The role of sensitivity to abscisic acid and gibberellin in pre-maturity α-amylase formation in wheat grains

To study the role of abscisic acid (ABA) and gibberellin (GA) sensitivity in regulating pre-maturity α-amylase (PMA) in wheat grains, plants were grown in a glasshouse under cold-shock and ambient conditions. α-amylase activity in response to applied ABA and GA was measured in detached-grains with the embryo removed (in vitro) and in intact-grains attached to the plant (in situ). The in vitro experiment was conducted using Spark (low PMA-susceptible genotype) and Rialto (highly PMA-susceptible genotype), with the aim of defining the time point for GA-sensitivity. The results showed an increase in GA-sensitivity at about 640 degree days after anthesis (DAA) in Rialto. There was no evidence for a change in ABA-sensitivity in either variety. The in situ experiments were conducted using genotypes from a Spark × Rialto doubled haploid population segregating for the Rht-D1a (tall) or Rht-D1b allele and for the presence or absence of 1BS/1RS. For Rht-D1a (tall) or Rht-D1b genotypes with or without 1BS/1RS, the cold-shock significantly increased GA-sensitivity, whereas there was no significant change in ABA-sensitivity. These results show PMA is related to an increase in GA-sensitivity that occurs in the aleurone at around 640 degree DAA, and can be enhanced by environmental factors (e.g. cold-shock).     

The role of sensitivity to abscisic acid and gibberellin in pre-maturity α-amylase formation in wheat grains

To study the role of abscisic acid (ABA) and gibberellin (GA) sensitivity in regulating pre-maturity α-amylase (PMA) in wheat grains, plants were grown in a glasshouse under cold-shock and ambient conditions. α-amylase activity in response to applied ABA and GA was measured in detached-grains with the embryo removed (in vitro) and in intact-grains attached to the plant (in situ). The in vitro experiment was conducted using Spark (low PMA-susceptible genotype) and Rialto (highly PMA-susceptible genotype), with the aim of defining the time point for GA-sensitivity. The results showed an increase in GA-sensitivity at about 640 degree days after anthesis (DAA) in Rialto. There was no evidence for a change in ABA-sensitivity in either variety. The in situ experiments were conducted using genotypes from a Spark × Rialto doubled haploid population segregating for the Rht-D1a (tall) or Rht-D1b allele and for the presence or absence of 1BS/1RS. For Rht-D1a (tall) or Rht-D1b genotypes with or without 1BS/1RS, the cold-shock significantly increased GA-sensitivity, whereas there was no significant change in ABA-sensitivity. These results show PMA is related to an increase in GA-sensitivity that occurs in the aleurone at around 640 degree DAA, and can be enhanced by environmental factors (e.g. cold-shock).     

Use of the hormone-biosynthesis inhibitors fluridone and paclobutrazol to determine the effects of altered abscisic acid and gibberellin levels on pre-maturity α-amylase formation in wheat grains

During germination of cereal grain, α-amylase formation is known to be inhibited by abscisic acid (ABA) and stimulated by gibberellins (GA). The role of these hormones in pre-maturity α-amylase (PMA) formation in wheat grains is less well understood. Our previous work with ABA and GA exogenously applied to grains demonstrated a clear stimulatory effect of GA, with little effect of ABA. Here, in glasshouse experiments, fluridone (ABA biosynthesis inhibitor; FD [20 μM]) or paclobutrazol (GA biosynthesis inhibitor; PB [20 μM]) were applied to intact, developing grains of the PMA-susceptible variety Rialto at 480 days after anthesis (DAA) to assess if a reduction in endogenous ABA and/or GA alters PMA formation. The experiments were conducted under non-PMA-inducing (ambient) and PMA-inducing (cold-shock) conditions. In solvent-only treated grains, a cold-shock significantly reduced the ABA content but increased GA and α-amylase activity. FD increased GA levels and α-amylase activity under ambient conditions, but decreased GA levels and α-amylase activity under cold-shock conditions, with no effect on ABA levels under either condition. PB had no effect under ambient conditions, but reduced GA levels and α-amylase under cold-shock conditions. These results indicate an association between GA levels at mid-grain development and PMA formation in wheat.     

The heterogeneous distribution of α-amylase and endoxylanase activity over a population of preharvest sprouted wheat kernels and their localization in individual kernels

To develop targeted approaches to improve the quality of preharvest sprouted (PHS) wheat as a raw material for food manufacturing, knowledge on the nature and distribution of hydrolytic enzymes in PHS wheat is crucial. Results of the present study indicate that α-amylase and endoxylanase activities are heterogeneously distributed among a population of PHS kernel. Within individual severely sprouted kernels, the enzyme activities are heterogeneously distributed throughout the different tissues. α-Amylase activity, almost exclusively of endogenous nature, is mainly detected in the germ region and to a lesser extent in the aleurone layer. Endoxylanase activity is predominantly of microbial origin and located on the kernel surface. In spite of this, light and epifluorescence microscopy show decreased kernel integrity and cell wall breakdown in the crushed cells layer, the endosperm, and the aleurone layer in a selection of kernels upon preharvest sprouting. This knowledge offers opportunities for the development of treatments to reduce the enzyme load in PHS wheat at postharvest level to improve its flour quality.     

Polymorphism and pedigree analysis of β-amylase isozymes in Australian wheat

β-Amylase encoded by multiple loci on chromosomes of triticeae crop plants is a starch metabolic enzyme with a significant physiological role in flour functionality. In this study, wheat grain β-amylase isozymes were characterized using an extensive Australian wheat population and genetic breeding/mapping lines. β-amylase isozymes were fractionated by high-resolution native polyacrylamide gel electrophoresis (PAGE) and validated by zymogram and peptide sequencing using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Specific antibodies were generated against the β-amylase purified through protein chromatography and used for β-amylase identification. Further characterization of wheat β-amylases was carried out with immunoblotting and 2-dimensional electrophoresis (2-DE). The enzyme was found to be polymorphic among the Australian and foreign wheat cultivars, with a total of six isozymes: two common and four varying isozymes, the latter group including one novel β-amylase isozyme of fast-mobility in native PAGE, According to pedigree analysis, the origin of this particular isozyme was from the ancestral genotype, Ciano F 67.     

Effects of fungal α-amylase on chemically leavened wheat flour doughs

Chemical leaveners are used in doughs to generate carbon dioxide, as an alternative to yeast, in making a range of bakery products. In this study, the effects of fungal α-amylase and ascorbic acid on chemically leavened doughs were followed by measuring dough extensibility, true rheological properties, the amount of free liquid in doughs following ultracentrifugation and the quality of baked products. As with yeasted doughs, the bake qualities of chemically leavened doughs also improved in the presence of fungal α-amylases. The bake qualities were not affected when the equivalent amount of ascorbic acid was added. The differences in dough formulations were detected from measurements of true rheological properties, not from extensibilities of doughs. The amount of free liquid was larger and of lower viscosity in doughs containing α-amylases. The properties of the continuous liquid phase were found to be important in defining the rheological and baking qualities of doughs.     

Content of starch and sugars and in vitro digestion of starch by α-amylase in five minor millets

Five varieties of minor millets were studied for their amylose, soluble amylose, amylopectin, soluble amylopectin, reducing sugar, total sugar and starch contents. Pure starch was isolated from each variety and the enzymic degradation of starch by porcine pancreatic -amylase were examined with and without gelatinisation. Gelatinised sample ofEchinochloa frumentacea (var. K2) showed minimal hydrolysis and gelatinised sample ofPanicum miliaceum (var. CO3) showed maximum hydrolysis of starch by porcine pancreatic -amylase. Gelatinised starch was highly susceptible to enzymic digestion when compared to ungelatinised starch. The extent of starch degradation varied from 71 to 85 percent in gelatinised samples and starch degradation in ungelatinised sample varied from 10 to 18 percent.     

Inhibition of α-amylase purified fromOctopus vulgaris Lam. by albumin inhibitors from wheat flour

Octopus -amylase has been purified to homogeneity by single-step affinity chromatography on Sepharose-bound wheat albumin inhibitors. Two electrophoretically distinguishable isoamylases are obtained, both consisting of a single polypeptide chain with molecular weight 45,000. Octopus -amylase is more effectively inhibited by a monomeric than by a dimeric protein inhibitor from wheat. The inhibition is dependent on pH, temperature, and time of preincubation. Properties are compared with those of other animal -amylases.     

A rapid,automated method for measuring α-amylase in pre-harvest sprouted (sprout damaged) wheat

The quality of wheat for baking is critically dependent on the level of α-amylase (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1), which can be present as “late maturity α-amylase” (LMA), or due to pre-harvest sprouting due to high rainfall and humidity at the time of harvesting. The most commonly used method to measure α-amylase in wheat grain is the Hagberg Falling Number method, but values are also influenced by rheological properties of starch in the grain. In this study we describe a simple, rapid, automated method (Amylase SD) for measurement of α-amylase in pre-harvest sprouted (sprout damaged) wheat grain. The method (Amylase SD) measures the release of p-nitrophenol from 4,6-O-ethylidene-α-4-nitrophenyl-maltoheptaoside by α-amylase in the presence of α-glucosidase. The absorbance of p-nitrophenolate measured at 405 nm in a ChemWell^®-T auto-analyser is directly related to the level of α-amylase activity present in the milled wheat grain extract. The Amylase SD method generated <6%CV and correlation to the Falling Number method was represented by an inflection point at ∼160 s. The precision, sensitivity and speed of this method provides an ideal alternative to the Falling Number method for measurement of α-amylase (sprout damage) in wheat grain in wheat breeding programmes or at grain receival points.     

RNAi-mediated suppression of the abscisic acid catabolism gene Os ABA8ox1 increases abscisic acid content and tolerance to saline–alkaline stress in rice(Oryza sativa L.)

Saline–alkaline(SA) stress is characterized by high salinity and high alkalinity(high p H), which severely inhibit plant growth and cause huge losses in crop yields worldwide. Here we show that a moderate elevation of endogenous abscisic acid(ABA) levels by RNAi-mediated suppression of Os ABA8 ox1(Os ABA8 ox1-kd), a key ABA catabolic gene, significantly increased tolerance to SA stress in rice plants. We produced Os ABA8 ox1-kd lines in two different japonica cultivars, Dongdao 4 and Nipponbare. Compared with nontransgenic control plants(WT), the Os ABA8 ox1-kd seedlings accumulated 25.9%–55.7% higher levels of endogenous ABA and exhibited reduced plasmalemma injury, ROS accumulation and Na;/K;ratio, and higher survival rates, under hydroponic alkaline conditions simulated by 10, 15, and 20 mmol L-1 of Na;CO;. In pot trials using SA field soils of different alkali levels(p H 7.59, 8.86, and 9.29), Os ABA8 ox1–kd plants showed markedly higher seedling survival rates and more vigorous plant growth, resulting in significantly higher yield components including panicle number(85.7%–128.6%), spikelets per panicle(36.9%–61.9%), branches(153.9%–236.7%), 1000–kernel weight(20.0%–28.6%), and percentage of filled spikelets(96.6%–1340.8%) at harvest time. Under severe SA soil conditions(p H = 9.29, EC = 834.4 μS cm-1),Os ABA8 ox1-kd lines showed an 194.5%–1090.8% increase in grain yield per plant relative to WT plants.These results suggest that suppression of Os ABA8 ox1 to increase endogenous ABA levels provides a new molecular approach for improving rice yield in SA paddies.     

Inhibitory Activity of Cinnamon Bark Species and their Combination Effect with Acarbose against Intestinal α-glucosidase and Pancreatic α-amylase

Inhibition of α-glucosidase and pancreatic α-amylase is one of the therapeutic approaches for delaying carbohydrate digestion, resulting in reduced postprandial glucose. The aim of this study was to evaluate the phytochemical analysis and the inhibitory effect of various cinnamon bark species against intestinal α-glucosidase and pancreatic α-amylase. The results showed that the content of total phenolic, flavonoid, and condensed tannin ranged from 0.17 to 0.21 g gallic acid equivalent/g extract, from 48.85 to 65.52 mg quercetin equivalent/g extract, and from 0.12 to 0.15 g catechin equivalent/g extract, respectively. The HPLC fingerprints of each cinnamon species were established. Among cinnamon species, Thai cinnamon extract was the most potent inhibitor against the intestinal maltase with the IC₅₀ values of 0.58 ± 0.01 mg/ml. The findings also showed that Ceylon cinnamon was the most effective intestinal sucrase and pancreatic α-amylase inhibitor with the IC₅₀ values of 0.42 ± 0.02 and 1.23 ± 0.02 mg/ml, respectively. In addition, cinnamon extracts produced additive inhibition against intestinal α-glucosidase and pancreatic α-amylase when combined with acarbose. These results suggest that cinnamon bark extracts may be potentially useful for the control of postprandial glucose in diabetic patients through inhibition of intestinal α-glucosidase and pancreatic α-amylase.     

The location of disulphide bonds in α-type gliadins

Gliadin prepared from gluten of the cultivar Rektor by extraction with 70% (v/v) aqueous ethanol adjusted to pH 5.5 was separated by RP-HPLC. Amongst 23 components obtained, two α-type gliadins (α3- and α8-gliadin) were selected for the determination of disulphide bonds. After both proteins were digested with thermolysin, differential RP-HPLC (chromatography prior to and after reduction of disulphide bonds) was used for the detection of cystine peptides. Two cystine peptides from α3-gliadin and three cystine peptides from α8-gliadin were isolated by RP-HPLC. The resulting peptides were reduced and alkylated with 4-vinylpyridine, separated by RP-HPLC and their amino acid sequences determined. The cystine peptides from both α-type gliadins had similar structures, and the corresponding fragments had homologous sequences. One cystine peptide of each gliadin was composed of three fragments linked by two disulphide bonds. The second cystine peptide consisted of two fragments linked by one disulphide bond. The third cystine peptide derived from α8-gliadin was different from the second peptide in one position of the sequences (glutamic acid instead of glutamine). Comparing complete sequences of α-type gliadins described in the literature, the cystine peptides from α3- and α8-gliadins were identical with corresponding sequences of clones A1235 and A212, respectively¹¹. The structures of the cystine peptides analysed indicate one intramolecular disulphide bond within domain III of α-type gliadins and two disulphide bonds between domains III and V. The linkages found correspond to homologous linkages determined for low M_r subunits of glutenin and glutenin-bound γ-type gliadins⁶. Obviously, these intramolecular disulphide bonds are not linked randomly, but are strongly directed.     

Relative abundance and damage of some insect pests of wheat under different tillage practices in rice–wheat cropping in India

Tillage changes the physical and chemical properties of soil and can also inhibit or enhance useful and harmful fauna. In agriculture, different tillage technologies are being tried to enhance crop productivity, but little concrete information seems to exist on their effects on pest abundance and damage. To address this lack of information, sowing of wheat was investigated under different tillage systems. In order to monitor pest abundance and damage in altered tillage systems, the present studies on the relative abundance and damage due to insect pests viz. pink stem borer (PSB, Sesamia inferens Walker), termites (Microtermes obesi Holmgren and Odontotermes obesus Rambur) and root aphid (Rhopalosiphum rufiabdominalis Sasaki) were undertaken in a rice–wheat cropping system during 2010–11 and 2011–12. Pest abundance and damage was monitored in four tillage systems i.e. conventional tillage (CT), zero tillage (ZT), ZT + mulch and rotary tillage (RT) under insecticide protected and unprotected conditions. The application of insecticide did not affect root aphid incidence or termite damage. However, significant differences in PSB damage in insecticide protected (0.9%) and unprotected (1.2%) conditions were observed. The investigations demonstrated that in CT, damage by PSB (0.6%) was minimum; however termite damage (2.2%) was maximum as compared to all other tillage conditions. In ZT, PSB damage (1.4%) was maximum and root aphid incidence (3.1 aphids/tiller) was minimum in comparison to other tillage conditions. ZT + mulch resulted in inter-mediate insect pest incidence/damage; however, RT was the least effective practice which showed relatively high incidence/damage of these three insects (1.2% PSB damage, 1.9% termite damage and 5.1 aphids/tiller). The insecticide × tillage interaction indicated that insecticide application is needed only in ZT and RT for PSB management.     

Associations between caryopsis dormancy, α-amylase activity,and pre-harvest sprouting in barley

Pre-harvest sprouting (PHS) is a concern for barley (Hordeum vulgare L.) producers, grain processors, and researchers worldwide. Pre-harvest sprouting has been mainly attributed to low dormancy, which is determined by genotype, stage of plant maturation, and environmental conditions during caryopsis development. Fourteen barley genotypes were sown in field experiments at two sites in North Dakota in 2004 and 2005. Spikes were harvested at four different stages: ≈500 g kg⁻¹ moisture content, physiological maturity, harvest maturity, and post-harvest maturity. Results indicated that barley genotypes were released from dormancy at different rates. The 14 barley genotypes were divided into three classes based on their dormancy loss rate during caryopsis development. C93-3230-24 was highly dormant, and ‘Stander’ and ‘Legacy’ were highly susceptible to PHS due to lack of dormancy from as early as 20 d after heading date. All other genotypes fell into the third group that had intermediate dormancy loss rate. No significant correlation was detected between barley α-amylase activity and germination percentage. A moderate association between malt α-amylase activity and caryopsis dormancy suggested that cultivars with increased malt α-amylase activity tend to have low dormancy and may be more prone to PHS.     

Optimisation of droplet digital PCR for determining copy number variation of α-gliadin genes in mutant and gene-edited polyploid bread wheat

Targeted and random mutagenesis of gene families require accurate quantification. Droplet digital PCR (ddPCR) enables high-throughput screening of copy number variation (CNV). We tested the accuracy of ddPCR for CNV analysis in the large α-gliadin gene family, using degenerate primers. First, duplex ddPCR assays measured α-gliadins in diploid (15–17 copies) and tetraploid (70–76 copies) wheat accessions and a corresponding number in resulting Synthetic Hexaploid Wheat, demonstrating linear amplification up to 86–95 genes. Second, we amplified 61 α-gliadin genes in Chinese Spring. Most α-gliadins of the homoeologous chromosomes 6A and 6D were correctly amplified, as corroborated using deletion and nullisomic-tetrasomic lines, but one group of genes from 6B were not amplified with these primers. Third, in Paragon we amplified 61 α-gliadin genes while selected γ-irradiated mutant lines revealed reductions of 25–50%. Finally, using two duplex ddPCR assays, we showed that CRISPR/Cas9-targeting of α-gliadins in Fielder produced indels (1–50 bp) in up to 10 α-gliadin genes plus large deletions (>300 bp) in 20 of 87 amplified α-gliadin genes. ddPCR is suitable for high-throughput screening of CNV and gene-editing-induced mutations in large gene families, in polyploids. In wheat, ddPCR enables screening of gliadins in breeding programs towards hypoimmunogenic gluten for coeliac patients.     

Genetics and chemistry of pigments in wheat grain – A review

Cereal grains contain many phytochemicals, some of which significantly influence the grain colour. Anthocyanins are accumulated in the aleurone or pericarp layer and give blue, purple or combination of these colours. Flavonoids, such as yellow C-glycosides of flavones, flavonols, flavanonols, proanthocyanidins and reddish-coloured phlobaphenes are mainly present in the outer layer of grains while carotenoids that are responsible for yellow grain are in the endosperm. Therefore, accumulation of these pigments in the grain can represent an important target in breeding programmes aimed at increasing the concentrations of bioactive components in grain and products. This review therefore summarises our current knowledge of anthocyanin and carotenoid pigments, their genetic control and variation in levels in different wheat lines.     

Investigating the impact of α-amylase, α-glucosidase and glucoamylase action on yeast-mediated bread dough fermentation and bread sugar levels

Wheat flour is generally supplemented with α-amylases to increase maltose levels in bread dough and increase loaf volume. While the preference of yeast for glucose and fructose over maltose as substrate for fermentation is well documented, the impact of maltose versus glucose producing enzymes on bread dough fermentation kinetics and bread sugar levels is ill documented. Hence the impact of α-amylase, α-glucosidase and glucoamylase action on both aspects was investigated. Glucoamylase and α-amylase increase the total fermentable sugar content of dough, while α-glucosidase only affects the glucose/maltose ratio. Due to their effect on total fermentable sugar levels, addition of α-amylase or glucoamylase prolongs the total productive fermentation time, while this is not the case for α-glucosidase. In contrast to α-amylase, both glucoamylase and α-glucosidase supplementation leads to higher CO₂ production rates during the initial stages of fermentation. In the final bread product, different sugar levels are observed depending on the dosage and type of starch-degrading enzyme. The results of this study imply that long and short fermentation processes benefit from α-amylase and α-glucosidase addition, respectively, while glucoamylase supplementation is suitable for both long and short fermentation times.     

Studies on α-amylase and trypsin inhibitors in legume seeds using agar diffusion and isoelectric focusing techniques

Amylolytic and tryptic inhibitors of faba bean extracts were determined by an agar diffusion test. The amylolytic inhibitor had protein characters. Furthermore, water-soluble trypsin inhibitors ofCicer arietinum, Lens esculenta, Lupinus termis, Phaseolus vulgaris, Pisum sativum, Trigonella foenum-graecum andVicia faba which were separated by polyacrylamide gel isoelectric focusing (PAGIF) in thin-layers, showed species specific patterns. Negative staining showed 10 bands for French beans, 9 for fenugreek seeds, 8 for lentils and chickpeas, 7 for peas and 6 for faba beans. Lupin seeds were free from trypsin inhibitors. Treatments (soaking, germination and heat processing) of faba beans reduced the number of trypsin inhibitors in PAGIF patterns, less after soaking and germination, but more after roasting and frying. No inhibitors were detected after cooking.