Effects of Different Gluten Proteins on Starch's Structural and Physicochemical Properties during Heating and Their Molecular Interactions.

Starch-gluten interactions are affected by biopolymer type and processing. However, the differentiation mechanisms for gluten-starch interactions during heating have not been illuminated. The effects of glutens from two different wheat flours (a weak-gluten (Yangmai 22, Y22) and a medium-strong gluten (Yangmai 16, Y16)) on starch's (S) structural and physicochemical properties during heating and their molecular interactions were investigated in this study. The results showed that gluten hindered the gelatinization and swelling of starch during heating when temperature was below 75 °C, due to competitive hydration and physical barriers of glutens, especially in Y22. Thus, over-heating caused the long-range molecular order and amylopectin branches of starch to be better preserved in the Y22-starch mixture (Y22-S) than in the Y16-starch mixture (Y16-S). Meanwhile, the starch's degradation pattern during heating in turn influenced the polymerization of both glutens. During heating, residual amylopectin branching points restricted the aggregation and cross-linking of gluten proteins due to steric hindrance. More intense interaction between Y16 and starch during heating mitigated the steric hindrance in starch-gluten networks, which was due to more residual short-range ordered starch and hydrogen bonds involved in the formation of starch-gluten networks in Y16-S during heating.

Modification of starch composition, structure and properties through editing of TaSBEIIa in both winter and spring wheat varieties by CRISPR/Cas9.

Foods high in amylose content and resistant starch (RS) offer great potential to improve human health and lower the risk of serious noninfectious diseases. Common wheat (Triticum aestivum L.) is a major staple food crop globally. However, the RS contents in the grains of modern wheat varieties are low. Here, we report the generation of high-amylose wheat through targeted mutagenesis of TaSBEIIa in a modern winter wheat cv Zhengmai 7698 (ZM) and a spring wheat cv Bobwhite by CRISPR/Cas9, respectively. We generated a series of transgene-free mutant lines either with partial or triple-null TasbeIIa alleles in ZM and Bobwhite, respectively. Analyses of starch composition, structure and properties revealed that the effects of partial or triple-null alleles were dosage dependent with triple-null lines demonstrated more profound impacts on starch composition, fine structures of amylopectin and physiochemical and nutritional properties. The flours of triple-null lines possessed significantly increased amylose, RS, protein and soluble pentosan contents which benefit human health. Baking quality analyses indicated that the high-amylose flours may be used as additives or for making cookies. Collectively, we successfully modified the starch composition, structure and properties through targeted mutagenesis of TaSBEIIa by CRISPR/Cas9 in both winter and spring wheat varieties and generated transgene-free high-amylose wheat. Our finding provides deep insights on the role of TaSBEIIa in determining starch composition, structure, properties and end-use quality in different genetic backgrounds and improving RS content with multiple breeding and end-use applications in cereal crop species through genome editing for health benefits.

Expression of starch-binding factor CBM20 in barley plastids controls the number of starch granules and the level of CO2 fixation.

The biosynthesis of starch granules in plant plastids is coordinated by the orchestrated action of transferases, hydrolases, and dikinases. These enzymes either contain starch-binding domain(s) themselves, or are dependent on direct interactions with co-factors containing starch-binding domains. As a means to competitively interfere with existing starch-protein interactions, we expressed the protein module Carbohydrate-Binding Motif 20 (CBM20), which has a very high affinity for starch, ectopically in barley plastids. This interference resulted in an increase in the number of starch granules in chloroplasts and in formation of compound starch granules in grain amyloplasts, which is unusual for barley. More importantly, we observed a photosystem-independent inhibition of CO2 fixation, with a subsequent reduced growth rate and lower accumulation of carbohydrates with effects throughout the metabolome, including lower accumulation of transient leaf starch. Our results demonstrate the importance of endogenous starch-protein interactions for controlling starch granule morphology and number, and plant growth, as substantiated by a metabolic link between starch-protein interactions and control of CO2 fixation in chloroplasts.

Protein Targeting to Starch 1 is essential for starchy endosperm development in barley.

Plant starch is the main energy contributor to the human diet. Its biosynthesis is catalyzed and regulated by co-ordinated actions of several enzymes. Recently, a factor termed Protein Targeting to Starch 1 (PTST1) was identified as being required for correct granule-bound starch synthase (GBSS) localization and demonstrated to be crucial for amylose synthesis in Arabidopsis. However, the function of its homologous protein in storage tissues (e.g. endosperm) is unknown. We identified a PTST1 homolog in barley and it was found to contain a crucial coiled-coil domain and carbohydrate-binding module. We demonstrated the interaction between PTST1 and GBSS1 by fluorescence resonance energy transfer (FRET) in barley endosperm. By tagging PTST1 with the fluorophore mCherry, we observed that it is localized in the stroma of barley endosperm amyloplasts. PTST1 overexpression in endosperm increased endogenous gbss1a gene expression and amylose content. Gbss1a and ptst1 mutants were generated using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-related protein 9 (Cas9)-based targeted mutagenesis. Homozygous gbss1a mutants showed a waxy phenotype. Grains of ptst1 mutants did not accumulate any starch. These grains dried out during the desiccation stage and were unable to germinate, suggesting that PTST1 is essential for development of starchy endosperm and viable grains.

Nitrogen topdressing timing modifies free amino acids profiles and storage protein gene expression in wheat grain.

BACKGROUND: Nitrogen is one basic element of amino acids and grain protein in wheat. In field experiments, wheat plants were subjected to different timing of nitrogen topdressing treatments: at the stages of emergence of the top fifth leaf (TL5), top third leaf (TL3) and top first leaf (TL1) to test the regulatory effects of nitrogen topdressing timing on grain protein quality. The underlying mechanisms were elucidated by clarifying the relationship between proteolysis in vegetative organs and accumulation of amino acids in the endosperm cavity, conversion of amino acids, and storage protein synthesis in endosperm of wheat grain. RESULTS: Delayed nitrogen topdressing up-regulated gene expression related to nitrogen metabolism and protease synthesis in the flag leaf, followed by more free amino acids being transported to both the cavity and the endosperm from 7 days after anthesis (DAA) to 13 DAA in TL1. TL1 enhanced the conversion between free amino acids in endosperm and upregulated the expression of genes encoding high molecular weight (HMW) and low molecular weight (LMW) subunits and protein disulfide isomerases-like (PDIL) proteins, indicating that the synthesis and folding of glutenin were enhanched by delayed nitrogen topdressing. As a consequense, the content of glutenin macropolymers (GMP) and glutenin increased with delaying nitrogen topdressing. CONCLUSIONS: The results highlight the relationship between nitrogen remobilization and final grain protein production and suggest that the nitrogen remobilization processes could be a potential target for improving the quality of wheat grain. Additionally, specific gene expression related to nitrogen topdressing was identified, which conferred more detailed insights into underlying mechanism on the modification protein quality.

Metabolomic Analysis Reveals Patterns of Whole Wheat and Pearling Fraction Flour Quality Response to Nitrogen in Two Wheat Lines with Contrasting Protein Content.

Nitrogen (N) application increases wheat yield and protein content and affects the nutritional quality of the grain. Analysis of N use efficiency revealed that N uptake efficiency is a key factor affecting protein content. Two wheat lines with significant differences in protein content were used to investigate the response of differential accumulation of metabolites to N levels and the spatial variation pattern of metabolites related to nutritional quality in wheat grains using widely targeted metabolomics analysis. The results showed that amino acids, nucleic acids, and phytohormones and their derivatives and glycolytic processes are the crucial factors affecting protein content in two wheat lines. Amino acids and derivatives, lipids, and flavonoids are the main contributors to metabolite spatial variation of grains, which were interactively regulated by nitrogen and genotypes. N application significantly increased the relative accumulation of beneficial bioactive substances in the inner layer (P3 to P5), but excessive N application may inhibit this effect and lead to poor nutritional quality.

Effect of High-Molecular Weight Glutenin Subunits (HMW-GSs) on Gluten Polymerization during Biscuit Making: Insights from Experimental and Molecular Dynamics Simulation Study.

The effect of high-molecular weight glutenin subunits (HMW-GSs) on gluten polymerization during biscuit making was investigated using a set of HMW-GS deletion lines. Results showed that the deletion of HMW-GSs improved the biscuit quality compared with the wild type (WT), especially in x-type HMW-GS deletion lines. Slight gluten depolymerization was observed during dough mixing, while progressive gluten polymerization occurred during biscuit baking. The deletion of HMW-GSs suppressed the polymerization of glutenin and gliadin compared with the WT during biscuit baking, especially in x-type HMW-GS deletion lines. These actions resulted in less elevation of the intermolecular ß-sheet and ordered α-helix and altering the disulfide (SS) conformation to a less stable conformation in HMW-GS deletion lines compared with the WT during baking. Molecular dynamics simulation analysis further demonstrated that x-type HMW-GSs had higher thermal stability compared with y-type HMW-GSs during heating.

Influence of High-Molecular-Weight Glutenin Subunit on Components and Multiscale Structure of Gluten and Dough Quality in Soft Wheat.

A set of high-molecular-weight glutenin subunit (HMW-GS) deletion lines were used to investigate the influences of HMW-GS on wheat gluten, and dough properties were investigated using a set of HMW-GS deletion lines. Results showed that HMW-GS deletion significantly decreased the dough stability time, as well as viscoelastic moduli (G' and G″), compared with the wild type, where the deletion of x-type HMW-GSs (Ax1d, Bx7d, and Dy12d) decreased more than y-type HMW-GSs (By8d and Dy12d). The deletion of HMW-GS significantly decreased HMW-GS contents and increased α-/γ-gliadin contents. A proteomic study showed that the HMW-GS deletion down-regulated the HMW-GS, ß-amylase, serpins, and protein disulfide isomerase and up-regulated the LMW-GS, α/γ-gliadin, and α-amylase inhibitor. Meanwhile, HMW-GS deletion significantly decreased contents of ß-turn and ß-sheet. In addition, less energetically stable disulfide conformations (trans-gauche-gauche and trans-gauche-trans) were abundant in HMW-GS deletion lines. Furthermore, analysis of five HMW-GSs based on amino acid sequences proved that Dx2 and Bx7 had a more stable structure, followed by Ax1, then Dy12, and finally By8.

Insights into the Functional Components in Wheat Grain: Spatial Pattern, Underlying Mechanism and Cultivation Regulation.

Wheat is a staple crop; its production must achieve both high yield and good quality due to worldwide demands for food security and better quality of life. It has been found that the grain qualities vary greatly within the different layers of wheat kernels. In this paper, the spatial distributions of protein and its components, starch, dietary fiber, and microelements are summarized in detail. The underlying mechanisms regarding the formation of protein and starch, as well as spatial distribution, are discussed from the views of substrate supply and the protein and starch synthesis capacity. The regulating effects of cultivation practices on gradients in composition are identified. Finally, breakthrough solutions for exploring the underlying mechanisms of the spatial gradients of functional components are presented. This paper will provide research perspectives for producing wheat that is both high in yield and of good quality.

DNA methylation levels of TaP5CS and TaBADH are associated with enhanced tolerance to PEG-induced drought stress triggered by drought priming in wheat.

Drought priming is a promising strategy to enhance tolerance to recurred drought in wheat. However, the underlying mechanisms of priming-induced tolerance are far from clear. Here, three different priming intensities (P1D, P2D, P3D) and two varieties with different sensitivities to drought priming were used to investigate the effects and mechanisms of drought priming. Results showed light (P1D) or moderate (P2D) drought priming intensity induced positive effects for the drought sensitive variety (YM16), while high (P3D) priming intensity brought a negative impact on the plant drought resistant. For drought insensitive one (XM33), light priming intensity had no significant effect on tolerance to drought, while moderate or high intensity showed better priming effects. Moderate priming induced higher leaf water potential and also the osmolytes levels. Consistent with the proline and betaine, the related synthetic enzymatic activities, as well as the expression of TaP5CS and TaBADH were higher in P2D in YM16 and P3D in XM33. The contents of proline and betaine showed a positive correlation with activities of SOD, CAT, GR, AsA, and GSH contents, and a negative correlation with O2.-, H2O2, and MDA contents. Further analysis revealed CG demethylation of ATG-proximal regions in the promoter of TaP5CS and TaBADH were involved in promoting the synthesis of proline and betaine in primed plants. Collectively, these findings demonstrate drought priming effect was variety independent but depended on the priming severity, and demethylation of TaP5CS and TaBADH involved in the accumulation of osmolytes which contribute to the enhanced drought tolerance induced by priming.

Effects of Cysteine and Inorganic Sulfur Applications at Different Growth Stages on Grain Protein and End-Use Quality in Wheat.

The aim of this study was to test the significant effects of inorganic sulfur and cysteine on grain protein and flour quality in wheat and to provide a theoretical basis of wheat cultivation techniques with high yield and quality. In the field experiment, a winter wheat cultivar, Yangmai 16, was used, and five treatments were established, i.e., S0 (no sulfur fertilizer application during the whole wheat growth period), S(B)60 (60 kg ha-1 inorganic sulfur fertilizer was applied as the basal fertilizer), Cys(B)60 (60 kg ha-1 cysteine sulfur fertilizer was applied as the basal fertilizer), S(J)60 (60 kg ha-1 inorganic sulfur fertilizer was applied as the jointing fertilizer), and Cys(J)60 (60 kg ha-1 cysteine sulfur fertilizer was applied as the jointing fertilizer). The fertilizer application at jointing stage showed a better influence than basal fertilizer application on protein quality; for the content of albumin, gliadin, and high molecular weight glutenin (HMW-GS), Cys(J)60 was the best among these treatments. An increase of 7.9%, 24.4%, 43.5%, 22.7% and 36.4% was found in grain yield, glutenin content, glutenin macro-polymer (GMP), low molecular weight glutenin (LMW-GS), and S content under Cys(J)60, in relation to the control, respectively. A similar trend was found in the end-use quality, as exemplified by an increase of 38.6%, 10.9%, 60.5%, and 109.8% in wet gluten content, dry gluten content, sedimentation volume, and bread-specific volume, respectively; a decrease of 69.3% and 69.1% in bread hardness and bread chewiness was found under Cys(J)60. In terms of application period, topdressing at jointing stage is compared with base fertilizer, the sulfur fertilizer application at jointing stage showed larger effects on grain protein and flour quality, from the different types of sulfur fertilizer, the application of cysteine performed better than the use of inorganic sulfur. The Cys(J)60 exhibited the best effects on protein and flour quality. It was suggested that sufficient sulfur application at jointing stage has the potential to enhance the grain protein and flour quality.

Comparative study on bread quality and starch digestibility of normal and waxy wheat (Triticum aestivum L.) modified by maltohexaose producing α-amylases.

It is highly desirable to produce bread with both acceptable texture and health benefits. In this study, maltohexaose (G6) producing amylase AmyM and its truncation AmyM-TR2 from Corallococcus sp. strain EGB were used to determine their effects to bread quality and starch physicochemical properties. During bread fermentation, AmyM or AmyM-TR2 continuously degraded the starch, resulting in more obvious decrease in relative crystallinity, the ordered structure, pasting viscosities and gelatinization enthalpy of starch than in control. The dough treated with AmyM or AmyM-TR2 increased bread volume and slowly digestible starch content, decreased bread hardness, and extended bread shelf life and as compared with control, and the dough treated with AmyM-TR2 had better improvement effects than AmyM. The volume and slowly digestible starch content of bread from the treatment of AmyM-TR2 increased by 9.74% and 7.56% in normal wheat, 1.42% and 10.28% in waxy wheat as compared with AmyM, respectively. AmyM-TR2 affected the substrate targeting, proximity and structure disruption effects, which contributed to the degradation of more starch than AmyM.

CRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley Grain.

The use of plants as heterologous hosts to produce recombinant proteins has some intriguing advantages. There is, however, the potential of overloading the endoplasmic reticulum (ER) capacity when producing recombinant proteins in the seeds. This leads to an ER-stress condition and accumulating of unfolded proteins. The unfolded protein response (UPR) is activated to alleviate the ER-stress. With the aim to increase the yield of human epidermal growth factor (EGF) and mouse leukemia inhibitory factor (mLIF) in barley, we selected genes reported to have increased expression during ER-induced stress. The selected genes were calreticulin (CRT), protein disulfide isomerase (PDI), isopentenyl diphosphate isomerase (IPI), glutathione-s-transferase (GST), HSP70, HSP26, and HSP16.9. These were knocked out using CRISPR/Cas9 or overexpressed by conventional transgenesis. The generated homozygous barley lines were crossed with barley plants expressing EGF or mLIF and the offspring plants analyzed for EGF and mLIF protein accumulation in the mature grain. All manipulated genes had an impact on the expression of UPR genes when plantlets were subjected to tunicamycin (TN). The PDI knockout plant showed decreased protein body formation, with protein evenly distributed in the cells of the endosperm. The two genes, GST and IPI, were found to have a positive effect on recombinant protein production. mLIF expression was increased in a F2 homozygous GST knockout mutant background as compared to a F2 GST wild-type offspring. The overexpression of IPI in a F1 cross showed a significant increase in EGF expression. We demonstrate that manipulation of UPR related genes can have a positive effect on recombinant protein accumulation.

Changes in spatiotemporal protein and amino acid gradients in wheat caryopsis after N-topdressing.

Wheat grain nitrogen content displays large variations within different pearling fractions of grains because of radial gradients in the protein content. We identified how spatiotemporal mechanisms regulate this. The protein gradients emerged clearly at 19 days after anthesis, with the highest N content in aleurone and seed coat, followed by outer endosperm, whereas the lowest was in middle and inner endosperm. Laser microdissection, qRT-PCR and LC-MS were used to dissect tissue from aleurone, outer endosperm, middle endosperm, inner endosperm and transfer cells, measure gene expression and levels of free and protein-bound amino acids, respectively. The results showed that different FAA transportation pathways worked in parallel during grain filling stage while the grain protein gradient did not follow spatial expression of storage proteins. Additionally, two nitrogen (N) topdressing timings were conducted, either at the emergence of top third leaf (standard timing) or top first leaf (delayed timing), finding that delayed N topdressing enhanced both amino acids supply and protein synthesis capacity. The results provide insight into protein synthesis and amino acid transport pathways in endosperm and suggest targets for the enhancement of specialty pearled wheat with higher quality.

Nitrogen topdressing timing modifies the gluten quality and grain hardness related protein levels as revealed by iTRAQ.

Nitrogen fertilization regimes significantly affect both grain quality and yield. Wheat plants were subjected to different application timing of topdressed nitrogen at the emergence of the top fifth (TL5), top third (TL3) and top first leaf (TL1), respectively. The iTRAQ (isobaric tag for relative and absolute quantitation) technology was adopted to obtain the complete proteome of wheat flour and to identify the differentially expressed proteins (DEPs) as regulated by nitrogen topdressing timing. Collectively, 591 proteins into 17 functional categories in flour of mature grains were identified. In comparison to TL3, 50 and 63 DEPs were identified in TL5 and TL1, respectively. Nine of the DEPs commonly dependent on nitrogen topdressing timing are the γ-gliadins or high-molecular-weight glutenin subunits. Additionally, delaying nitrogen topdressing modified the grain hardness and allergic protein content. The results suggested that altering nitrogen topdressing timing is a potential strategy for pursuing targeted processing quality of wheat flour.