Starch Branching Enzyme IIa Research Articles

Effects of Starch Synthesis-Related Genes Polymorphism on Quality of Glutinous Rice.

Glutinous rice (Oryza sativa L.) quality includes thermal properties, retrogradation and pasting viscosity properties, and so on, which have little or no amylose. However, the genetic network regulation of different quality indices has not been systematically studied. The aim was to investigate the relationship between starch synthesis-related genes (SSRGs) and the physicochemical properties of glutinous rice by targeted-gene association analysis (TGAS). The genotypes of 17 SSRGs were analyzed using 46 gene-specific molecular markers in 63 glutinous rice accessions. TGAS and gene interactions analysis indicated that soluble starch synthase (SS) IIa, SSI, starch branching enzyme (BE) IIa, and pullulanase (PUL) had significant genetic effects on glutinous rice quality. SSI and SSIIa were the major genes that regulated thermal properties and retrogradation properties (RP). PUL was central in the regulation of gel consistency (GC), and it participated in the regulation of pasting viscosity parameters (PVP) except for the pasting time and the pasting temperature. BEIIb, ISA1, SSIVb, BEIIa, SSIVa, and their interactions with SSIIa regulated gelatinization temperature (GT) and PVP. The starch properties of glutinous rice are mainly controlled by SSIIa, SSI, PUL, and their interactions, but SSIIa is central among them. These findings indicate that starch properties in glutinous rice have a complex genetic system. It provides crucial information for promoting glutinous rice quality.

Effect of semolina pudding prepared from starch branching enzyme IIa and b mutant wheat on glycaemic response in vitro and in vivo: a randomised controlled pilot study.

Refined starchy foods are usually rapidly digested, leading to poor glycaemic control, but not all starchy foods are the same. Complex carbohydrates like resistant starch (RS) have been shown to reduce the metabolic risk factors for chronic diseases such as hyperglycaemia and overweight. The aim of the project was to develop a semolina-based food made from a starch branching enzyme II (sbeIIa/b-AB) durum wheat mutant with a high RS content and to measure its glycaemic index using a double-blind randomised pilot study. We report here the amylose, RS and non-starch polysaccharide concentration of raw sbeIIa/b-AB and wild-type control (WT) semolina. We measured RS after cooking to identify a model food for in vivo testing. Retrograded sbeIIa/b-AB semolina showed a higher RS concentration than the WT control (RS = 4.87 ± 0.6 g per 100 g, 0.77 ± 0.34 g per 100 g starch DWB, respectively), so pudding was selected as the test food. Ten healthy participants consumed ∼50 g of total starch from WT and sbeIIa/b-AB pudding and a standard glucose drink. Capillary blood glucose concentrations were measured in the fasting and postprandial state (2 h): incremental area-under-the-curve (iAUC) and GI were calculated. We found no evidence of difference in GI between sbeIIa/b-AB pudding and the WT control, but the starch digestibility was significantly lower in sbeIIa/b-AB pudding compared to the WT control in vitro (C90 = 33.29% and 47.38%, respectively). Based on these results, novel sbeIIa/b-AB wheat foods will be used in future in vivo studies to test the effect of different RS concentrations and different food matrices on glycaemia.

Sugary Endosperm is Modulated by Starch Branching Enzyme IIa in Rice (Oryza sativa L.)

BackgroundStarch biosynthesis is one of the most important pathways that determine both grain quality and yield in rice (Oryza sativa L.). Sugary endosperm, sugary-1 (sug-1), is a mutant trait for starch biosynthesis. Rice plants carrying sug-1 produce grains that accumulate water-soluble carbohydrates instead of starch, even after maturity. Although this trait enhances the diversity of grain quality, sugary endosperm rice has hardly been commercialized due to the severely wrinkled grains and subsequent problems in milling. This study was conducted to identify the genes responsible for the sug-h phenotype through a map-based cloning technology.ResultsWe induced a mild sugary mutant, sugary-h (sug-h) through the chemical mutagenesis on the Korean japonica cultivar Hwacheong. Grains of the sug-h mutant were translucent and amber-colored, and the endosperm appeared less wrinkled than sug-1, whereas the soluble sugar content was fairly high. These characteristics confer greater marketability to the sug-h mutant. Genetic analyses indicated that the sug-h mutant phenotype was controlled by a complementary interaction of two recessive genes, Isoamylase1 (OsISA1), which was reported previously, and Starch branching enzyme IIa (OsBEIIa), which was newly identified in this study. Complementation tests indicated that OsBEIIa regulated the properties of sugary endosperm.ConclusionsComplementary interactions between the starch biosynthesis genes OsISA1 and OsBEIIa determine the mild sugary endosperm mutant, sugary-h, in rice. Our finding may facilitate the breeding of sugaryendosperm rice for commercial benefit.

TILLING mutants of durum wheat result in a high amylose phenotype and provide information on alternative splicing mechanisms

The amylose/amylopectin ratio has a major influence over the properties of starch and determines its optimal end use. Here, high amylose durum wheat has been bred by combining knock down alleles at the two homoelogous genes encoding starch branching enzyme IIa (SBEIIa-A and SBEIIa-B). The complete silencing of these genes had a number of pleiotropic effects on starch synthesis: it affected the transcriptional activity of SBEIIb, ISA1 (starch debranching enzyme) and all of the genes encoding starch synthases (SSI, SSIIa, SSIII and GBSSI). The starch produced by grain of the double SBEIIa mutants was high in amylose (up to ∼1.95 fold that of the wild type) and contained up to about eight fold more resistant starch. A single nucleotide polymorphism adjacent to the splice site at the end of exon 10 of the G364E mutant copies of both SBEIIa-A and SBEIIa-B resulted in the loss of a conserved exonic splicing silencer element. Its starch was similar to that of the SBEIIa double mutant. G364E SBEIIa pre-mRNA was incorrectly processed, resulting in the formation of alternative, but non-functional splicing products.

Reduced expression of starch branching enzyme IIa and IIb in maize endosperm by RNAi constructs greatly increases the amylose content in kernel with nearly normal morphology.

RNAi technology was applied to suppress the expression of starch branching enzyme IIa and IIb and to increase amylose content in maize endosperm, and stably inherited high-amylose maize lines were obtained. Amylose is an important material for industries and in the human diet. Maize varieties with endosperm amylose content (AC) of greater than 50 % are termed amylomaize, and possess high industrial application value. The high-amylose trait is controlled by multi-enzyme reaction and intricate gene-environment interaction. Starch branching enzymes are key factors for regulating the branching profiles of starches. In this paper, we report the successful application of RNAi technology for improving amylose content in maize endosperm through the suppression of the ZmSBEIIa and ZmSBEIIb genes by hairpin SBEIIRNAi constructs. These SBEIIRNAi transgenes led to the down-regulation of ZmSBEII expression and SBE activity to various degrees and altered the morphology of starch granules. Transgenic maize lines with AC of up to 55.89 % were produced, which avoided the significant decreases in starch content and grain yield that occur in high-amylose ae mutant. Novel maize lines with high AC offer potential benefits for high-amylose maize breeding. A comparison of gene silencing efficiency among transgenic lines containing different hpSBEIIRNA constructs demonstrated that (1) it was more efficient to use both ZmSBEIIa and ZmSBEIIb specific regions than to use the conserved domain as the inverted repeat arms; (2) the endosperm-specific promoter of the 27-kDa γ-zein provided more efficient inhibition than the CaMV 35S promoter; and (3) inclusion of the catalase intron in the hpSBEIIRNA constructs provided a better silencing effect than the chalcone synthase intron in the hpRNA construct design for suppression of the SBEII subfamily in endosperm.

Characterization of SBEIIa homoeologous genes in bread wheat

To elucidate some of the molecular mechanisms involved in genome differentiation and evolution of cultivated wheats, we compared orthologous genes encoding starch branching enzyme IIa (SBEIIa). Bread wheat is an allohexaploid species comprising the three genomes A, B and D, each of which contributes a copy of the SBEIIa gene, involved in starch biosynthesis and known to control important quality traits related to technological and nutritional value of wheat-based food products. Alignment of the nucleotide sequences of these three genes revealed variation, both at the level of single nucleotides and indels. Multiple transposon elements were identified in the intragenic regions, some of which appear to have inserted before the divergence of the wheat diploid genomes. The B genome homoeologue was the most divergent of the three genes. Two MITE transposon insertions were detected within the intronic sequence of SBEIIa-B and two other transposons within SBEIIa-D. The presence/absence of these transposons in a panel of diploid and polyploid Triticum and Aegilops species provided some insights into the phylogeny of wheat.

Development of high amylose wheat through TILLING.

BackgroundWheat (Triticum spp.) is an important source of food worldwide and the focus of considerable efforts to identify new combinations of genetic diversity for crop improvement. In particular, wheat starch composition is a major target for changes that could benefit human health. Starches with increased levels of amylose are of interest because of the correlation between higher amylose content and elevated levels of resistant starch, which has been shown to have beneficial effects on health for combating obesity and diabetes. TILLING (Targeting Induced Local Lesions in Genomes) is a means to identify novel genetic variation without the need for direct selection of phenotypes.ResultsUsing TILLING to identify novel genetic variation in each of the A and B genomes in tetraploid durum wheat and the A, B and D genomes in hexaploid bread wheat, we have identified mutations in the form of single nucleotide polymorphisms (SNPs) in starch branching enzyme IIa genes (SBEIIa). Combining these new alleles of SBEIIa through breeding resulted in the development of high amylose durum and bread wheat varieties containing 47-55% amylose and having elevated resistant starch levels compared to wild-type wheat. High amylose lines also had reduced expression of SBEIIa RNA, changes in starch granule morphology and altered starch granule protein profiles as evaluated by mass spectrometry.ConclusionsWe report the use of TILLING to develop new traits in crops with complex genomes without the use of transgenic modifications. Combined mutations in SBEIIa in durum and bread wheat varieties resulted in lines with significantly increased amylose and resistant starch contents.

High resolution melting analysis for the detection of EMS induced mutations in wheat SBEIIa genes.

BackgroundManipulation of the amylose-amylopectin ratio in cereal starch has been identified as a major target for the production of starches with novel functional properties. In wheat, silencing of starch branching enzyme genes by a transgenic approach reportedly caused an increase of amylose content up to 70% of total starch, exhibiting novel and interesting nutritional characteristics.In this work, the functionality of starch branching enzyme IIa (SBEIIa) has been targeted in bread wheat by TILLING. An EMS-mutagenised wheat population has been screened using High Resolution Melting of PCR products to identify functional SNPs in the three homoeologous genes encoding the target enzyme in the hexaploid genome.ResultsThis analysis resulted in the identification of 56, 14 and 53 new allelic variants respectively for SBEIIa-A, SBEIIa-B and SBEIIa-D. The effects of the mutations on protein structure and functionality were evaluated by a bioinformatic approach. Two putative null alleles containing non-sense or splice site mutations were identified for each of the three homoeologous SBEIIa genes; qRT-PCR analysis showed a significant decrease of their gene expression and resulted in increased amylose content. Pyramiding of different single null homoeologous allowed to isolate double null mutants showing an increase of amylose content up to 21% compared to the control.ConclusionTILLING has successfully been used to generate novel alleles for SBEIIa genes known to control amylose content in wheat. Single and double null SBEIIa genotypes have been found to show a significant increase in amylose content.

The barley amo1 locus is tightly linked to the starch synthase IIIa gene and negatively regulates expression of granule-bound starch synthetic genes.

In this study of barley starch synthesis, the interaction between mutations at the sex6 locus and the amo1 locus has been characterized. Four barley genotypes, the wild type, sex6, amo1, and the amo1sex6 double mutant, were generated by backcrossing the sex6 mutation present in Himalaya292 into the amo1 ‘high amylose Glacier’. The wild type, amo1, and sex6 genotypes gave starch phenotypes consistent with previous studies. However, the amo1sex6 double mutant yielded an unexpected phenotype, a significant increase in starch content relative to the sex6 phenotype. Amylose content (as a percentage of starch) was not increased above the level observed for the sex6 mutation alone; however, on a per seed basis, grain from lines containing the amo1 mutation (amo1 mutants and amo1sex6 double mutants) synthesize significantly more amylose than the wild-type lines and sex6 mutants. The level of granule-bound starch synthase I (GBSSI) protein in starch granules is increased in lines containing the amo1 mutation (amo1 and amo1sex6). In the amo1 genotype, starch synthase I (SSI), SSIIa, starch branching enzyme IIa (SBEIIa), and SBEIIb also markedly increased in the starch granules. Genetic mapping studies indicate that the ssIIIa gene is tightly linked to the amo1 locus, and the SSIIIa protein from the amo1 mutant has a leucine to arginine residue substitution in a conserved domain. Zymogram analysis indicates that the amo1 phenotype is not a consequence of total loss of enzymatic activity although it remains possible that the amo1 phenotype is underpinned by a more subtle change. It is therefore proposed that amo1 may be a negative regulator of other genes of starch synthesis.

Starch-branching enzyme IIa is required for proper diurnal cycling of starch in leaves of maize.

Starch-branching enzyme (SBE), a glucosyl transferase, is required for the highly regular pattern of α-1,6 bonds in the amylopectin component of starch. In the absence of SBEIIa, as shown previously in the sbe2a mutant of maize (Zea mays), leaf starch has drastically reduced branching and the leaves exhibit a severe senescence-like phenotype. Detailed characterization of the maize sbe2a mutant revealed that SBEIIa is the primary active branching enzyme in the leaf and that in its absence plant growth is affected. Both seedling and mature sbe2a mutant leaves do not properly degrade starch during the night, resulting in hyperaccumulation. In mature sbe2a leaves, starch hyperaccumulation is greatest in visibly senescing regions but also observed in green tissue and is correlated to a drastic reduction in photosynthesis within the leaf. Starch granules from sbe2a leaves observed via scanning electron microscopy and transmission electron microscopy analyses are larger, irregular, and amorphous as compared with the highly regular, discoid starch granules observed in wild-type leaves. This appears to trigger premature senescence, as shown by an increased expression of genes encoding proteins known to be involved in senescence and programmed cell death processes. Together, these results indicate that SBEIIa is required for the proper diurnal cycling of transitory starch within the leaf and suggest that SBEIIa is necessary in producing an amylopectin structure amenable to degradation by starch metabolism enzymes.

Control of starch branching in barley defined through differential RNAi suppression of starch branching enzyme IIa and IIb.

The roles of starch branching enzyme (SBE, EC 2.4.1.18) IIa and SBE IIb in defining the structure of amylose and amylopectin in barley (Hordeum vulgare) endosperm were examined. Barley lines with low expression of SBE IIa or SBE IIb, and with the low expression of both isoforms were generated through RNA-mediated silencing technology. These lines enabled the study of the role of each of these isoforms in determining the amylose content, the distribution of chain lengths, and the frequency of branching in both amylose and amylopectin. In lines where both SBE IIa and SBE IIb expression were reduced by >80%, a high amylose phenotype (>70%) was observed, while a reduction in the expression of either of these isoforms alone had minor impact on amylose content. The structure and properties of the high amylose starch resulting from the concomitant reduction in the expression of both isoforms of SBE II in barley were found to approximate changes seen in amylose extender mutants of maize, which result from lesions eliminating expression of the SBE IIb gene. Amylopectin chain length distribution analysis indicated that both SBE IIa and SBE IIb isoforms play distinct roles in determining the fine structure of amylopectin. A significant reduction in the frequency of branches in amylopectin was noticed only when both SBE IIa and SBE IIb were reduced, whereas there was a significant increase in the branching frequency of amylose when SBE IIb alone was reduced. Functional interactions between SBE isoforms are suggested, and a possible inhibitory role of SBE IIb on other SBE isoforms is discussed.

Mechanistic Investigation of a Starch-Branching Enzyme Using Hydrodynamic Volume SEC Analysis

Two linear alpha-(1,4)-D-glucans substrates, of degrees of polymerization DP approximately 150 and 6000, were exposed to maize starch-branching enzyme IIa (mSBEIIa) in vitro. The resulting branched alpha-glucans and their constituent chains (obtained by debranching) were analyzed by nuclear magnetic resonance (NMR) and size-exclusion chromatography (SEC). SEC data for the debranched species are presented as chain-length distributions, while those for branched species are presented as hydrodynamic volume distributions (HVDs), which is the most meaningful way to present such data (because SEC separates by size, not molar mass, and a sample of branched polymers with the same size can have a range of molar masses). A rigorous interpretation of the HVDs of the substrate and its branched product show that at least part of the branching is an interchain transfer mechanism in both the short- and long-chain substrate cases. A bimodal HVD of the in vitro branched alpha-glucan derived from the short-chain substrate was observed, and it is postulated that the divergence of the two populations is due to very small chains being unable to undergo branching. In the case of the in vitro branching of the long-chain substrate, the formation of maltohexaose during the reaction and the presence of a monomodal HVD were observed, suggesting a distinct mode of action of mSBEIIa on this substrate. Quantification of the branching level by NMR showed the branched glucans from both substrates had substantial amounts of branching (2.1-4.5%), ascribed to the intrinsic nature of the action of mSBEIIa on the two substrates. It is postulated that differences in the degrees of substrate association affect the pattern of branching catalyzed by the enzyme, and a putative active site structure is proposed based on the appearance of maltohexaose. The molar mass distribution of the constituent chains of the in vitro branched alpha-glucans obtained by isoamylase treatment reveals the transfer of chains of specific size and supports the supposition given in the literature that mSBEIIa is responsible for short-chain branching in amylopectin. It is suggested that hydrodynamic volume SEC analysis should be used as a tool for the mechanistic investigation of SBEs, allowing SEC data of in vitro branched alpha-glucans to be both comparable and quantitative.

Isolation, identification and characterisation of starch‐interacting proteins by 2‐D affinity electrophoresis

A 2-D affinity electrophoretic technique (2-DAE) has been used to isolate proteins that interact with various starch components from total barley endosperm extracts. In the first dimension, proteins are separated by native PAGE. The second-dimensional gel contains polysaccharides such as amylopectin and glycogen. The migration of starch-interacting proteins in this dimension is determined by their affinity towards a particular polysaccharide and these proteins are therefore spatially separated from the bulk of proteins in the crude extract. Four distinct proteins demonstrate significant affinity for amylopectin and have been identified as starch branching enzyme I (SBEI), starch branching enzyme IIa (SBEIIa), SBEIIb and starch phosphorylase using polyclonal antibodies and zymogram activity analysis. In the case of starch phosphorylase, a protein spot was excised from a 2-DAE polyacrylamide gel and analysed using Q-TOF MS/MS, resulting in the alignment of three internal peptide sequences with the known sequence of the wheat plastidic starch phosphorylase isoform. This assignment was confirmed by the determination of the enzyme's function using zymogram analysis. Dissociation constants (Kd) were calculated for the three enzymes at 4 degrees C and values of 0.20, 0.21 and 1.3 g/L were determined for SBEI, SBEIIa and starch phosphorylase, respectively. Starch synthase I could also be resolved from the other proteins in the presence of glycogen and its identity was confirmed using a polyclonal antibody and by activity analysis. The 2-DAE method described here is simple, though powerful, enabling protein separation from crude extracts on the basis of function.

The two genes encoding starch-branching enzymes IIa and IIb are differentially expressed in barley.

The sbeIIa and sbeIIb genes, encoding starch-branching enzyme (SBE) IIa and SBEIIb in barley (Hordeum vulgare L.), have been isolated. The 5' portions of the two genes are strongly divergent, primarily due to the 2064-nucleotide-long intron 2 in sbeIIb. The sequence of this intron shows that it contains a retro-transposon-like element. Expression of sbeIIb but not sbeIIa was found to be endosperm specific. The temporal expression patterns for sbeIIa and sbeIIb were similar and peaked around 12 d after pollination. DNA gel-blot analysis demonstrated that sbeIIa and sbeIIb are both single-copy genes in the barley genome. By fluorescence in situ hybridization, the sbeIIa and sbeIIb genes were mapped to chromosomes 2 and 5, respectively. The cDNA clones for SBEIIa and SBEIIb were isolated and sequenced. The amino acid sequences of SBEIIa and SBEIIb were almost 80% identical. The major structural difference between the two enzymes was the presence of a 94-amino acid N-terminal extension in the SBEIIb precursor. The (beta/alpha)8-barrel topology of the alpha-amylase superfamily and the catalytic residues implicated in branching enzymes are conserved in both barley enzymes.

Characterization of dull1, a Maize Gene Coding for a Novel Starch Synthase

The maize dull1 (du1) gene is a determinant of the structure of endosperm starch, and du1- mutations affect the activity of two enzymes involved in starch biosynthesis, starch synthase II (SSII) and starch branching enzyme IIa (SBEIIa). Six novel du1- mutations generated in Mutator-active plants were identified. A portion of the du1 locus was cloned by transposon tagging, and a nearly full-length Du1 cDNA sequence was determined. Du1 codes for a predicted 1674-residue protein, comprising one portion that is similar to SSIII of potato, as well as a large unique region. Du1 transcripts are present in the endosperm during the time of starch biosynthesis, but the mRNA was undetectable in leaf or root tissue. The predicted size of the Du1 gene product and its expression pattern are consistent with those of maize SSII. The Du1 gene product contains two repeated regions in its unique N terminus. One of these contains a sequence identical to a conserved segment of SBEs. We conclude that Du1 codes for a starch synthase, most likely SSII, and that secondary effects of du1- mutations, such as reduction of SBEIIa, result from the primary deficiency in this starch synthase.