Engineering high α-amylase levels in wheat grain lowers Falling Number but improves baking properties.

Late maturity α-amylase (LMA) and preharvest sprouting (PHS) are genetic defects in wheat. They are both characterized by the expression of specific isoforms of α-amylase in particular genotypes in the grain prior to harvest. The enhanced expression of α-amylase in both LMA and PHS results in a reduction in Falling Number (FN), a test of gel viscosity, and subsequent downgrading of the grain, along with a reduced price for growers. The FN test is unable to distinguish between LMA and PHS; thus, both defects are treated similarly when grain is traded. However, in PHS-affected grains, proteases and other degradative process are activated, and this has been shown to have a negative impact on end product quality. No studies have been conducted to determine whether LMA is detrimental to end product quality. This work demonstrated that wheat in which an isoform α-amylase (TaAmy3) was overexpressed in the endosperm of developing grain to levels of up to 100-fold higher than the wild-type resulted in low FN similar to those seen in LMA- or PHS-affected grains. This increase had no detrimental effect on starch structure, flour composition and enhanced baking quality, in small-scale 10-g baking tests. In these small-scale tests, overexpression of TaAmy3 led to increased loaf volume and Maillard-related browning to levels higher than those in control flours when baking improver was added. These findings raise questions as to the validity of the assumption that (i) LMA is detrimental to end product quality and (ii) a low FN is always indicative of a reduction in quality. This work suggests the need for a better understanding of the impact of elevated expression of specific α-amylase on end product quality.

Suppression of glucan, water dikinase in the endosperm alters wheat grain properties, germination and coleoptile growth.

Starch phosphate ester content is known to alter the physicochemical properties of starch, including its susceptibility to degradation. Previous work producing wheat (Triticum aestivum) with down-regulated glucan, water dikinase, the primary gene responsible for addition of phosphate groups to starch, in a grain-specific manner found unexpected phenotypic alteration in grain and growth. Here, we report on further characterization of these lines focussing on mature grain and early growth. We find that coleoptile length has been increased in these transgenic lines independently of grain size increases. No changes in starch degradation rates during germination could be identified, or any major alteration in soluble sugar levels that may explain the coleoptile growth modification. We identify some alteration in hormones in the tissues in question. Mature grain size is examined, as is Hardness Index and starch conformation. We find no evidence that the increased growth of coleoptiles in these lines is connected to starch conformation or degradation or soluble sugar content and suggest these findings provide a novel means of increasing coleoptile growth and early seedling establishment in cereal crop species.

A genetic strategy generating wheat with very high amylose content.

Resistant starch (RS), a type of dietary fibre, plays an important role in human health; however, the content of RS in most modern processed starchy foods is low. Cereal starch, when structurally manipulated through a modified starch biosynthetic pathway to greatly increase the amylose content, could be an important food source of RS. Transgenic studies have previously revealed the requirement of simultaneous down-regulation of two starch branching enzyme (SBE) II isoforms both located on the long arm of chromosome 2, namely SBEIIa and SBEIIb, to elevate the amylose content in wheat from ~25% to ~75%. The current study revealed close proximity of genes encoding SBEIIa and SBEIIb isoforms in wheat with a genetic distance of 0.5 cM on chromosome 2B. A series of deletion and single nucleotide polymorphism (SNP) loss of function alleles in SBEIIa, SBEIIb or both was isolated from two different wheat populations. A breeding strategy to combine deletions and SNPs generated wheat genotypes with altered expression levels of SBEIIa and SBEIIb, elevating the amylose content to an unprecedented ~85%, with a marked concomitant increase in RS content. Biochemical assays were used to confirm the complete absence in the grain of expression of SBEIIa from all three genomes in combination with the absence of SBEIIb from one of the genomes.

Influence of in situ progressive N-terminal is still controversial truncation of glycogen branching enzyme in Escherichia coli DH5α on glycogen structure, accumulation, and bacterial viability.

BACKGROUND: Glycogen average chain length (ACL) has been linked with bacterial durability, but this was on the basis of observations across different species. We therefore wished to investigate the relationship between bacterial durability and glycogen ACL by varying glycogen average chain length in a single species. It has been shown that progressive shortening of the N-terminus of glycogen branching enzyme (GBE) leads to a lengthening of oligosaccharide inter-α-1,6-glycosidic chain lengths, so we sought to harness this to create a set of Escherichia coli DH5α strains with a range of glycogen average chain lengths, and assess these strains for durability related attributes, such as starvation, cold and desiccation stress resistance, and biofilm formation. RESULTS: A series of Escherichia coli DH5α mutants were created with glgB genes that were in situ progressively N-terminus truncated. N-terminal truncation shifted the distribution of glycogen chain lengths from 5-11 DP toward 13-50 DP, but the relationship between glgB length and glycogen ACL was not linear. Surprisingly, removal of the first 270 nucleotides of glgB (glgBΔ270) resulted in comparatively high glycogen accumulation, with the glycogen having short ACL. Complete knockout of glgB led to the formation of amylose-like glycogen containing long, linear α1,4-glucan chains with significantly reduced branching frequency. Physiologically, the set of mutant strains had reduced bacterial starvation resistance, while minimally increasing bacterial desiccation resistance. Finally, although there were no obvious changes in cold stress resistance or biofilm forming ability, one strain (glgBΔ180) had significantly increased biofilm formation in favourable media. CONCLUSIONS: Despite glgB being the first gene of an operon, it is clear that in situ mutation is a viable means to create more biologically relevant mutant strains. Secondly, there was the suggestion in the data that impairments of starvation, cold and desiccation resistance were worse for the strain lacking glgB, though the first of these was not statistically significant. The results provide prima facie evidence linking abiotic stress tolerance with shorter glycogen ACL. However, further work needs to be done, perhaps in a less labile species. Further work is also required to tease out the complex relationship between glycogen abundance and glycogen structure.

The different effects of starch synthase IIa mutations or variation on endosperm amylose content of barley, wheat and rice are determined by the distribution of starch synthase I and starch branching enzyme IIb between the starch granule and amyloplast stroma.

KEY MESSAGE: The distribution of starch synthase I and starch branching enzyme IIb between the starch granule and amyloplast stroma plays an important role in determining endosperm amylose content of cereal grains. Starch synthase IIa (SSIIa) catalyses the polymerisation of intermediate length glucan chains of amylopectin in the endosperm of cereals. Mutations of SSIIa genes in barley and wheat and inactive SSIIa variant in rice induce similar effects on the starch structure and the amylose content, but the severity of the phenotypes is different. This study compared the levels of transcripts and partitioning of proteins of starch synthase I (SSI) and starch branching enzyme IIb (SBEIIb) inside and outside the starch granules in the developing endosperms of these ssIIa mutants and inactive SSIIa variant. Pleiotropic effects on starch granule-bound proteins suggested that the different effects of SSIIa mutations on endosperm amylose content of barley, wheat and rice are determined by the distribution of SSI and SBEIIb between the starch granule and amyloplast stroma in cereals. Regulation of starch synthesis in ssIIa mutants and inactive SSIIa variant may be at post-translational level or the altered amylopectin structure deprives the affinity of SSI and SBEIIb to amylopectin.

Suppression of starch synthase I expression affects the granule morphology and granule size and fine structure of starch in wheat endosperm.

Studies in Arabidopsis and rice suggest that manipulation of starch synthase I (SSI) expression in wheat may lead to the production of wheat grains with novel starch structure and properties. This work describes the suppression of SSI expression in wheat grains using RNAi technology, which leads to a low level of enzymatic activity for SSI in the developing endosperm, and a low abundance of SSI protein inside the starch granules of mature grains. The amylopectin fraction of starch from the SSI suppressed lines showed an increased frequency of very short chains (degree of polymerization, dp 6 and 7), a lower proportion of short chains (dp 8-12), and more intermediate chains (dp 13-20) than in the grain from their negative segregant lines. In the most severely affected line, amylose content was significantly increased, the morphology of starch granules was changed, and the proportion of B starch granules was significantly reduced. The change of the fine structure of the starch in the SSI-RNAi suppression lines alters the gelatinization temperature, swelling power, and viscosity of the starch. This work demonstrates that the roles of SSI in the determination of starch structure and properties are similar among different cereals and Arabidopsis.

Characterization of starch phosphorylases in barley grains.

BACKGROUND: Starch is synthesized in both leaves and storage tissues of plants. The role of starch syntheses and branching enzymes is well understood; however, the role of starch phosphorylase is not clear. RESULTS: A gene encoding Pho1 from barley was characterized and starch phosphorylases from both developing and germinating grain were characterized and purified. Two activities were detected: one with a molecular mass of 110 kDa and the other of 95 kDa. It was demonstrated through the use of antisera that the 110 kDa activity was located in the amyloplast and could correspond to the polypeptide encoded by the Pho1 gene cloned. The 95 kDa activity was localized to the cytoplasm, most strongly expressed in germinating grain, and was classified as a Pho2-type sequence. Using RNAi technology to reduce the content of Pho1 in the grain to less than 30% of wild type did not lead to any visible phenotype, and no dramatic alterations in the structure of the starch were observed. CONCLUSION: Two starch phosphorylase activities were identified and characterized in barley grains, and shown to be present during starch synthesis. However, their role in starch synthesis still remains to be elucidated.

Functional characterization of barley betaglucanless mutants demonstrates a unique role for CslF6 in (1,3;1,4)-ß-D-glucan biosynthesis.

(1,3;1,4)-ß-D-glucans (mixed-linkage glucans) are found in tissues of members of the Poaceae (grasses), and are particularly high in barley (Hordeum vulgare) grains. The present study describes the isolation of three independent (1,3;1,4)-ß-D-glucanless (betaglucanless; bgl) mutants of barley which completely lack (1,3;1,4)-ß-D-glucan in all the tissues tested. The bgl phenotype cosegregates with the cellulose synthase like HvCslF6 gene on chromosome arm 7HL. Each of the bgl mutants has a single nucleotide substitution in the coding region of the HvCslF6 gene resulting in a change of a highly conserved amino acid residue of the HvCslF6 protein. Microsomal membranes isolated from developing endosperm of the bgl mutants lack detectable (1,3;1,4)-ß-D-glucan synthase activity indicating that the HvCslF6 protein is inactive. This was confirmed by transient expression of the HvCslF6 cDNAs in Nicotiana benthamiana leaves. The wild-type HvCslF6 gene directed the synthesis of high levels of (1,3;1,4)-ß-D-glucans, whereas the mutant HvCslF6 proteins completely lack the ability to synthesize (1,3;1,4)-ß-D-glucans. The fine structure of the (1,3;1,4)-ß-D-glucan produced in the tobacco leaf was also very different from that found in cereals having an extremely low DP3/DP4 ratio. These results demonstrate that, among the seven CslF and one CslH genes present in the barley genome, HvCslF6 has a unique role and is the key determinant controlling the biosynthesis of (1,3;1,4)-ß-D-glucans. Natural allelic variation in the HvCslF6 gene was found predominantly within introns among 29 barley accessions studied. Genetic manipulation of the HvCslF6 gene could enable control of (1,3;1,4)-ß-D-glucans in accordance with the purposes of use.

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.

Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing.

The inactivation of starch branching IIb (SBEIIb) in rice is traditionally associated with elevated apparent amylose content, increased peak gelatinization temperature, and a decreased proportion of short amylopectin branches. To elucidate further the structural and functional role of this enzyme, the phenotypic effects of down-regulating SBEIIb expression in rice endosperm were characterized by artificial microRNA (amiRNA) and hairpin RNA (hp-RNA) gene silencing. The results showed that RNA silencing of SBEIIb expression in rice grains did not affect the expression of other major isoforms of starch branching enzymes or starch synthases. Structural analyses of debranched starch showed that the doubling of apparent amylose content was not due to an increase in the relative proportion of amylose chains but instead was due to significantly elevated levels of long amylopectin and intermediate chains. Rices altered by the amiRNA technique produced a more extreme starch phenotype than those modified using the hp-RNA technique, with a greater increase in the proportion of long amylopectin and intermediate chains. The more pronounced starch structural modifications produced in the amiRNA lines led to more severe alterations in starch granule morphology and crystallinity as well as digestibility of freshly cooked grains. The potential role of attenuating SBEIIb expression in generating starch with elevated levels of resistant starch and lower glycaemic index is discussed.

A survey of ß-glucan and arabinoxylan content in wheat.

BACKGROUND: Dietary fibre lowers the risk of coronary heart disease and colorectal cancer. This survey quantifies mixed link ß-glucan (MBG) and arabinoxylan (AX) in wheat and investigates relationships between the grain carbohydrates. MBG and AX contents were measured in 500 and 200 wheat accessions respectively, including diploid, tetraploid and hexaploid genotypes comprising primitive, synthetic and elite lines. RESULTS: Overall, MBG contents ranged between 1.8 and 18.0 g kg(-1) grain dry weight. In wheat-barley addition lines and triticale hexaploids the levels were 9.0-11.3 and 3.5-9.6 g kg(-1) respectively. The amounts in synthetic wheats were nearer their tetraploid parents than their diploid parents. AX and total non-starch polysaccharide (NSP) contents ranged from 23.7 to 107.5 g kg(-1) and from 31.7 to 136.7 g kg(-1) respectively. Linear regressions showed that the relationships of starch and grain weight with NSP glucose were stronger than those with AX. CONCLUSION: The results indicated insufficient genetic diversity in the germplasm surveyed to initiate a breeding programme to increase the amount of MBG in wheat grain to 20 g kg(-1) , a level considered high enough to confer a 10-15% reduction in blood cholesterol.

Sponge and dough bread making: genetic and phenotypic relationships with wheat quality traits.

The genetic and phenotypic relationships among wheat quality predictors and sponge and dough bread making were evaluated in a population derived from a cross between an Australian cultivar 'Chara' and a Canadian cultivar 'Glenlea'. The genetic correlation across sites for sponge and dough loaf volume was high; however, phenotypic correlations across sites for loaf volume were relatively low compared with rheological tests. The large difference between sites was most likely due to temperature differences during grain development reflected in a decrease in the percentage of unextractable polymeric protein and mixing time. Predictive tests (mixograph, extensograph, protein content and composition, micro-zeleny and flour viscosity) showed inconsistent and generally poor correlations with end-product performance (baking volume and slice area) at both sites, with no single parameter being effective as a predictor of end-product performance. The difference in the relationships between genetic and phenotypic correlations highlights the requirement to develop alternative methods of selection for breeders and bakers in order to maximise both genetic gain and predictive assessment of grain quality.

Oil Accumulation in Transgenic Potato Tubers Alters Starch Quality and Nutritional Profile.

Plant storage compounds such as starch and lipids are important for human and animal nutrition as well as industry. There is interest in diverting some of the carbon stored in starch-rich organs (leaves, tubers, and cereal grains) into lipids in order to improve the energy density or nutritional properties of crops as well as providing new sources of feedstocks for food and manufacturing. Previously, we generated transgenic potato plants that accumulate up to 3.3% triacylglycerol (TAG) by dry weight in the tubers, which also led to changes in starch content, starch granule morphology and soluble sugar content. The aim of this study was to investigate how TAG accumulation affects the nutritional and processing properties of high oil potatoes with a particular focus on starch structure, physical and chemical properties. Overall, TAG accumulation was correlated with increased energy density, total nitrogen, amino acids, organic acids and inorganic phosphate, which could be of potential nutritional benefit. However, TAG accumulation had negative effects on starch quality as well as quantity. Starch from high oil potatoes had lower amylose and phosphate content, reduced peak viscosity and higher gelatinization temperature. Interestingly, starch pasting properties were disproportionately affected in lines accumulating the highest levels of TAG (>2.5%) compared to those accumulating only moderate levels (0.2-1.6%). These results indicate that optimized engineering of specialized crops for food, feed, fuel and chemical industries requires careful selection of traits, and an appropriate level of transgene expression, as well as a better understanding of starch structure and carbon partitioning in plant storage organs.

Molecular, mesoscopic and microscopic structure evolution during amylase digestion of extruded maize and high amylose maize starches.

Extrusion processing of cereal starch granules with high (>50%) amylose content is a promising approach to create nutritionally desirable resistant starch, i.e. starch that escapes digestion in the small intestine. Whilst high amylose content seems to be required, the structural features responsible for the slow digestion of extrudates are not fully understood. We report the effects of partial enzyme digestion of extruded maize starches on amylopectin branch length profiles, double and single helix contents, crystallinity and lamellar periodicity. Comparing results for three extruded maize starches (27, 57, and 84% apparent amylose) that differ in amylase-sensitivity allows conclusions to be drawn concerning the rate-determining features operating under the digestion conditions used. Enzyme resistance is shown to originate from a combination of molecular and mesoscopic factors, including both recrystallization and an increase in very short branches during the digestion process. This is in contrast to the behaviour of the same starches in the granular form (Shrestha et al., 2012) where molecular and mesoscopic factors are secondary to microscopic structures in determining enzyme susceptibility. Based on the structure of residual material after long-time digestion (>8h), a model for resistant starch from processed high amylose maize starches is proposed based on a fringed micelle structure with lateral aggregation and enzyme susceptibility both limited by attached clusters of branch points.

Molecular, mesoscopic and microscopic structure evolution during amylase digestion of maize starch granules.

Cereal starch granules with high (>50%) amylose content are a promising source of nutritionally desirable resistant starch, i.e. starch that escapes digestion in the small intestine, but the structural features responsible are not fully understood. We report the effects of partial enzyme digestion of maize starch granules on amylopectin branch length profiles, double and single helix contents, gelatinisation properties, crystallinity and lamellar periodicity. Comparing results for three maize starches (27, 57, and 84% amylose) that differ in both structural features and amylase-sensitivity allows conclusions to be drawn concerning the rate-determining features operating under the digestion conditions used. All starches are found to be digested by a side-by-side mechanism in which there is no major preference during enzyme attack for amylopectin branch lengths, helix form, crystallinity or lamellar organisation. We conclude that the major factor controlling enzyme susceptibility is granule architecture, with shorter length scales not playing a major role as inferred from the largely invariant nature of numerous structural measures during the digestion process (XRD, NMR, SAXS, DSC, FACE). Results are consistent with digestion rates being controlled by restricted diffusion of enzymes within densely packed granular structures, with an effective surface area for enzyme attack determined by external dimensions (57 or 84% amylose - relatively slow) or internal channels and pores (27% amylose - relatively fast). Although the process of granule digestion is to a first approximation non-discriminatory with respect to structure at molecular and mesoscopic length scales, secondary effects noted include (i) partial crystallisation of V-type helices during digestion of 27% amylose starch, (ii) preferential hydrolysis of long amylopectin branches during the early stage hydrolysis of 27% and 57% but not 84% amylose starches, linked with disruption of lamellar repeating structure and (iii) partial B-type recrystallisation after prolonged enzyme incubation for 57% and 84% amylose starches but not 27% amylose starch.

Differential effects of genetically distinct mechanisms of elevating amylose on barley starch characteristics.

The relationships between starch structure and functionality are important in underpinning the industrial and nutritional utilisation of starches. In this work, the relationships between the biosynthesis, structure, molecular organisation and functionality have been examined using a series of defined genotypes in barley with low (<20%), standard (20-30%), elevated (30-50%) and high (>50%) amylose starches. A range of techniques have been employed to determine starch physical features, higher order structure and functionality. The two genetic mechanisms for generating high amylose contents (down-regulation of branching enzymes and starch synthases, respectively) yielded starches with very different amylopectin structures but similar gelatinisation and viscosity properties driven by reduced granular order and increased amylose content. Principal components analysis (PCA) was used to elucidate the relationships between genotypes and starch molecular structure and functionality. Parameters associated with granule order (PC1) accounted for a large percentage of the variance (57%) and were closely related to amylose content. Parameters associated with amylopectin fine structure accounted for 18% of the variance but were less closely aligned to functionality parameters.

Multiple effects of the starch synthase II mutation in developing wheat endosperm.

A line of wheat (Triticum aestivum L.), sgp-1, that does not express starch synthase II (SSII, also known as SGP-1) has previously been reported. In this study, F1 derived doubled haploid lines with homozygous wild type or mutant alleles for SGP-1 genes were identified from a cross between the original mutant and a wild type Australian cultivar. Analysis of the starch granules showed that in the mutant lines they are markedly distorted from 15 days postanthesis during grain development. Starch branching patterns showed an increase in the proportion of short chains (DP 6-10) at an earlier stage, but this increase became much more pronounced at 15 days postanthesis and persisted until maturity. There was also a consistent and drastic reduction throughout seed development in the relative amounts of starch branching enzyme II (SBEII, comprising SBEIIa and SBEIIb) and starch synthase I (SSI) bound to the starch granules. In the soluble phase, however, there was relatively little change in the amount of SBEIIb, SBEIIa or SSI protein. Therefore loss of SSII specifically leads to the loss of SBEIIb, SBEIIa and SSI protein in the granule-bound phase and the effect of this mutation is clearly manifest from the mid-stage of endosperm development in wheat.

Effects of starch synthase IIa gene dosage on grain, protein and starch in endosperm of wheat.

Starch synthases (SS) are responsible for elongating the alpha-1,4 glucan chains of starch. A doubled haploid population was generated by crossing a line of wheat, which lacks functional ssIIa genes on each genome (abd), and an Australian wheat cultivar, Sunco, with wild type ssIIa alleles on each genome (ABD). Evidence has been presented previously indicating that the SGP-1 (starch granule protein-1) proteins present in the starch granule in wheat are products of the ssIIa genes. Analysis of 100 progeny lines demonstrated co-segregation of the ssIIa alleles from the three genomes with the SGP-1 proteins, providing further evidence that the SGP-1 proteins are the products of the ssIIa genes. From the progeny lines, 40 doubled haploid lines representing the eight possible genotypes for SSIIa (ABD, aBD, AbD, ABd, abD, aBd, Abd, abd) were characterized for their grain weight, protein content, total starch content and starch properties. For some properties (chain length distribution, pasting properties, swelling power, and gelatinization properties), a progressive change was observed across the four classes of genotypes (wild type, single nulls, double nulls and triple nulls). However, for other grain properties (seed weight and protein content) and starch properties (total starch content, granule morphology and crystallinity, granule size distribution, amylose content, amylose-lipid dissociation properties), a statistically significant change only occurred for the triple nulls, indicating that all three genes had to be missing or inactive for a change to occur. These results illustrate the importance of SSIIa in controlling grain and starch properties and the importance of amylopectin fine structure in controlling starch granule properties in wheat.

Contrasting effects of chronic heat stress and heat shock on kernel weight and flour quality in wheat.

Phytotron studies were conducted to compare the potential effects of chronic high-temperatures (daily maxima of over 20˚C) and heat-shock conditions (a few days of over 32˚C), on wheat (Triticum aestivum L.) yield and quality, to form a basis for the selection of improved high-temperature tolerance in wheat. The series of heat-shock treatments were designed to provide similar heat loads, by varying the duration (number of days) of each treatment. Studies involved two cultivars, Lyallpur and Trigo 1. Both showed a reduction in kernel weight in response to chronic high day temperatures (i.e. above 18˚C), with Trigo 1 more tolerant than Lyallpur. Kernel weight of both cultivars was also reduced by short periods of heat shock, and this was most evident at day/night temperatures above 30/25˚C. There was no reduction in the germination of the lighter weight kernels formed under either chronic high temperature or heat-shock conditions. Dough strength, as judged by mixing time, declined in both genotypes with prolonged chronic high temperature, and also following the most extreme of the heat-shock treatments - Trigo 1 showed an ability to resist these changes better than Lyallpur. The heat-related decreases in dough strength were associated with decreases in the proportion of the larger molecular size glutenin (most 'unextractable'). This change in quality was not however, associated with changes in flour protein content.

Multiple isoforms of starch branching enzyme-I in wheat: lack of the major SBE-I isoform does not alter starch phenotype.

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.