Foods high in resistant starch have the potential to improve human health and lower the risk of serious noninfectious diseases. RNA interference was used to down-regulate the two different isoforms of starch-branching enzyme (SBE) II (SBEIIa and SBEIIb) in wheat endosperm to raise its amylose content. Suppression of SBEIIb expression alone had no effect on amylose content; however, suppression of both SBEIIa and SBEIIb expression resulted in starch containing >70% amylose. When the >70% amylose wheat grain was fed to rats in a diet as a wholemeal, several indices of large-bowel function, including short-chain fatty acids, were improved relative to standard wholemeal wheat. These results indicate that this high-amylose wheat has a significant potential to improve human health through its resistant starch content.

ABSTRACT A role for the Escherichia coli glgX gene in bacterial glycogen synthesis and/or degradation has been inferred from the sequence homology between the glgX gene and the genes encoding isoamylase-type debranching enzymes; however, experimental evidence or definition of the role of the gene has been lacking. Construction of E. coli strains with defined deletions in the glgX gene is reported here. The results show that the GlgX gene encodes an isoamylase-type debranching enzyme with high specificity for hydrolysis of chains consisting of three or four glucose residues. This specificity ensures that GlgX does not generate an extensive futile cycle during glycogen synthesis in which chains with more than four glucose residues are transferred by the branching enzyme. Disruption of glgX leads to overproduction of glycogen containing short external chains. These results suggest that the GlgX protein is predominantly involved in glycogen catabolism by selectively debranching the polysaccharide outer chains that were previously recessed by glycogen phosphorylase.

The role of starch branching enzyme-I (SBE-I) in determining starch structure in the endosperm has been investigated. Null mutations of SBE-I at the A, B and D genomes of wheat were identified in Australian wheat varieties by immunoblotting. By combining individual null mutations at the B and D genomes through hybridisation, a double-null mutant wheat, which lacks the B and D isoforms of SBE-I, was developed. Wheat mutants lacking all the three isoforms of SBE-I were generated from a doubled haploid progeny of a cross between the BD double-null mutant line and a Chinese Spring (CS) deletion line lacking the A genome isoform. Comparison of starch from this mutant wheat to that from wild type revealed no substantial alteration in any of the structural or functional properties analysed. Further analysis of this triple-null mutant line revealed the presence of another residual peak of SBE-I activity, referred to as SBE-Ir, in wheat endosperm representing < 3% of the activity of SBE-I in wild type endosperm.

Protein phosphorylation in amyloplasts and chloroplasts of Triticum aestivum (wheat) was investigated after the incubation of intact plastids with gamma-(32)P-ATP. Among the soluble phosphoproteins detected in plastids, three forms of starch branching enzyme (SBE) were phosphorylated in amyloplasts (SBEI, SBEIIa, and SBEIIb), and both forms of SBE in chloroplasts (SBEI and SBEIIa) were shown to be phosphorylated after sequencing of the immunoprecipitated (32)P-labeled phosphoproteins using quadrupole-orthogonal acceleration time of flight mass spectrometry. Phosphoamino acid analysis of the phosphorylated SBE forms indicated that the proteins are all phosphorylated on Ser residues. Analysis of starch granule-associated phosphoproteins after incubation of intact amyloplasts with gamma-(32)P-ATP indicated that the granule-associated forms of SBEII and two granule-associated forms of starch synthase (SS) are phosphorylated, including SSIIa. Measurement of SBE activity in amyloplasts and chloroplasts showed that phosphorylation activated SBEIIa (and SBEIIb in amyloplasts), whereas dephosphorylation using alkaline phosphatase reduced the catalytic activity of both enzymes. Phosphorylation and dephosphorylation had no effect on the measurable activity of SBEI in amyloplasts and chloroplasts, and the activities of both granule-bound forms of SBEII in amyloplasts were unaffected by dephosphorylation. Immunoprecipitation experiments using peptide-specific anti-SBE antibodies showed that SBEIIb and starch phosphorylase each coimmunoprecipitated with SBEI in a phosphorylation-dependent manner, suggesting that these enzymes may form protein complexes within the amyloplast in vivo. Conversely, dephosphorylation of immunoprecipitated protein complex led to its disassembly. This article reports direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation and protein-protein interactions in the control of starch anabolism and catabolism.

Analysis of barley shrunken grain mutants has identified lines with a novel high amylose starch phenotype. The causal mutation is located at the sex6 locus on chromosome 7H, suggesting the starch synthase IIa (ssIIa) gene as a candidate gene altered by the mutation. Consistent with this hypothesis, no evidence of SSIIa protein expression in either the starch granule or soluble fractions of the endosperm was found. Sequences of the starch synthase IIa gene, ssIIa, from three independent sex6 lines showed the presence of a stop codon preventing translation of the ssIIa transcript in each line. Perfect segregation of the starch phenotype with the presence of stop codons in the ssIIa gene was obtained, providing strong evidence for the lesion in the ssIIa gene being the causal mutation for the sex6 phenotype. The loss of SSIIa activity in barley leads to novel and informative phenotypes. First, a decrease in amylopectin synthesis to less than 20% of the wild-type levels indicates that SSIIa accounts for the majority of the amylopectin polymer elongation activity in barley. Secondly, in contrast to high amylose starches resulting from branching enzyme downregulation, the sex6 starches have a shortened amylopectin chain length distribution and a reduced gelatinisation temperature. Thirdly, the mutation leads to pleiotropic effects on other enzymes of the starch biosynthesis pathway, abolishing the binding of SSI, branching enzyme IIa and branching enzyme IIb to the starch granules of sex6 mutants, while not significantly altering their expression levels in the soluble fraction.

Genes and cDNAs for starch-branching enzyme II (SBEII) have been isolated from libraries constructed from Aegilops tauschii and wheat (Triticum aestivum) endosperm, respectively. One class of genes has been termed wSBEII-DA1 and encodes the N terminus reported for an SBEII from wheat endosperm. On the basis of phylogenetic comparisons with other branching enzyme sequences, wSBEII-DA1 is considered to be a member of the SBEIIa class. The wSBEII-DA1 gene consists of 22 exons with exons 4 to 21 being identical in length to the maize (Zea mays) SBEIIb gene, and the gene is located in the proximal region of the long arm of chromosome 2 at a locus designated sbe2a. RNA encoding SBEIIa can be detected in the endosperm from 6 d after flowering and is at its maximum level from 15 to 18 d after anthesis. Use of antibodies specific for SBEIIa demonstrated that this protein was present in both the soluble and granule bound fractions in developing wheat endosperm. We also report a cDNA sequence for SBEIIa that could arise by variant transcription/splicing. A second gene, termed wSBEII-DB1, was isolated and encodes an SBEII, which shows greater sequence identity with SBEIIb-type sequences than with SBEIIa-type sequences. Comparisons of SBEII gene structures among wheat, maize, and Arabidopsis indicate the lineage of the SBEII genes.

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.

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.

A cDNA clone, and a corresponding genomic DNA clone, containing full-length sequences encoding wheat starch synthase I, were isolated from a cDNA library of hexaploid wheat (Triticum aestivum) and a genomic DNA library of Triticum tauschii, respectively. The entire sequence of the starch synthase-I cDNA (wSSI-cDNA) is 2591 bp, and it encodes a polypeptide of 647 amino-acid residues that shows 81% and 61% identity to the amino-acid sequences of SSI-type starch synthases from rice and potato, respectively. In addition, the putative N-terminal amino-acid sequence of the encoded protein is identical to that determined for the N-terminal region of the 75-kDa starch synthase present in the starch granule of hexaploid wheat. Two prominent starch synthase activities were demonstrated to be present in the soluble fraction of wheat endosperm by activity staining of the non-denaturing PAGE gels. The most anodal band (wheat SSI) shows the highest staining intensity and results from the activity of a 75-kDa protein. The wheat SSI mRNA is expressed in the endosperm during the early to mid stages of wheat grain development but was not detected by Northern blotting in other tissues from the wheat plant. The gene encoding the wheat SSI (SsI-D1) consists of 15 exons and 14 introns, similar to the structure of the rice starch synthase-I gene. While the exons of wheat and rice are virtually identical in length, the wheat SsI-D1 gene has longer sequences in introns 1, 2, 4 and 10, and shorter sequences in introns 6, 11 and 14, than the corresponding rice gene.

Three forms of starch branching enzyme (BE) from developing hexaploid wheat (Triticum aestivum) endosperm have been partially purified and characterized. Immunological cross-reactivities indicate that two forms (WBE-IAD, 88 kD, and WBE-IB, 87 kD) are related to the maize BE I class and that WBE-II (88 kD) is related to maize BE II. Comparison of the N-terminal sequences from WBE-IAD and WBE-II with maize and rice BEs confirms these relationships. Evidence is presented from the analysis of nullisomic-tetrasomic wheat lines demonstrating that WBE-IB is located on chromosome 7B and that the WBE-IAD fraction contains polypeptides that are encoded on chromosomes 7A and 7D. The wheat endosperm BE classes are differentially expressed during endosperm development. WBE-II is expressed at a constant level throughout mid and late endosperm development. In contrast, WBE-IAD and WBE-IB are preferentially expressed in late endosperm development. Differences are also observed in the kinetic characteristics of the enzymes. The WBE-I isoforms have a 2- to 5-fold higher affinity for amylose than does WBE-II, and the WBE-I isoforms are activated up to 5-fold by phosphorylated intermediates and inorganic phosphate, whereas WBE-II is activated only 50%. The potential implications of this activation of BE I for starch biosynthesis are discussed.

Wheat starch contains two classes of associated proteins: proteins which are embedded within the granule and loosely associated surface proteins. The characterisation of the major proteins that are embedded in the granule are described. Gel electrophoresis on the basis of size resolved these proteins into five bands of molecular weights 60, 75, 85, 100 and 105 kDa. These polypeptides were demonstrated to be within the granule by their resistance to proteinase K digestion when granules were ungelatinised. The N-terminal sequences of these polypeptides are reported. The most prominent polypeptide is the 60 kDa granule-bound starch synthase. The N-terminal sequence obtained from the 75 kDa polypeptide shows homology to rice soluble starch synthase. The 85 kDa band was resolved into at least two types of polypeptides, one of which reacted with polyclonal antiserum to the maize branching enzyme IIb. The 100 and 105 kDa polypeptides were located only in the granule and are related, on the basis of N-terminal sequence similarity and cross-reactivity to monoclonal antibodies. SDS-PAGE and monoclonal antibody cross-reactivity experiments suggest that the 100 and 105 kDa polypeptides are absent from starch granules from all other species examined, including other cereals. It is speculated that all the major granule proteins are involved in starch biosynthesis.