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Wheat remains a critical global food source, pressured by climate change and the need to maximize yield, improve processing and nutritional quality and ensure safety. An enormous amount of research has been conducted to understand gluten protein composition and structure in relation to end-use quality, yet progress has become stagnant. This is mainly due to the need and inability to biochemically characterize the intact functional glutenin polymer in order to correlate to quality, necessitating reduction to monomeric subunits and a loss of contextual information. While some individual gluten proteins might have a positive or negative influence on gluten quality, it is the sum total of these proteins, their relative and absolute expression, their sub-cellular trafficking, the amount and size of glutenin polymers, and ratios between gluten protein classes that define viscoelasticity of gluten. The sub-cellular trafficking of gluten proteins during seed maturation is still not completely clear and there is evidence of dual pathways and therefore different destinations for proteins, either constitutively or temporally. The trafficking of proteins is also unclear in endosperm cells as they undergo programmed cell death; Golgi disappear around 12 DPA but protein filling continues at least to 25 DPA. Modulation of the timing of cellular events will invariably affect protein deposition and therefore gluten strength and function. Existing and emerging proteomics technologies such as proteoform profiling and top-down proteomics offer new tools to study gluten protein composition as a whole system and identify compositional patterns that can modify gluten structure with improved functionality.

Fusarium infection is a worldwide agricultural problem of billion dollar proportions globally, and it has increasingly threatened entire regional food supplies. In addition to the toxin deoxynivalenol (DON), Fusarium species express digestive enzymes that degrade starch and protein, affecting the quality of infected grains, especially wheat processing performance which depends largely on gluten proteins. In this study, the impact of Fusarium protease on the functionality of Canada Western Red Spring (CWRS) wheat was assessed by adding Fusarium-damaged kernels (FDK) to a FDK-free base wheat sample. Digestion of beta-casein by extracts of flours, milled from sound and FDK-spiked wheat samples, demonstrated elevated cleavage in FDK-spiked flour extracts as follows: N-terminal to lysine (eight-fold), N- and C-terminal to isoleucine (four-fold and three-fold, respectively), N-terminal to tyrosine (three-fold) and C-terminal to arginine at P1′ (five-fold). Comparison of abbreviated (45 min) and standard (135 min) extensigraph test results indicated that desirable increases in dough resistance to extension (Rmax) due to gluten re-polymerization after longer resting were partially to completely counteracted in FDK-spiked flours in a dose-dependent manner. Baking tests confirmed that while loaf volume is similar, proofed dough from FDK-spiked samples caused detectable loaf collapse at 3% FDK. Extensigraph Rmax and Fusarium protease levels were inversely related, and effected by both the extent and severity of infection. While the current FDK tolerances for grading Canadian wheat can effectively control protease damage, prevalence of deoxynivalenol (DON) weak- and non-producing Fusarium strains/species (e.g., F. avenaceum) in some growing regions must be considered to protect functionality if grading is solely based on DON content.

Fusarium infection is a worldwide agricultural problem of billion dollar proportions globally, and it has increasingly threatened entire regional food supplies. In addition to the toxin deoxynivalenol (DON), Fusarium species express digestive enzymes that degrade starch and protein, affecting the quality of infected grains, especially wheat processing performance which depends largely on gluten proteins. In this study, the impact of Fusarium protease on the functionality of Canada Western Red Spring (CWRS) wheat was assessed by adding Fusarium-damaged kernels (FDK) to a FDK-free base wheat sample. Digestion of beta-casein by extracts of flours, milled from sound and FDK-spiked wheat samples, demonstrated elevated cleavage in FDK-spiked flour extracts as follows: N-terminal to lysine (eight-fold), N- and C-terminal to isoleucine (four-fold and three-fold, respectively), N-terminal to tyrosine (three-fold) and C-terminal to arginine at P1′ (five-fold). Comparison of abbreviated (45 min) and standard (135 min) extensigraph test results indicated that desirable increases in dough resistance to extension (Rmax) due to gluten re-polymerization after longer resting were partially to completely counteracted in FDK-spiked flours in a dose-dependent manner. Baking tests confirmed that while loaf volume is similar, proofed dough from FDK-spiked samples caused detectable loaf collapse at 3% FDK. Extensigraph Rmax and Fusarium protease levels were inversely related, and effected by both the extent and severity of infection. While the current FDK tolerances for grading Canadian wheat can effectively control protease damage, prevalence of deoxynivalenol (DON) weak- and non-producing Fusarium strains/species (e.g., F. avenaceum) in some growing regions must be considered to protect functionality if grading is solely based on DON content.

The objective of this study was to clarify the role of (4 R )-(+)-4-methyl-1-nonanol (MNol), a female-produced sex pheromone, in mediating the mating behavior of male Tenebrio molitor L. beetles. The abilities of MNol and authentic female extract (FE) to attract virgin males and to elicit copulatory behavior (CB) from the males were determined by bioassay. Hollow glass rods were used to mimic females. The samples of interest were assayed by coating each either on the outside or on the inside surface of the hollow rods, in order to determine if the responses were due to volatile pheromones (which would be perceived whether coated on the inside or the outside of the rods) or to “contact” pheromones (which would be perceived only when coated on the outside of the rods). The responses of virgin male beetles (6–11 days post-emergence) to FE, MNol, or pentane (control) were compared at both high and low concentrations (n = 10 for each treatment). MNol and the FE showed a similar ability to attract males, at either high or low concentrations. At high concentrations, both MNol and the FE elicited CB from the males. However, unlike the FE, at low concentrations MNol was unable to elicit CB from the males. Thus the FE must contain at least one separate “copulation pheromone” that can elicit the CB at low concentrations. Furthermore, the observed “copulation pheromone” must be volatile, since no difference in response was observed whether the FE was coated on the inside or on the outside surface of the hollow rods. Thus the biological role of MNol is likely to attract males to the females and, once the male is in the immediate vicinity of the female, to act in concert with other pheromones (of which at least one is volatile) to facilitate copulation.

The fungal pathogens Cochliobolus sativus and Fusarium graminearum are frequently isolated from diseased barley spikes in western Canada. When seeds co-infected with these two species were plated on solid nutrient media, C. sativus strongly inhibited the growth of F. graminearum and at least five other Fusarium species associated with Fusarium head blight (FHB). An inhibitory substance was secreted into the media in advance of C. sativus hyphae, creating a clear zone of inhibition which was devoid of any hyphae. The size of this zone of inhibition varied between C. sativus isolates. C. sativus inhibited F. graminearum and FHB development when it was inoculated onto spikes of ‘AC Metcalfe’, ‘Manley’ or ‘Stander’ barley before inoculation with F. graminearum, whereas co-inoculation or inoculation with C. sativus after F. graminearum did not reduce FHB symptoms. A putative toxic compound was enriched from a liquid culture of C. sativus and shown to retain activity in vitro. The compound was partially...

By applying a high-resolution LC-MS method, proteoform profiling of wheat glutenin proteins has revealed numerous protein pairs differing by an average mass of 163 ± 1 Da. The most abundant of 21 such pairs, 40 344 Da and 40 181 Da (coded P40344 and P40181, respectively), were purified and subjected to peptide mapping. P40344 matched the calculated mature protein mass for Uniprot B2Y2Q7, an s-type low-molecular weight glutenin subunit (LMW-GS). The P40344 and P40181 digests differed by a single digest peptide, unambiguously matched to the C-terminus of the protein and differing by the loss of a tyrosine. Of 327 unique mature wheat LMW-GS sequences from 369 UNIPROT accessions, tyrosine is at the C-terminus in all but one accession with evidence of transcription. The C-terminal LMW-GS sequences are also highly conserved, with only 10 variants of the last six amino acids. This is the first observation of cleavage of a C-terminal tyrosine other than alpha-tubulin, and the first evidence of this post-translational modification in plants. The identification of this novel post-translational modifications in gluten proteins may provide new insights on the differential modulation of biological processes involved in seed maturation and their effect on functional properties.

Methodology for the quantitative analysis of intact glutenin proteins by ESI-LC-MS was developed with 1 Da mass resolution, constituting the first published application of proteoform profiling to plant biology. Two parent lines and 28 F5 crosses were analyzed in two blocks, 14 months apart. Control sample data were used to align retention times and normalize abundance between sample sets. A total of 4622 observations of 347 distinct proteoforms between 17 899 and 88 744 Da were observed. Proteoform abundances spanned a 1000-fold range and were linear (r2 > 0.990) with dilution. A novel method for the objective elimination of low intensity, noise-dominated data using replicate variability within the dataset is presented. Two abundant PTMs were detected; one known but uncharacterized Bx and Dy high-molecular-weight glutenin subunit (HMWGS) PTM and the other in 24 low molecular weight proteoform pairs. Finally, 16 abundant proteoforms were detected in progeny but not in either parent. This application should increase the statistical power of correlations between gluten complement and functional data and drive the detection of novel PTMs that may indicate differential regulation of the cellular processes related to quality.

A reference method (higher accuracy) and a routine method (higher throughput) were developed for the extraction of cyanogenic glycosides from flaxseed. Conditions of (essentially) complete extraction were identified by comparing grinding methods and extraction solvent composition, and optimizing solvent-to-meal ratio, extraction time, and repeat extraction. The reference extraction method consists of sample grinding using a high-speed impact plus sieving mill at 18 000 rpm with a 1.0 mm sieve coupled with triple-pooled extraction in a sonicating water bath (40C, 30 min) using 75 methanol. The routine method differs by the use of a coffee mill to grind samples and a single extraction. The 70 and 80 methanol solutions were equal and superior to other combinations from 50 to 100 aqueous ethanol or methanol. The extraction efficiencies of the routine method (relative to the reference method) was 87.9 2.0 SD (linustatin) and 87.6 1.9 SD (neolinustatin) using four composite samples that were generated from seeds of multiple cultivars over two crop years and locations across Western Canada. Ground flaxseed was stable after storage at room temperature, refrigeration, or freezing for up to 7 days, and frozen for at least 2 weeks but less than 2 months. Extracts were stable for up to 1 week at room temperature and at least 2 weeks when refrigerated or frozen.

The development of well-characterized rapid methodology for the extraction and gas chromatographic analysis of the cyanogenic glycosides linustatin and neolinustatin from flaxseed (Linum usitatissimum L.) is reported. Two quantitation methods using phenyl-β-d-glucopyranoside as an internal standard are described: direct quantitation using linustatin and neolinustatin external standard curves standard curve slope variabilities of 2.6 and 5.7% relative standard deviation (RSD), respectively, over 7 days or by use of methyl-α-d-glucopyranoside as a surrogate external standard, with conversion factors to convert to linustatin and neolinustatin concentration 1.109 ± 0.015 (SD) mg linustatin/mg methyl-α-d-glucopyranoside and 1.180 ± 0.067 (SD) mg neolinustatin/mg methyl-α-d-glucopyranoside. The former method is direct, thereby contributing less uncertainty to the method, and the latter adds a small degree of uncertainty coupled with considerable cost savings. Limits of detection for all standards were in the low- to sub-nanogram level and were 10-100 times lower than the lower limit of quantitation (LOQ). Repeatability precision was performed on 2 separate days at the lower and upper LOQs, with the RSD in peak response being 1% or lower in all cases. Extraction methods were evaluated for their ability to extract linustatin and neolinustatin from flaxseed using several combinations of aqueous ethanol, and recoveries were determined against the highest yielding method. Recoveries were as low as 82%, indicating that optimized extraction methodology is critical for the accuracy of results.

A reference method (higher accuracy) and a routine method (higher throughput) were developed for the extraction of cyanogenic glycosides from flaxseed. Conditions of (essentially) complete extraction were identified by comparing grinding methods and extraction solvent composition, and optimizing solvent-to-meal ratio, extraction time, and repeat extraction. The reference extraction method consists of sample grinding using a high-speed impact plus sieving mill at 18 000 rpm with a 1.0 mm sieve coupled with triple-pooled extraction in a sonicating water bath (40 degrees C, 30 min) using 75% methanol. The routine method differs by the use of a coffee mill to grind samples and a single extraction. The 70 and 80% methanol solutions were equal and superior to other combinations from 50 to 100% aqueous ethanol or methanol. The extraction efficiencies of the routine method (relative to the reference method) was 87.9 +/- 2.0% SD (linustatin) and 87.6 +/- 1.9% SD (neolinustatin) using four composite samples that were generated from seeds of multiple cultivars over two crop years and locations across Western Canada. Ground flaxseed was stable after storage at room temperature, refrigeration, or freezing for up to 7 days, and frozen for at least 2 weeks but less than 2 months. Extracts were stable for up to 1 week at room temperature and at least 2 weeks when refrigerated or frozen.

The development of well-characterized rapid methodology for the extraction and gas chromatographic analysis of the cyanogenic glycosides linustatin and neolinustatin from flaxseed (Linum usitatissimum L.) is reported. Two quantitation methods using phenyl-beta-D-glucopyranoside as an internal standard are described: direct quantitation using linustatin and neolinustatin external standard curves [standard curve slope variabilities of 2.6 and 5.7% relative standard deviation (RSD), respectively, over 7 days] or by use of methyl-alpha-D-glucopyranoside as a surrogate external standard, with conversion factors to convert to linustatin and neolinustatin concentration [1.109 +/- 0.015 (SD) mg linustatin/mg methyl-alpha-D-glucopyranoside and 1.180 +/-0.067 (SD) mg neolinustatin/mg methyl-alpha-D-glucopyranoside]. The former method is direct, thereby contributing less uncertainty to the method, and the latter adds a small degree of uncertainty coupled with considerable cost savings. Limits of detection for all standards were in the low- to sub-nanogram level and were 10-100 times lower than the lower limit of quantitation (LOQ). Repeatability precision was performed on 2 separate days at the lower and upper LOQs, with the RSD in peak response being 1% or lower in all cases. Extraction methods were evaluated for their ability to extract linustatin and neolinustatin from flaxseed using several combinations of aqueous ethanol, and recoveries were determined against the highest yielding method. Recoveries were as low as 82%, indicating that optimized extraction methodology is critical for the accuracy of results.