Amylolysis of wheat starches. I. Digestion kinetics of starches with varying functional properties

The susceptibility of wheat (Triticum aestivum L.) starches to hydrolysis by pancreatic α-amylase in vitro was investigated using a series of 35 starches with slightly enriched amylose content within a narrow range (36–43%), but widely differing functional properties. After 2 h of incubation with α-amylase, native starch granules were digested to different extents, but there were no differences between any of the starches once they were gelatinized. Cooling the starch for 72 h at 4 °C after cooking reduced the susceptibility of all of the starches to enzymic digestion by a similar extent, whereas addition of monopalmitin decreased the digestibility of the starches that contained amylose, but did not affect the digestibility of waxy starches that were also included in the study. Amylopectin chain length distribution of partly digested starch granules displayed increased proportion of short and medium chains and decreased proportion of long chains in comparison to native granules. Separated large (A) and small (B) starch granules from three of the starches differed significantly in their susceptibility to in-vitro digestion. A predictive model of the susceptibility of starch in the different forms was developed from the physico-chemical and functional properties of the starches.     

Detection of mutations in the 7A allele of wheat (Triticum aestivum) granule-bound starch synthase (Wx-7A) with a monoclonal antibody produced by targeted peptide immunisation

A peptide sequence unique for the granule-bound starch synthase (GBSS1) of hexaploid wheat (Triticum aestivum) was identified by sequence alignment of the three isoforms. Multiple antigenic peptides (MAP) of different length (16 and 20 AA) were synthesised against the selected sequence. The assessment of the immune response in mice against differently presented forms (resin-bound, free) and two types of adjuvants indicated that the free MAP with the longer peptide is more immunogenic. A 20AA MAP that elicited a strong immune response was used to produce a monoclonal antibody for the 7A isoform of GBSS 1. Epitope mapping of the selected Mab (F5-1F2) revealed that a 12mer partial sequence of the immunising peptide was specifically detected. Based on this Mab, a simple high throughput ELISA was developed that allows the quick identification of wheat lines carrying the 7A allele of GBSS 1 with minute amounts of sample.     

Effect of the three waxy null alleles on enzymes associated to wheat starch granules using proteomic approach

In bread wheat (Triticum aestivum L.), waxy genes are present at three loci (Wx-A1, Wx-B1 and Wx-D1) and are responsible for amylose synthesis in the grain. Near isogenic lines (NILs) of the agronomic cultivar Tremie with either one, two or three waxy null alleles were used to study the genome response to these null alleles through proteomic analysis of starch granule associated proteins of mature grain. Among the 352 spots that were revealed, 86 varied significantly between NILs. The main differences were observed for the granule bound starch synthase (GBSS), product of the waxy genes. A strong relation (R² = 0.94) between the amount of GBSS per gram of starch granule and the amylose quantity in the grain was revealed through quantification of the GBSS in each NIL. The analysis of GBSS quantity for each NIL invalidated the additive hypothesis for the expression of this enzyme in (Wx-A1a Wx-D1a Wx-B1b) (Wx-D1a Wx-B1a Wx-A1b) and the normal form of Tremie. In addition, four proteins identified as inactive GBSS were increased (+144.9%) in the triple null isogenic form compared to the normal form of Tremie. The quantitative variations of the proteins present in starch granules support the idea of regulatory mechanisms between genomes.     

Starch granules size distribution in superior and inferior grains of wheat is related to enzyme activities and their gene expressions during grain filling

Mature wheat endosperm contains A-, B-, C-type starch granules, and each class has unique physiochemical properties which determine the quality of starch. The dynamics of the starch granule size distribution, activities of starch synthases and expression of starch synthase encoding genes were studied in superior and inferior grains during grain filling. Compared with inferior grains, superior grains showed higher grain weight, contents of starch, amylose and amylopectin. The formation of A-, B-, C-type starch granules initiated at 4, 8, 20 DAF, respectively, and was well consistent with the temporally change patterns of starch synthase activities and relative gene expression levels. For instance, activities of soluble and granule-bound starch synthases (designated SSS and GBSS) peaked at 20 and 24 DAF. Genes encoding isoforms of starch synthases expressed at different grain filling periods. In addition, SS I was generally expressed over the grain filling stage; the SS II and SS III were expressed over the early and mid grain filling stage, and the GBSS I was expressed during the mid to late grain filling stage. In addition, the time-course changes in activities of starch synthases and expression of starch synthase encoding genes explained well the dynamics of the starch granule size distribution.     

Analysis of starch swelling power in Australian breeding lines of hexaploid wheat (Triticum aestivum L.)

Starch is the major component of wheat (Triticum aestivum L.) grain and is composed of two large glucan molecules, amylose and amylopectin. The ratio between the two polymers types influences the water absorbing properties of starch upon heating, and thus affects the end-use of grain and purified starch. In this study, we evaluated the starch swelling power (SSP) values in seven wheat populations developed from crosses involving low-SSP lines. Analysis of starch produced by the F₂ generation plants showed that the largest SSP variation (11.4–16.2) and lowest SSP mean (13.9) was obtained for a population derived from doubled haploid lines SM1028 (SSP = 14.5) and VK306 (SSP = 13.6). The population of 360 lines was advanced by single seed descent to the following generations for further studies. Starch analysis of grain produced by F₄ generation lines in two field locations during 2006 and in a greenhouse environment during 2005 showed that SSP values were relatively stably inherited. The average broad-sense heritability was 73% and significant (P < 0.001) genotype × genotype and genotype × environment interactions were seen. Starches with the highest and lowest SSP values were inversely related to amylose concentration determined by high pressure liquid chromatography (HPLC)–size exclusion chromatography (SEC) of debranched starch. Developed lines with the lowest SSP values surpassed 40% in apparent amylose concentration. The study illustrates that screening for SSP in early generations can be used to develop wheat lines with desired starch swelling characteristics.     

In vitro pullulanase activity of wheat (Triticum aestivum L.) limit-dextrinase type starch debranching enzyme is modulated by redox conditions

Expression of a limit-dextrinase (LD) type starch debranching enzyme (EC 3.2.1.41) was observed in developing wheat (Triticum aestivum L.) endosperm and germinating grains, indicating a role for the enzyme in both biosynthesis and degradation of starch. A full-length cDNA, TaLD1, encoding LD in wheat developing kernels was isolated and predicted to encode a 98.6 kDa mature protein active in amyloplasts. Isolated cDNA was expressed in Escherichia coli to produce a recombinant His-tagged LD, which mainly accumulated in inclusion bodies as an inactive enzyme. Extraction of His-tagged LD from the inclusion bodies followed by dialysis under thiol/disulphide redox conditions allowed partial refolding of the protein and detection of pullulanase specific activities by zymogram analysis and enzyme assays. Several active conformations were demonstrated by the recombinant TaLD1 and pullulanase activity could be modulated by redox conditions in vitro. The results suggest that cellular redox conditions may regulate the level of LD activity in wheat tissues.     

Simulating multiple pest damage in varying winter wheat production situations

The production situation–injury profile paradigm can be used as a framework to assess the harmfulness of multiple-pest complexes in a changing agriculture. A mechanistic simulation model for wheat, WHEATPEST, was developed to incorporate drivers of (i) variable production situations and (ii) their related injury profiles. The model simulates the harmful effects of pathogens, pests, and weeds in a simple, open, generic manner. Simulation drivers were derived from published reports, in particular through a meta-analysis of highly detailed farmers’ field surveys in the United Kingdom and the Netherlands. Preliminary analysis of the model's performances indicates that WHEATPEST conforms with available published reports in a range of production situations and injury profiles. While improvement on some components of the model are discussed, this work points at the need for the collection of cross-disciplinary, reasonably accurate, and standardised data at a system's level, and at the usefulness of modelling tools for basic research and policy.     

Genetic analysis of field resistance to tan spot in spring wheat

Tan spot, caused by Pyrenophora tritici-repentis (Died.) Drechs., is an important constraint to wheat (Triticum aestivum L.) yield in many countries. Since the inheritance of field resistance to tan spot is poorly understood, this study was conducted to determine the genetic control of resistance in the field. Resistance was measured as disease severity caused by P. tritici-repentis race 1 in four crosses involving five wheat parents: parent 1 (P₁) = catbird; parent 2 (P₂) = Milan/Shanghai-7; parent 3 (P₃) = Alondra/Coc//Ures; parent 4 (P₄) = Bcb//Dundee/Gul/3/Gul); parent 5 (P₅) = ND/VG9144//Kal/BB/3/Yaco/4/Chil. P₁, P₂ and P₃ were resistant and P₄ was moderately resistant, whereas P₅ was susceptible to tan spot. The F₂-derived F₃ families and the parents were field evaluated at El Batán, Mexico, in 1996. When all the plants within a F₃ family expressed low levels of disease severity similar to that of the resistant parent it was classified resistant (R), otherwise the progeny was classified as susceptible (S). The progeny of the three crosses of the susceptible parent with the resistant and moderately resistant parents P₂, P₃, and P₄ segregated as 3R:13S whereas the progeny in the cross with P₁ showed a segregation ratio of 1R:15S. This suggests that each resistant parent possessed two genes conditioning resistance to tan spot severity caused by P. tritici-repentis race 1. Information on the inheritance of resistance measured as disease severity on adult plants under field conditions is of practical importance for wheat breeding programs seeking improvement in tan spot resistance.     

Analyses of albumins, globulins and amphiphilic proteins by proteomic approach give new insights on waxy wheat starch metabolism

Starch is composed of two types of glucose polymers: amylose and amylopectin. The Waxy (Wx) locus controls amylose synthesis in the wheat kernel. Hexaploid wheat has three Wx loci located on chromosomes 7A (Wx-A1), 4A (Wx-B1), and 7D (Wx-D1). Eight near isogenic lines (NILs) of Triticum aestivum cv. Tremie with one, two or three Wx null alleles were used. The albumin–globulin fraction, and amphiphilic proteins were separated using 2-dimensional electrophoresis (2DE) allowing the changes in the waxy kernel to be identified. Albumin–globulin fraction showed overexpression of sucrose synthases in the waxy NILs compared to the normal form of Tremie and a decrease in many proteins related to stress and defence metabolism such as serpins. A subunit of ADP-glucose pyrophosphorylase (AGPase), which is known to play a major role in starch synthesis, was also shown to be down regulated in the waxy NILs. Amphiphilic proteins confirmed the observations made on the albumin–globulin fraction with a decrease in a stress-related protein. These different regulations linked to observations made on wheat kernel (thousand kernel weight (TKW), protein amount per grain, size and distribution of the starch granules) led to formulation of the hypothesis that waxy endosperm does not reach maturity of the wild-type endosperm.     

The contribution of ear photosynthesis to grain filling in bread wheat (Triticum aestivum L.)

The contribution of ear photosynthesis to grain filling in wheat (Triticum aestivum L.) is not well known. The main objective of this work was to evaluate this contribution through three different experimental approaches: (1) ear photosynthesis was reduced by removing awns or shading the ears (in combination with a defoliation treatment), (2) grain weight per ear was compared in an ‘all shaded’ crop versus plants where only the vegetative parts were shaded (‘ear emerging’), and (3) ear photosynthesis was reduced with DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), a specific inhibitor of photosystem II.     

Transgenic wheats with elevated levels of Dx5 and/or Dy10 high-molecular-weight glutenin subunits yield doughs with increased mixing strength and tolerance

To test the effects of independently increasing the in vivo levels of high-molecular-weight glutenin subunits (HMW-GS) Dx5 and Dy10 on wheat flour properties, we increased the copy numbers of their corresponding genes by genetic transformation. Thirteen transformants with increases in one or both subunits were chosen for biochemical and functional characterization by solvent fractionation, RP-HPLC, SDS-sedimentation, and micro-mixing. Increases in Dx5 and Dy10 contents ranged from 1.4- to 3.5-fold and 1.2- to 5.4-fold, respectively, and generally resulted in increased polymeric protein, increased mixing times and tolerances, and lower peak resistances. Increases in Dx5 content had larger effects on most parameters than comparable increases in Dy10. Flours with more than 2.6-times the native levels of Dx5 could not be mixed under standard 2-g mixograph conditions, while flours with 5.4 times the native levels of Dy10 could be mixed if sufficient time was allowed. Increases in Dx5 and Dy10 had additive effects on mixing behavior. These experiments demonstrate that dough mixing strength and tolerance can be increased by raising the levels of native HMW-GS Dx5 or Dy10, but that the effects of the two subunits are quantitatively and qualitatively different.     

Waxy endosperm accompanies increased fat and saccharide contents in bread wheat (Triticum aestivum L.) grain

The contents of fat, starch, pentosan, fructan, β-glucan and several mono- and oligosaccharides in grain were evaluated to find out the possible effects of the Wx-D1 gene of bread wheat using two sets of near-isogenic waxy and non-waxy lines and two low-amylose mutant lines with a common genetic background of Kanto 107. These materials have two non-functional Wx-A1b and Wx-B1b alleles in common. Waxy near-isogenic lines with a non-functional Wx-D1d allele showed consistently increased contents of fat, total fructan, β-glucan, glucose, fructose, sucrose, 1-kestose, 6-kestose, neokestose, nystose and bifurcose compared with non-waxy lines with a functional Wx-D1a allele throughout three growing/harvest seasons. Starch and total pentosan contents were inconsistently influenced by the allelic status of the Wx-D1 locus, while water-soluble pentosan and raffinose contents were not affected. The compositional changes of a low-amylose mutant line with an almost non-functional Wx-D1f allele were closely similar to those of waxy near-isogenic lines, while significantly different changes were barely observed in another low-amylose mutant line with a partly functional Wx-D1g allele in two seasons. These results showed that the Wx-D1 gene has pleiotropic effects on the fat and saccharide contents of bread wheat grain.     

Kinetics of starch digestion and functional properties of twin-screw extruded sorghum

The time-course of starch digestion in twin-screw extruded milled sorghum grain was investigated using an in-vitro procedure based on glucometry. The sorghum grains were hammer-milled, and extruded at three levels each of moisture and screw speed. Irrespective of the extrusion conditions, extruded and non-extruded milled sorghum grain exhibited monophasic digestograms, and the modified first-order kinetic and Peleg models adequately described the digestograms. Extrusion increased the rate of digestion by about ten times compared with non-extrudates. Starch gelatinisation varied in the extrudates, and microscopy revealed a mixture of raw, gelatinised and destructured starch and protein components in the extrudates. Starch digestion parameters significantly (p < 0.05) correlated with extruder response and various functional properties of the extrudates. Extrusion conditions for maximum starch gelatinisation in milled sorghum grain for fastest digestion as an efficient animal feed were interpolated, as well as the conditions for directly-expanded extrudates with potential for human food, where minimum starch digestion is desired.     

Effects of transition season management on soil N dynamics and system N balances in rice–wheat rotations of Nepal

In the low-input rice–wheat production systems of Nepal, the N nutrition of both crops is largely based on the supply from soil pools. Declining yield trends call for management interventions aiming at the avoidance of native soil N losses. A field study was conducted at two sites in the lowland and the upper mid-hills of Nepal with contrasting temperature regimes and durations of the dry-to-wet season transition period between the harvest of wheat and the transplanting of lowland rice. Technical options included the return of the straw of the preceding wheat crop, the cultivation of short-cycled crops during the transition season, and combinations of both. Dynamics of soil N_min, nitrate leaching, nitrous oxide emissions, and crop N uptake were studied throughout the year between 2004 and 2005 and partial N balances of the cropping systems were established. In the traditional system (bare fallow between wheat and rice) a large accumulation of soil nitrate N and its subsequent disappearance upon soil saturation occurred during the transition season. This nitrate loss was associated with nitrate leaching (6.3 and 12.8 kg ha⁻¹ at the low and high altitude sites, respectively) and peaks of nitrous oxide emissions (120 and 480 mg m⁻² h⁻¹ at the low and high altitude sites, respectively). Incorporation of wheat straw at 3 Mg ha⁻¹ and/or cultivation of a nitrate catch crop during the transition season significantly reduced the build up of soil nitrate and subsequent N losses at the low altitude site. At the high altitude site, cumulative grain yields increased from 2.35 Mg ha⁻¹ with bare fallow during the transition season to 3.44 Mg ha⁻¹ when wheat straw was incorporated. At the low altitude site, the cumulative yield significantly increased from 2.85 Mg ha⁻¹ (bare fallow) to between 3.63 and 6.63 Mg ha⁻¹, depending on the transition season option applied. Irrespective of the site and the land use option applied during the transition season, systems N balances remained largely negative, ranging from −37 to −84 kg N ha⁻¹. We conclude that despite reduced N losses and increased grain yields the proposed options need to be complemented with additional N inputs to sustain long-term productivity.     

Genetic characterization of kernel polyphenol oxidases in wheat and related species

Polyphenol oxidase (PPO) activity causes undesirable darkening of raw Asian noodles and other wheat products. In this study we investigate the genetic origins and diversity of wheat kernel PPO. PPO was characterized via activity assays, antigenic staining, and Southern blots in Triticum aestivum, Triticum dicoccoides, Triticum durum, Triticum dicoccum, Triticum monococcum, Triticum urartu, Aegilops speltoides, and Aegilops tauschii. Among these species, PPO activity was well-correlated with antigenic staining intensity toward a wheat kernel-type PPO antibody. High PPO activity was observed in all three T. monococcum accessions (A^m genome), one Ae. speltoides accession, one T. durum accession, and two hexaploid wheat cultivars. Southern blots suggested the presence of two or more kernel-type PPO genes in diploid progenitors of the hexaploid A, B, and D genomes. Whole-kernel PPO activity was evaluated in disomic substitution lines derived from three T. dicoccoides accessions in the background of T. durum ‘Langdon’. PPO activity was primarily associated with chromosome 2A and to a much lower degree with chromosome 2B. DNA sequence comparisons showed that the intron associated with the high PPO allele on chromosome 2AL of hexaploid wheat had 94% nucleotide identity with the homeologous intron found in T. monococcum, a species with high kernel PPO activity. This implies that the ancestral PPO allele on the A genome is one of the high activity, and the low PPO allele found in hexaploid wheat represents a relatively recent genetic alteration. Results confirm the presence of multiple kernel-type PPO genes in the diploid and tetraploid progenitors and relatives of hexaploid wheat. However, it is likely that relatively few of the many kernel-type PPO genes present in wheat contribute substantially to kernel PPO activity. A single genetic locus on homeologous group 2 chromosomes may be the primary cause of high PPO activity in wheat kernels.     

Water use and yield of wheat/maize intercropping under alternate irrigation in the oasis field of northwest China

A field experiment was carried out to investigate the effects of alternate irrigation (AI) on the yield, water use and water use efficiency (WUE) of wheat (Triticum aestivum L.)/maize (Zea mays L.) intercropping system in an oasis region of northwest China in 2006-2008. Three planting patterns, i.e., sole wheat, sole maize and wheat/maize intercropping. Three irrigation levels were applied for each treatment during 3 years. Results showed that land use efficiency of wheat and maize was significantly enhanced by intercropping system; land equivalent ratio (LER) of wheat/maize intercropping system in different treatments was all greater than 1.0. Moreover, significant difference in grain yield was observed between intercropping treatment and sole cropping treatment, in which the yield of intercropped wheat was 55.37-74.88% of sole wheat, and intercropped maize was 66.63-78.87% of sole maize. Wheat/maize intercropping treatments increased water use by 1.8-16.4% than half of the total water use of sole-cropping wheat and maize. Compared to sole cropping wheat treatments, wheat/maize intercropping with alternate irrigation significantly improved water use efficiency (WUE) by 30.5-57.7%, 55.5-71.4% and 12.0-19.8%, and increased by 32.7-37.8%, 9.5-15.8% and 4.0-20.8% than sole cropping maize treatments in 2006-2008, respectively. Our results suggest that AI should be a useful water-saving irrigation method on wheat/maize intercropping in arid oasis field where intercropping planting is decreased because of limited water resource.     

Effect of hydrostatic pressure and temperature on the chemical and functional properties of wheat gluten II. Studies on the influence of additives

Cysteine, N-ethylmaleinimide, radical scavengers, various salts or urea were added to wheat gluten. After treatment at increasing pressure (0.1–800 MPa) and temperature (30–80 °C) the resulting material was analysed by micro-extension tests and an extraction/HPLC method to measure protein solubility. Furthermore, cysteine was added to isolated gliadin and glutenin prior to high-pressure treatment and protein solubility was determined. The resistance to extension of gluten strongly increased and the solubility of gliadin in aqueous ethanol decreased with increasing pressure and temperature. As compared to experiments without additive the observed effects were much stronger. Isolated gliadin turned largely insoluble in aqueous ethanol when cysteine was added prior to high-pressure treatment. The S-rich α- and γ-gliadins were much more strongly affected than the S-poor ω-gliadins pointing to a disulphide related mechanism. Monomeric gliadin components were completely recovered after reduction of the aggregates with dithioerythritol. In contrast, samples without free thiol groups such as isolated gliadins or with SH groups, which had been blocked by N-ethylmaleinimide, were hardly affected by high-pressure treatment. The addition of radical scavengers to gluten showed no effect in comparison to the control experiment, indicating that a radical mechanism of the high-pressure effect can be excluded. The observed effects can be explained by thiol-/disulphide interchange reactions, which require the presence of free thiol groups in the sample. The addition of salts and urea showed that unfolding of the protein due to weakening of interprotein hydrogen bonds is strongest for ions with a high radius (e.g. thiocyanate). This leads to weakening of gluten at ambient pressure but it facilitates high pressure induced reactions, e.g. of disulphide bonds.