'Raising the Pulse': The environmental, nutritional and health benefits of pulse-enhanced foods.

Diet is a key modulator of non-communicable diseases, and food production represents a major cause of environmental degradation and greenhouse gas emissions. Yet, 'nudging' people to make better food choices is challenging, as factors including affordability, convenience and taste often take priority over the achievement of health and environmental benefits. The overall 'Raising the Pulse' project aim is to bring about a step change in the nutritional value of the UK consumers' diet, and to do so in a way that leads to improved health and greater sustainability within the UK food system. To achieve our objectives, UK-specific faba bean production systems that optimise both end users' diets and environmental and economic sustainability of production will be implemented in collaboration with key stakeholders (including industry, the retail sector and government). Palatable faba bean flours will be produced and used to develop 'Raising the Pulse' food products with improved nutritional profile and environmental value. Consumer focus groups and workshops will establish attitudes, preferences, drivers of and barriers to increased consumption of such products. They will inform the co-creation of sensory testing and University-wide intervention studies to evaluate the effects of pulses and 'Raising the Pulse' foods on diet quality, self-reported satiety, nutritional knowledge, consumer acceptance and market potential. Nutrient bioavailability and satiety will be evaluated in a randomised-controlled postprandial human study. Finally, a system model will be developed that predicts changes to land use, environment, business viability, nutrition and human health after substitution of existing less nutritionally beneficial and environmentally sustainable ingredients with pulses. Government health and sustainability priorities will be addressed, helping to define policy-relevant solutions with significant beneficial supply chain economic impacts and transformed sustainable food systems to improve consumer diet quality, health and the environment.

Seed Development and Protein Accumulation Patterns in Faba Bean (Vicia faba, L.).

A major objective in faba bean breeding is to improve its protein quality by selecting cultivars with enhanced desirable physicochemical properties. However, the protein composition of the mature seed is determined by a series of biological processes occurring during seed growth. Thus, any attempt to explain the final seed composition must consider the dynamics of the seed proteome during seed development. Here, we investigated the proteomic profile of developing faba bean seeds across 12 growth stages from 20 days after pollination (DAP) to full maturity. We analyzed trypsin-digested total protein extracts from the seeds at different growth stages by liquid chromatography-tandem mass spectrometry (LC-MS/MS), identifying 1217 proteins. The functional clusters of these proteins showed that, in early growth stages, proteins related to cell growth, division, and metabolism were most abundant compared to seed storage proteins that began to accumulate from 45 DAP. Moreover, label-free quantification of the relative abundance of seed proteins, including important globulin proteins, revealed several distinct temporal accumulation trends among the protein classes. These results suggest that these proteins are regulated differently and require further understanding of the impact of the different environmental stresses occurring at different grain filling stages on the expression and accumulation of these seed storage proteins.

Accumulation and deposition of triacylglycerols in the starchy endosperm of wheat grain.

A combination of lipidomics, transcriptomics and bioimaging has been used to study triacylglycerol synthesis and deposition in the developing starchy endosperm of wheat. The content of TAG increased between 14 and 34 days after anthesis, from 50 to 115 mg/100 g dry wt and from about 35 to 175 mg/100 g dry wt in two experiments. The major fatty acids were C16 (palmitic C16:0 and palmitoleic C16:1) and C18 (stearic C18:0, oleic C18:1, linoleic C18:2 and linolenic C18:3), with unsaturated fatty acids accounting for about 75-80% of the total throughout development. Linoleic acid (C18:2) was the major component at all stages and the proportion increased during development. Transcript profiling indicated that predominant route to TAG synthesis and oil accumulation is via the Kennedy pathway and diacylglycerol acyltransferase (DGAT) activity. Confocal microscopy of stained tissue sections showed that TAG accumulated in droplets which are associated with protein and concentrated in the starchy endosperm cells below the sub-aleurone cells. Transcripts encoding 16kd oleosins were also expressed, indicating that the oil droplets are in part stabilised by oleosin proteins.

The contribution of fiber components to water absorption of wheat grown in the UK.

BACKGROUND AND OBJECTIVES: The water absorption (WA) of white wheat flour is a major factor affecting processing quality, and millers, therefore, process their wheat to achieve the required level. Although it is likely that WA is determined by the amounts and compositions of three major grain components, starch, protein, and arabinoxylan, the contribution of the latter is not agreed and not recognized in the widely used Farrand equation. FINDINGS: We have measured a range of parameters related to fiber amount and composition and tested the ability of these to improve the prediction of WA using a modified Farrand equation. The addition of a range of single fiber traits improved the prediction of WA from a baseline of 82.98% to a maximum of 86.78%, but inclusion of all fiber traits as PCs resulted in a further improvement to 90%. Inclusion of the PCs also accounted for variation in WA between harvest years. The greatest improvement from inclusion of a single trait was observed with ß-glucan, the inclusion of arabinogalactan peptide (AGP) also resulted in improved prediction of WA. CONCLUSIONS: The study shows that fiber components contribute to variation in WA, including differences between harvest years, but that ß-glucan and AGP have similar or greater impacts than AX. SIGNIFICANCE AND NOVELTY: The study dissects the contributions of AX amount and composition to WA and demonstrates a contribution of b-glucan for the first time.

Identification and Quantification of Major Faba Bean Seed Proteins.

Faba bean (Vicia faba L.) holds great importance for human and animal nutrition for its high protein content. However, better understanding of its seed protein composition is required in order to develop cultivars that meet market demands for plant proteins with specific quality attributes. In this study, we screened 35 diverse Vicia faba genotypes by employing the one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE) method, and 35 major protein bands obtained from three genotypes with contrasting seed protein profiles were further analyzed by mass spectrometry (MS). Twenty-five of these protein bands (MW range: ∼ 9-107 kDa) had significant (p ≤ 0.05) matches to polypeptides in protein databases. MS analysis showed that most of the analyzed protein bands contained more than one protein type and, in total, over 100 proteins were identified. These included major seed storage proteins such as legumin, vicilin, and convicilin, as well as other protein classes like lipoxygenase, heat shock proteins, sucrose-binding proteins, albumin, and defensin. Furthermore, seed protein extracts were separated by size-exclusion high-performance liquid chromatography (SE-HPLC), and percentages of the major protein classes were determined. On average, legumin and vicilin/convicilin accounted for 50 and 27% of the total protein extract, respectively. However, the proportions of these proteins varied considerably among genotypes, with the ratio of legumin:vicilin/convicilin ranging from 1:1 to 1:3. In addition, there was a significant (p < 0.01) negative correlation between the contents of these major fractions (r = -0.83). This study significantly extends the number of identified Vicia faba seed proteins and reveals new qualitative and quantitative variation in seed protein composition, filling a significant gap in the literature. Moreover, the germplasm and screening methods presented here are expected to contribute in selecting varieties with improved protein content and quality.

High post-anthesis temperature effects on bread wheat (Triticum aestivum L.) grain transcriptome during early grain-filling.

BACKGROUND: High post-anthesis (p.a) temperatures reduce mature grain weights in wheat and other cereals. However, the causes of this reduction are not entirely known. Control of grain expansion by the maternally derived pericarp of the grain has previously been suggested, although this interaction has not been investigated under high p.a. temperatures. Down-regulation of pericarp localised genes that regulate cell wall expansion under high p.a. temperatures may limit expansion of the encapsulated endosperm due to a loss of plasticity in the pericarp, reducing mature grain weight. Here the effect of high p.a. temperatures on the transcriptome of the pericarp and endosperm of the wheat grain during early grain-filling was investigated via RNA-Seq and is discussed alongside grain moisture dynamics during early grain development and mature grain weight. RESULTS: High p.a. temperatures applied from 6-days after anthesis (daa) and until 18daa reduced the grain's ability to accumulate water, with total grain moisture and percentage grain moisture content being significantly reduced from 14daa onwards. Mature grain weight was also significantly reduced by the same high p.a. temperatures applied from 6daa for 4-days or more, in a separate experiment. Comparison of our RNA-Seq data from whole grains, with existing data sets from isolated pericarp and endosperm tissues enabled the identification of subsets of genes whose expression was significantly affected by high p.a. temperature and predominantly expressed in either tissue. Hierarchical clustering and gene ontology analysis resulted in the identification of a number of genes implicated in the regulation of cell wall expansion, predominantly expressed in the pericarp and significantly down-regulated under high p.a. temperatures, including endoglucanase, xyloglucan endotransglycosylases and a ß-expansin. An over-representation of genes involved in the 'cuticle development' functional pathway that were expressed in the pericarp and affected by high p.a. temperatures was also observed. CONCLUSIONS: High p.a. temperature induced down-regulation of genes involved in regulating pericarp cell wall expansion. This concomitant down-regulation with a reduction in total grain moisture content and grain weight following the same treatment period, adds support to the theory that high p.a. temperatures may cause a reduction in mature grain weight as result of decreased pericarp cell wall expansion.

Spatial distribution of functional components in the starchy endosperm of wheat grains.

The starchy endosperm of the mature wheat grain comprises three major cell types, namely sub-aleurone cells, prismatic cells and central cells, which differ in their contents of functional components: gluten proteins, starch, cell wall polysaccharides (dietary fibre) and lipids. Gradients are established during grain development but may be modified during grain maturation and are affected by plant nutrition, particularly nitrogen application, and environmental factors. Although the molecular controls of their formation are unknown, the high content of protein and low content of starch of sub-aleurone cells, compared to the other starchy endosperm cells types, may result from differences in developmental programming related to the cells having a separate origin (from anticlinal division of the aleurone cells). The gradients within the grain may be reflected in differences in the compositions of mill streams, particularly those streams enriched in the central and outer cells of the starchy endosperm, respectively, allowing the production of specialist flours for specific end uses.

Gradients in compositions in the starchy endosperm of wheat have implications for milling and processing.

BACKGROUND: Wheat is the major food grain consumed in temperate countries. Most wheat is consumed after milling to produce white flour, which corresponds to the endosperm storage tissue of the grain. Because the starchy endosperm accounts for about 80% of the grain dry weight, the miller aims to achieve flour yields approaching this value. SCOPE AND APPROACH: Bioimaging can be combined with biochemical analysis of fractions produced by sequential pearling of whole grains to determine the distributions of components within the endosperm tissue. KEY FINDINGS AND CONCLUSIONS: This reveals that endosperm is not homogeneous, but exhibits gradients in composition from the outer to the inner part. These include gradients in both amount and composition. For example, the content of gluten proteins decreases but the proportion of glutenin polymers increases from the outside to the centre of the tissue. However, the content of starch increases with changes in the granule size distribution, the proportions of amylose and amylopectin, and their thermal properties. Hence these parts of the endosperm differ in the functional properties for food processing. Gradients also exist in minor components which may affect health and processing, such as dietary fibre and lipids. The gradients in grain composition are reflected in differences in the compositions of the mill streams which are combined to give white flour (which may number over 20). These differences could therefore be exploited by millers and food processors to develop flours with compositions and properties for specific end uses.

Seed Storage Proteins of Faba Bean ( Vicia faba L): Current Status and Prospects for Genetic Improvement.

Faba bean ( Vicia faba L.) is one of the foremost candidate crops for simultaneously increasing both sustainability and global supply of plant protein. On a dry matter basis, its seeds contain about 29% protein of which more than 80% consists of globulin storage proteins (vicilin and legumin). However, to achieve optimum utilization of this crop for human and animal nutrition, both protein content and quality have to be improved. Though initial investigations on the heritability of these traits indicated the possibility for genetic improvement, little has been achieved so far, partly due to the lack of genetic information coupled with the complex relationship between protein content and grain yield. This review reports on the current knowledge on Vicia faba seed storage proteins, their structure, composition, and genetic control, and highlights key areas for further improvement of the content and composition of Vicia faba seed storage proteins on the basis of recent advances in Vicia faba genome knowledge and genetic tools.

Health and Nutrition Studies Related to Cereal Biodiversity: A Participatory Multi-Actor Literature Review Approach.

Recently, a large and growing body of literature has investigated the health potential of different wheat species. In particular, a considerable number of studies dealing with nutritional aspects has grown up around the theme of the recovery of ancient wheat varieties (species that have remained unchanged over the last hundred years). According to several studies, indeed, ancient varieties present a healthier nutritional profile than modern ones. In the framework of the European project "CERERE, CEreal REnaissance in Rural Europe: embedding diversity in organic and low-input food systems", this paper aimed to review recent research on the issue of health and nutritional cereal systems by adopting an innovative and participatory multi-actor approach which involved practitioners along with researchers. The participatory approach is the main innovation and peculiarity of this literature review. Nevertheless, the review highlights the many positive effects derived from eating whole and ancient grains such as a significant reduction in the risk of chronic diseases such as cancer, cardiovascular disease, and also a more favorable long-term weight management and increase in satiety. This review may be considered as a fruitful starting point that integrates research results to foster current and future healthier and sustainable practices in cereal systems.

Temperature and nitrogen supply interact to determine protein distribution gradients in the wheat grain endosperm.

Gradients exist in the distribution of storage proteins in the wheat (Triticum aestivum) endosperm and determine the milling properties and protein recovery rate of the grain. A novel image analysis technique was developed to quantify both the gradients in protein concentration, and the size distribution of protein bodies within the endosperm of wheat plants grown under two different (20 or 28 °C) post-anthesis temperatures, and supplied with a nutrient solution with either high or low nitrogen content. Under all treatment combinations, protein concentration was greater in the endosperm cells closest to the aleurone layer and decreased towards the centre of the two lobes of the grain, i.e. a negative gradient. This was accompanied by a decrease in size of protein bodies from the outer to the inner endosperm layers in all but one of the treatments. Elevated post-anthesis temperature had the effect of increasing the magnitude of the negative gradients in both protein concentration and protein body size, whilst limiting nitrogen supply decreased the gradients.

Intrinsic wheat lipid composition effects the interfacial and foaming properties of dough liquor.

Doughs were prepared from a single variety breadmaking flour (cv. Hereward), from three successive harvests (years; 2011, 2012 and 2013). A preparation of the aqueous phase from dough, known as dough liquor (DL), was prepared by ultracentrifugation and its physico-chemical properties were investigated. Surface tension and interfacial rheology, showed that the interface of DL was lipid-dominated and that 2013 DL had a different type of interface to 2011 and 2012 DL. This data was consistent with the improved foam stability observed for 2013 DL and with the types of lipids identified. All foams collapsed quickly, but the most stable foam was from 2013 DL with 89.2% loss in foam, followed by 2011 DL with 91.7% loss and 2012 had the least stable foam with a loss of 92.5% of the foam structure. Glycolipids (DGDG and MGDG) were enriched in 2013 DL, and were also present in DL foam, contributing towards improved stability. Neutral lipids, such as FFAs, were enriched in DL foams contributing towards instability and rapid foam collapse. Baking trials using 2012 and 2013 flour, showed increased loaf volumes and gas bubble diameter in 2013 bread compared to 2012 bread, highlighting the potential impact that surface active polar lipids, enriched in the aqueous phase of dough, could have on improving breadmaking quality.

The dynamics of protein body formation in developing wheat grain.

Wheat is a major source of protein in the diets of humans and livestock but we know little about the mechanisms that determine the patterns of protein synthesis in the developing endosperm. We have used a combination of enrichment with (15) N glutamine and NanoSIMS imaging to establish that the substrate required for protein synthesis is transported radially from its point of entrance in the endosperm cavity across the starchy endosperm tissues, before becoming concentrated in the cells immediately below the aleurone layer. This transport occurs continuously during grain development but may be slower in the later stages. Although older starchy endosperm cells tend to contain larger protein deposits formed by the fusion of small protein bodies, small highly enriched protein bodies may also be present in the same cells. This shows a continuous process of protein body initiation, in both older and younger starchy endosperm cells and in all regions of the tissue. Immunolabeling with specific antibodies shows that the patterns of enrichment are not related to the contents of gluten proteins in the protein bodies. In addition to providing new information on the dynamics of protein deposition, the study demonstrates the wider utility of NanoSIMS and isotope labelling for studying complex developmental processes in plant tissues.

Influence of temperature during grain filling on gluten viscoelastic properties and gluten protein composition.

BACKGROUND: The aim of this study was to investigate the effects of low to moderate temperatures on gluten functionality and gluten protein composition. Four spring wheat cultivars were grown in climate chambers with three temperature regimes (day/night temperatures of 13/10, 18/15 and 23/20 °C) during grain filling. RESULTS: The temperature strongly influenced grain weight and protein content. Gluten quality measured by maximum resistance to extension (Rmax ) was highest in three cultivars grown at 13 °C. Rmax was positively correlated with the proportion of sodium dodecyl sulfate-unextractable polymeric proteins (%UPP). The proportions of ω-gliadins and D-type low-molecular-weight glutenin subunits (LMW-GS) increased and the proportions of α- and γ-gliadins and B-type LMW-GS decreased with higher temperature, while the proportion of high-molecular-weight glutenin subunits (HMW-GS) was constant between temperatures. The cultivar Berserk had strong and constant Rmax between the different temperatures. CONCLUSION: Constant low temperature, even as low as 13 °C, had no negative effects on gluten quality. The observed variation in Rmax related to temperature could be explained more by %UPP than by changes in the proportions of HMW-GS or other gluten proteins. The four cultivars responded differently to temperature, as gluten from Berserk was stronger and more stable over a wide range of temperatures.

Distribution of Lipids in the Grain of Wheat (cv. Hereward) Determined by Lipidomic Analysis of Milling and Pearling Fractions.

Lipidomic analyses of milling and pearling fractions from wheat grain were carried out to determine differences in composition that could relate to the spatial distribution of lipids in the grain. Free fatty acids and triacylglycerols were major components in all fractions, but the relative contents of polar lipids varied, particularly those of lysophosphatidylcholine and digalactosyldiglyceride, which were enriched in flour fractions. By contrast, minor phospholipids were enriched in bran and offal fractions. The most abundant fatty acids in the analyzed acyl lipids were C16:0 and C18:2 and their combinations, including C36:4 and C34:2. Phospholipids and galactolipids have been reported to have beneficial properties for breadmaking, whereas free fatty acids and triacylglycerols are considered detrimental. The subtle differences in the compositions of fractions determined in the present study could therefore underpin the production of flour fractions with optimized compositions for different end uses.

Comparative in situ analyses of cell wall matrix polysaccharide dynamics in developing rice and wheat grain.

Cell wall polysaccharides of wheat and rice endosperm are an important source of dietary fibre. Monoclonal antibodies specific to cell wall polysaccharides were used to determine polysaccharide dynamics during the development of both wheat and rice grain. Wheat and rice grain present near synchronous developmental processes and significantly different endosperm cell wall compositions, allowing the localisation of these polysaccharides to be related to developmental changes. Arabinoxylan (AX) and mixed-linkage glucan (MLG) have analogous cellular locations in both species, with deposition of AX and MLG coinciding with the start of grain filling. A glucuronoxylan (GUX) epitope was detected in rice, but not wheat endosperm cell walls. Callose has been reported to be associated with the formation of cell wall outgrowths during endosperm cellularisation and xyloglucan is here shown to be a component of these anticlinal extensions, occurring transiently in both species. Pectic homogalacturonan (HG) was abundant in cell walls of maternal tissues of wheat and rice grain, but only detected in endosperm cell walls of rice in an unesterified HG form. A rhamnogalacturonan-I (RG-I) backbone epitope was observed to be temporally regulated in both species, detected in endosperm cell walls from 12 DAA in rice and 20 DAA in wheat grain. Detection of the LM5 galactan epitope showed a clear distinction between wheat and rice, being detected at the earliest stages of development in rice endosperm cell walls, but not detected in wheat endosperm cell walls, only in maternal tissues. In contrast, the LM6 arabinan epitope was detected in both species around 8 DAA and was transient in wheat grain, but persisted in rice until maturity.

The trafficking pathway of a wheat storage protein in transgenic rice endosperm.

BACKGROUND AND AIMS: The trafficking of proteins in the endoplasmic reticulum (ER) of plant cells is a topic of considerable interest since this organelle serves as an entry point for proteins destined for other organelles, as well as for the ER itself. In the current work, transgenic rice was used to study the pattern and pathway of deposition of the wheat high molecular weight (HMW) glutenin sub-unit (GS) 1Dx5 within the rice endosperm using specific antibodies to determine whether it is deposited in the same or different protein bodies from the rice storage proteins, and whether it is located in the same or separate phases within these. METHODS: The protein distribution and the expression pattern of HMW sub-unit 1Dx5 in transgenic rice endosperm at different stages of development were determined using light and electron microscopy after labelling with antibodies. KEY RESULTS: The use of HMW-GS-specific antibodies showed that sub-unit 1Dx5 was expressed mainly in the sub-aleurone cells of the endosperm and that it was deposited in both types of protein body present in the rice endosperm: derived from the ER and containing prolamins, and derived from the vacuole and containing glutelins. In addition, new types of protein bodies were also formed within the endosperm cells. CONCLUSIONS: The results suggest that the HMW 1Dx5 protein could be trafficked by either the ER or vacuolar pathway, possibly depending on the stage of development, and that its accumulation in the rice endosperm could compromise the structural integrity of protein bodies and their segregation into two distinct populations in the mature endosperm.

RNA interference suppression of genes in glycosyl transferase families 43 and 47 in wheat starchy endosperm causes large decreases in arabinoxylan content.

The cell walls of wheat (Triticum aestivum) starchy endosperm are dominated by arabinoxylan (AX), accounting for 65% to 70% of the polysaccharide content. Genes within two glycosyl transferase (GT) families, GT43 (IRREGULAR XYLEM9 [IRX9] and IRX14) and GT47 (IRX10), have previously been shown to be involved in the synthesis of the xylan backbone in Arabidopsis, and close homologs of these have been implicated in the synthesis of xylan in other species. Here, homologs of IRX10 TaGT47_2 and IRX9 TaGT43_2, which are highly expressed in wheat starchy endosperm cells, were suppressed by RNA interference (RNAi) constructs driven by a starchy endosperm-specific promoter. The total amount of AX was decreased by 40% to 50% and the degree of arabinosylation was increased by 25% to 30% in transgenic lines carrying either of the transgenes. The cell walls of starchy endosperm in sections of grain from TaGT43_2 and TaGT47_2 RNAi transgenics showed decreased immunolabeling for xylan and arabinoxylan epitopes and approximately 50% decreased cell wall thickness compared with controls. The proportion of AX that was water soluble was not significantly affected, but average AX polymer chain length was decreased in both TaGT43_2 and TaGT47_2 RNAi transgenics. However, the long AX chains seen in controls were absent in TaGT43_2 RNAi transgenics but still present in TaGT47_2 RNAi transgenics. The results support an emerging picture of IRX9-like and IRX10-like proteins acting as key components in the xylan synthesis machinery in both dicots and grasses. Since AX is the main component of dietary fiber in wheat foods, the TaGT43_2 and TaGT47_2 genes are of major importance to human nutrition.