southern and PCR analyses, at least eight alleles encoding granule-bound starch synthase I (GBSSI), which is responsible for amylose biosynthesis, have been identified in potato. These alleles were grouped into four classes, distinguishable by Southern analysis, and subdivided based on PCR. Despite the heterozygous and polyploid character of potato it was possible to assign variation in GBSSI activity to the allelic composition at the GBSSI loci within a large population of Solanum tuberosum cultivars and Solanum breeding lines. Moreover, the availability of an amf allele made it possible to reduce heterogeneity and enabled us to demonstrate an effect of GBSSI allelic composition on amylose content. The major difference between the alleles identified was the absence or presence of a 140-bp fragment at a site 0.5 kb upstream of the ATG start codon of the gene for GBSSI. The absence of this 140-bp fragment had a major effect on GBSSI activity and amylose content, while the presence of small deletions and simple sequence repeats had no obvious effect.

Algorithms↗
PubMed2000

The structure and expression of the wheat starch synthase III gene. Motifs in the expressed gene define the lineage of the starch synthase III gene family.

The endosperm of hexaploid wheat (Triticum aestivum [L.]) was shown to contain a high molecular weight starch synthase (SS) analogous to the product of the maize du1 gene, starch synthase III (SSIII; DU1). cDNA and genomic DNA sequences encoding wheat SSIII were isolated and characterized. The wheat SSIII cDNA is 5,346 bp long and contains an open reading frame that encodes a 1,628-amino acid polypeptide. A putative N-terminal transit peptide, a 436-amino acid C-terminal catalytic domain, and a central 470-amino acid SSIII-specific domain containing three regions of repeated amino acid similarity were identified in the wheat gene. A fourth region between the transit peptide and the SSIII-specific domain contains repeat motifs that are variable with respect to motif sequence and repeat number between wheat and maize. In dicots, this N-terminal region does not contain repeat motifs and is truncated. The gene encoding wheat SSIII, designated ss3, consists of 16 exons extending over 10 kb, and is located on wheat chromosome I. Expression of ss3 mRNA in wheat was detected in leaves, pre-anthesis florets, and from very early to middle stage of endosperm development. The entire N-terminal variable repeat region and the majority of the SSIII-specific domain are encoded on a single 2,703-bp exon. A gene encoding a class III SS from the Arabidopsis genome sequencing project shows a strongly conserved exon structure to the wheat ss3 gene, with the exception of the N-terminal region. The evolutionary relationships of the genes encoding monocot and dicot class III SSs are discussed.

Amino Acid Sequence↗
PubMed1999

The localization and expression of the class II starch synthases of wheat.

The starch granules of hexaploid wheat (Triticum aestivum) contain a group of three proteins known as SGP-1 (starch granule protein-1) proteins, which have apparent molecular masses of 100, 108, and 115 kD. The nature and role of these proteins has not been defined previously. We demonstrate that these polypeptides are starch synthases that are present in both the starch granule and the soluble fraction at the early stages of wheat endosperm development, but that are exclusively granule bound at mid and late endosperm development. A partial cDNA clone encoding a fragment of the 100-kD protein was obtained by screening a wheat endosperm cDNA expression library using monoclonal antibodies. Three classes of cDNA were subsequently isolated from a wheat endosperm cDNA library by nucleic acid hybridization and were shown to encode the 100-, 108-, and 115-kD proteins. The cDNA sequences are highly homologous to class II starch synthases and have the highest homology with the maize SSIIa (starch synthase IIa) gene. mRNA for the SGP-1 proteins was detected in the leaf, pre-anthesis florets, and endosperm of wheat and is highly expressed in the leaf and in the grain during the early to mid stages of development. We discuss the roles of the SGP-1 proteins in starch biosynthesis in wheat.

Amino Acid Sequence↗
PubMed1994

Toward an understanding of the biogenesis of the starch granule. Determination of granule-bound and soluble starch synthase functions in amylopectin synthesis.

Plant starch synthesis can be distinguished from those of bacterial, fungal, and animal glycogen by the presence of multiple elongation (starch synthases) and branching enzymes. This complexity has precluded genetic assignment of functions to the various soluble starch synthases in the building of amylopectin. In Chlamydomonas, we have recently shown that defects in the major soluble starch synthase lead to a specific decrease in the amount of a subset of amylopectin chains whose length ranges between 8 and 40 glucose residues (Fontaine, T., D'Hulst, C., Maddelein, M.-L., Routier, F., Marianne-Pepin, T., Decq, A., Wieruszeski, J. M., Delrue, B., Van Den Koornhuyse, N., Bossu, J.-P., Fournet, B., and Ball, S. G. (1993) J. Biol. Chem. 268, 16223-16230). We now demonstrate that granule-bound starch synthase, the enzyme that was thought to be solely responsible for amylose synthesis, is involved in amylopectin synthesis. Disruption of the Chlamydomonas granule-bound starch synthase structural gene establishes that synthesis of long chains by this enzyme can become an absolute requirement for amylopectin synthesis in particular mutant backgrounds. In the sole presence of soluble starch synthase I, Chlamydomonas directs the synthesis of a major water-soluble polysaccharide fraction and minute amounts of a new type of highly branched granular material, whose structure is intermediate between those of glycogen and amylopectin. These results lead us to propose that the nature of the elongation enzyme conditions the synthesis of distinct size classes of glucans in all starch fractions.

Amylopectin↗
PubMed2002

Role of granule-bound starch synthase in determination of amylopectin structure and starch granule morphology in potato.

Reductions in activity of SSIII, the major isoform of starch synthase responsible for amylopectin synthesis in the potato tuber, result in fissuring of the starch granules. To discover the causes of the fissuring, and thus to shed light on factors that influence starch granule morphology in general, SSIII antisense lines were compared with lines with reductions in the major granule-bound isoform of starch synthase (GBSS) and lines with reductions in activity of both SSIII and GBSS (SSIII/GBSS antisense lines). This revealed that fissuring resulted from the activity of GBSS in the SSIII antisense background. Control (untransformed) lines and GBSS and SSIII/GBSS antisense lines had unfissured granules. Starch analyses showed that granules from SSIII antisense tubers had a greater number of long glucan chains than did granules from the other lines, in the form of larger amylose molecules and a unique fraction of very long amylopectin chains. These are likely to result from increased flux through GBSS in SSIII antisense tubers, in response to the elevated content of ADP-glucose in these tubers. It is proposed that the long glucan chains disrupt organization of the semi-crystalline parts of the matrix, setting up stresses in the matrix that lead to fissuring.

Amylopectin↗
PubMed1995

Biochemical and molecular characterization of a novel starch synthase from potato tubers.

An isoform of starch synthase from potato tubers which is present both in the stroma of the plastid and tightly bound to starch granules has been identified biochemically and a cDNA has been isolated. The protein encoded by the cDNA is 79.9 kDa and has a putative transit peptide and a distinct N-terminal domain which is predicted to be highly flexible. It is similar in both amino acid sequence and predicted structure to the granule-bound starch synthase II (GBSSII) of pea embryos. When expressed in Escherichia coli, the mature protein has starch synthase activity. The importance of the isoform has been assessed by biochemical measurements and antisense transformation experiments in which the amount of the isoform in the tuber is severely and specifically reduced. Both approaches indicate that the isoform contributes a maximum of 15% of the total starch synthase activity of the tuber. It is suggested that this isoform and the GBSSII of pea embryos represent a widely distributed class of isoforms of starch synthase. The contribution to total starch synthase activity of members of this class probably varies considerably from one type of storage organ to another.

Amino Acid Sequence↗
PubMed1998

Isolation and characterization of the zSSIIa and zSSIIb starch synthase cDNA clones from maize endosperm.

Two starch synthase clones, zSSIIa and zSSIIb, were isolated from a cDNA library constructed from W64A maize endosperm. zSSIIa and zSSIIb are 3124 and 2480 bp in length, and contain open reading frames of 732 and 698 amino acid residues, respectively. The deduced amino acid sequences of the two clones share 58.1% sequence identity. Amino acid sequence identity between the zSSIIa and zSSIIb clones and the starch synthase II clones of potato and pea ranges between 45 to 51%. The predicted amino acid sequence from each SSII cDNA contains the KXGGL consensus motif at the putative ADP-Glc binding site. Both clones also contain putative transit peptides followed by the VRAA(E)A motif, the consensus cleavage site located at the C-terminus of chloroplast transit peptides. The identity of the zSSIIa and zSSIIb clones as starch synthases was confirmed by expression of enzyme activity in Escherichia coli. Genomic DNA blot analysis revealed two copies of zSSIIa and a single copy of zSSIIb. zSSIIa was expressed predominantly in the endosperm, while transcripts for zSSIIb were detected mainly in the leaf at low abundance. These findings establish that the zSSIIa and zSSIIb genes are characteristically distinct from genes encoding granule-bound starch synthase I (Waxy protein) and starch synthase I.

Amino Acid Sequence↗
PubMed1999

Specificity of starch synthase isoforms from potato.

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.

Amino Acid Sequence↗
PubMed1996

Cloning and functional analysis of a cDNA encoding a novel 139 kDa starch synthase from potato (Solanum tuberosum L.).

Three isoforms of starch synthase were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. An antibody directed against a domain conserved in starch synthases was used to clone a cDNA for one of these isoforms by screening a tuber-specific expression library. A partial cDNA of 2.6 kbp was obtained and used to isolate a full-length cDNA of 4167 bp. The deduced amino acid sequence identifies the protein as a novel type of starch synthase from potato with a molecular mass of 139.2 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SS III. An analysis of the expression pattern of the gene indicates that SS III is equally expressed in tubers of different developmental stages as well as in sink and source leaves. In several independent transgenic potato lines, where the expression of SS III was repressed using the antisense approach, the activity of a specific starch synthase isoform was reduced to non-detectable levels as determined through activity staining after native gel electrophoresis. The reduction of this isoform of starch synthase leads to the synthesis of a structurally modified starch in the transgenic plants: there is a drastic change in granule morphology and an increased level of covalently linked phosphate.

Amino Acid Sequence↗
PubMed1993

Identification, cDNA cloning, and gene expression of soluble starch synthase in rice (Oryza sativa L.) immature seeds.

Three forms of soluble starch synthase were resolved by anion-exchange chromatography of soluble extracts from immature rice (Oryza sativa L.) seeds, and each of these forms was further purified by affinity chromatograph. The 55-, 57-, and 57-kD proteins in the three preparations were identified as candidates for soluble starch synthase by western blot analysis using an antiserum against rice granule-bound starch synthase. It is interesting that the amino-terminal amino acid sequence was identical among the three proteins, except that the 55-kD protein lacked eight amino acids at the amino terminus. Thus, these three proteins are products of the same gene. The cDNA clones coding for this protein have been isolated from an immature rice seed library in lambda gt11 using synthetic oligonucleotides as probes. The deduced amino acid sequence of this protein contains a lysine-X-glycine-glycine consensus sequence for the ADP-glucose-binding site of starch and glycogen synthases. Therefore, we conclude that this protein corresponds to a form of soluble starch synthase in immature rice seeds. The precursor of the enzyme contains 626 amino acids, including a 113-residue transit peptide at the amino terminus. The mature form of soluble starch synthase shares a significant but low sequence identity with rice granule-bound starch synthase and Escherichia coli glycogen synthase. However, several regions, including the substrate-binding site, are highly conserved among these three enzymes. Blot hybridization analysis demonstrates that the gene encoding soluble starch synthase is a single-copy gene in the rice genome and is expressed in both leaves and immature seeds. These results suggest that soluble and granule-bound starch synthases play distinct roles in starch biosynthesis of plant.

Amino Acid Sequence↗
PubMed2000

Purification and characterization of soluble starch synthases from maize endosperm.

This study identified and characterized the soluble starch synthase of maize endosperm that was initially revealed as the SSII activity peak in anion exchange chromatography (J. L. Ozbun et al. (1971) Plant Physiol. 48, 765-769). At least six different genes coding for starch synthases are expressed in maize, although previously it was not known which of these is responsible for the SSII activity peak. The enzyme activity in the SSII peak was neutralized to a large extent by antibodies raised against the product of the Du1 gene, but was not affected by antibodies specific for the other highly expressed soluble starch synthase, zSSI, or for the zSSIIa or zSSIIb isoforms. These data provide direct evidence that Du1 codes for the starch synthase responsible for the SSII activity peak. This starch synthase was purified approximately 350-fold from endosperm extracts. The following enzymatic properties of the SSII activity were determined: temperature optimum, thermostability, pH effects, K(m) for different glucan primers and the glucosyl unit donor ADPGlc, V(max) using various primers, and stimulation by citrate. These properties were compared to those of zSSI purified over 1600-fold from maize endosperm by a parallel procedure. The major differences between the two enzymes were that the SSII activity displayed higher K(m) values for ADPGlc, a distinct temperature range for maximal activity, and different relative activities toward specific exogenous substrates. The purified SSI and SSII activities both were shown to be capable of elongating maltooligosaccharide primers in vitro.

Enzyme Stability↗
PubMed1996

Evidence that a 77-kilodalton protein from the starch of pea embryos is an isoform of starch synthase that is both soluble and granule bound.

In this paper we provide further evidence about the nature of a 77-kD starch synthase (SSII) that is both soluble and bound to the starch granules in developing pea (Pisum sativum L.) embryos. Mature SSII gives rise to starch synthase activity when expressed in a strain of Escherichia coli lacking glycogen synthase. In transgenic potatoes (Solanum tuberosum L.) expressing SSII, the protein is both soluble and bound to the starch granules. These results confirm that SSII is a starch synthase and indicate that partitioning between the soluble and granule-bound fraction of storage organs is an intrinsic property of the protein. A 60-kD isoform of starch synthase found both in the soluble and granule-bound fraction of the pea embryos is probably derived by the processing of SSII and is a different gene product from GBSSI, the exclusively granule-bound 59-kD isoform of starch synthase that is similar to starch synthases encoded by the waxy genes of cereals and the amf gene of potatoes. Consistent with this, expression in E. coli of an N-terminally truncated version of SSII gives rise to starch synthase activity.

Amino Acid Sequence↗
PubMed2002

Production of a freeze-thaw-stable potato starch by antisense inhibition of three starch synthase genes.

The use of unmodified starches in frozen foods is severely limited by the undesirable textural changes that occur after freezing and thawing. Retrogradation of glucan chains leads to syneresis, a separation of the starch gel and water phases. Stabilization of the starch structure is normally achieved by chemical modification to prevent these changes from occurring. We have now created a freeze-thaw-stable potato starch by alteration of starch composition and structure by genetic modification. An amylose-free starch with short-chain amylopectin was produced by simultaneous antisense downregulation of three starch synthase genes. This starch is extremely freeze-thaw-stable and shows no syneresis even after five freeze-thaw cycles. The use of this starch has potential for environmental and consumer benefits because its production requires no chemical modification.

Amylopectin↗