Characterisation of soluble and insoluble cell wall fractions from rye, wheat and hull-less barley endosperm flours

Within cereal endosperm flours, arabinoxylan and β-glucan molecules exist in either a soluble or an insoluble form. From a nutritional functionality viewpoint, soluble and insoluble forms offer different potential health advantages, so it is important to define both the features controlling solubilisation and the properties of each of the soluble and insoluble fractions. Factors known to affect the stability of arabinoxylan (AX) and β-glucan (BG) solutions include AX branching extent and type, and the ratio of cellotriose to cellotetraose units (DP3/DP4) in BG. Through studying the solubilisation of AX and BG from wheat, rye, and hull less barley endosperm under conditions that avoid the use of alkali or ethanol during the solubilisation process, we report (a) similar A/X ratios and fine structures for extracted soluble arabinoxylan and the corresponding insoluble AX within the cell walls for rye and wheat endosperm flours, (b) comparable DP3/DP4 ratios for soluble β-glucan, flour and insoluble β-glucan within the endosperm cell wall of hull less barley, and (c) evidence for enrichment of β-glucan at the exterior of residual insoluble cell walls. Therefore, the factors determining solubilisation of AX and BG from endosperm cell walls are different to those that determine the stability of aqueous solutions of the same polymers, and β-glucan may show limited solubilisation by being trapped within restraining cross-linked arabinoxylans in the cell wall

Differences in glycosyltransferase family 61 accompany variation in seed coat mucilage composition in Plantago spp.

Xylans are the most abundant non-cellulosic polysaccharide found in plant cell walls. A diverse range of xylan structures influence tissue function during growth and development. Despite the abundance of xylans in nature, details of the genes and biochemical pathways controlling their biosynthesis are lacking. In this study we have utilized natural variation within the Plantago genus to examine variation in heteroxylan composition and structure in seed coat mucilage. Compositional assays were combined with analysis of the glycosyltransferase family 61 (GT61) family during seed coat development, with the aim of identifying GT61 sequences participating in xylan backbone substitution. The results reveal natural variation in heteroxylan content and structure, particularly in P. ovata and P. cunninghamii, species which show a similar amount of heteroxylan but different backbone substitution profiles. Analysis of the GT61 family identified specific sequences co-expressed with IRREGULAR XYLEM 10 genes, which encode putative xylan synthases, revealing a close temporal association between xylan synthesis and substitution. Moreover, in P. ovata, several abundant GT61 sequences appear to lack orthologues in P. cunninghamii. Our results indicate that natural variation in Plantago species can be exploited to reveal novel details of seed coat development and polysaccharide biosynthetic pathways.Jana L. Phan, Matthew R. Tucker, Shi Fang Khor, Neil Shirley, Jelle Lahnstein, Cherie Beahan, Antony Bacic and Rachel A. Burto

Effects of diverse food processing conditions on the structure and solubility of wheat, barley and rye endosperm dietary fibre

The effects of archetypal food processing conditions (dough formation, baking, extrusion, and cooking/boiling) on dietary fibre structure and extractability from the endosperm flours of rye, hull less barley and wheat are reported. For all flours and processes, the distributions of soluble/insoluble cell wall dietary fibre as well as the chemical composition (arabinoxylan (AX) branching patterns, β-glucan DP3/DP4 (DP = degree of polymerisation) ratios) of solubilised fractions were characterised. The results show that overall the total amounts of AX and β-glucan (BG) were not significantly affected by processing but that there were similar increases in the soluble fibre fraction (20-29%) for baked, extruded, and boiled/cooked processes for each flour, with lower (10-15%) increases for all flours processed into dough. In all cases, solubilised fractions of AX and BG had very similar chemical structures to the starting flour, suggesting that increased solubilisation was not due to specific chemical fractions. Confocal images illustrate loosely-held associations of β-glucan with the cell walls of processed foods in contrast to some of the arabinoxylans which appear more tightly held within the residual cell walls. The similarities in behaviour across the three grains are consistent with mechanical treatments during food preparation resulting in similar extents of disentanglement of physically-constrained AX and BG leading to their partial solubilisation

Cell wall organic matrix composition and biomineralization across reef-building coralline algae under global change.

OnlinePublCrustose coralline algae (CCA) are one of the most important benthic substrate consolidators on coral reefs through their ability to deposit calcium carbonate on an organic matrix in their cell walls. Discrete polysaccharides have been recognized for their role in biomineralization, yet little is known about the carbohydrate composition of organic matrices across CCA taxa and whether they have the capacity to modulate their organic matrix constituents amidst environmental change, particularly the threats of ocean acidification (OA) and warming. We simulated elevated pCO₂ and temperature (IPCC RCP 8.5) and subjected four mid-shelf Great Barrier Reef species of CCA to two months of experimentation. To assess the variability in surficial monosaccharide composition and biomineralization across species and treatments, we determined the monosaccharide composition of the polysaccharides present in the cell walls of surficial algal tissue and quantified calcification. Our results revealed dissimilarity among species' monosaccharide constituents, which suggests that organic matrices are composed of different polysaccharides across CCA taxa. We also found that species differentially modulate composition in response to ocean acidification and warming. Our findings suggest that both variability in composition and ability to modulate monosaccharide abundance may play a crucial role in surficial biomineralization dynamics under the stress of OA and global warming.Ellie Bergstrom, Jelle Lahnstein, Helen Collins, Tessa M. Page, Vincent Bulone, and Guillermo Diaz-Pulid

Genetics and physiology of cell wall polysaccharides in the model C(4) grass, Setaria viridis spp

Published: 2 October 2015BACKGROUND: Setaria viridis has emerged as a model species for the larger C4 grasses. Here the cellulose synthase (CesA) superfamily has been defined, with an emphasis on the amounts and distribution of (1,3;1,4)-β-glucan, a cell wall polysaccharide that is characteristic of the grasses and is of considerable value for human health. METHODS: Orthologous relationship of the CesA and Poales-specific cellulose synthase-like (Csl) genes among Setaria italica (Si), Sorghum bicolor (Sb), Oryza sativa (Os), Brachypodium distachyon (Bradi) and Hordeum vulgare (Hv) were compared using bioinformatics analysis. Transcription profiling of Csl gene families, which are involved in (1,3;1,4)-β-glucan synthesis, was performed using real-time quantitative PCR (Q-PCR). The amount of (1,3;1,4)-β-glucan was measured using a modified Megazyme assay. The fine structures of the (1,3;1,4)-β-glucan, as denoted by the ratio of cellotriosyl to cellotetraosyl residues (DP3:DP4 ratio) was assessed by chromatography (HPLC and HPAEC-PAD). The distribution and deposition of the MLG was examined using the specific antibody BG-1 and captured using fluorescence and transmission electron microscopy (TEM). RESULTS: The cellulose synthase gene superfamily contains 13 CesA and 35 Csl genes in Setaria. Transcript profiling of CslF, CslH and CslJ gene families across a vegetative tissue series indicated that SvCslF6 transcripts were the most abundant relative to all other Csl transcripts. The amounts of (1,3;1,4)-β-glucan in Setaria vegetative tissues ranged from 0.2% to 2.9% w/w with much smaller amounts in developing grain (0.003% to 0.013% w/w). In general, the amount of (1,3;1,4)-β-glucan was greater in younger than in older tissues. The DP3:DP4 ratios varied between tissue types and across developmental stages, and ranged from 2.4 to 3.0:1. The DP3:DP4 ratios in developing grain ranged from 2.5 to 2.8:1. Micrographs revealing the distribution of (1,3;1,4)-β-glucan in walls of different cell types and the data were consistent with the quantitative (1,3;1,4)-β-glucan assays. CONCLUSION: The characteristics of the cellulose synthase gene superfamily and the accumulation and distribution of (1,3;1,4)-β-glucans in Setaria are similar to those in other C4 grasses, including sorghum. This suggests that Setaria is a suitable model plant for cell wall polysaccharide biology in C4 grasses.Riksfardini A. Ermawar, Helen M. Collins, Caitlin S. Byrt, Marilyn Henderson, Lisa A. O'Donovan, Neil J. Shirley, Julian G. Schwerdt, Jelle Lahnstein, Geoffrey B. Fincher and Rachel A. Burto

Overexpression of HvCslF6 in barley grain alters carbohydrate partitioning plus transfer tissue and endosperm development

In cereal grain sucrose is converted into storage carbohydrates; mainly starch, fructan and (1,3;1,4)-β-glucan (MLG). Previously, endosperm-specific overexpression of the HvCslF6 gene in hull-less barley resulted in high MLG and low starch content in mature grains. Morphological changes included inwardly elongated aleurone cells, irregular cell shapes of peripheral endosperm and smaller starch granules of starchy endosperm. Here we explored the physiological basis for these defects by investigating how changes in carbohydrate composition of developing grain impact mature grain morphology. Augmented MLG coincided with increased levels of soluble carbohydrates in the cavity and endosperm at the storage phase. Transcript levels of genes relating to cell wall, starch, sucrose and fructan metabolism were perturbed in all tissues. The cell walls of endosperm transfer cells (ETC) in transgenic grain were thinner and showed reduced mannan labelling relative to wild type. At the early storage phase rupture of the non-uniformly developed ETC and disorganization of adjacent endosperm cells was observed. Soluble sugars accumulated in the developing grain cavity, suggesting a disturbance of carbohydrate flow from the cavity towards the endosperm, resulting in a shrunken mature grain phenotype. Our findings demonstrate the importance of regulating carbohydrate partitioning in maintenance of grain cellularization and filling processes.Wai Li Lim, Helen M. Collins, Caitlin S. Byrt, Jelle Lahnstein, Neil J. Shirley, Matthew K. Aubert, Matthew R. Tucker, Manuela Peukert, Andrea Matros, and Rachel A. Burto

Youth environmental science learning and agency: A unifying lens across community and citizen science settings

This study addresses an existing gap in our understanding of how participation in environmental Community and Citizen Science (CCS) projects may impact young volunteers’ environmental science learning across a wide variety of settings. We examined youth learning across four settings which we represented as cases: 5 short-term field-based events (BioBlitzes), 3 longer-term field-based monitoring programs, fully online projects (Zooniverse), and a hybrid format that combines participation in the field and online spaces (iNaturalist). This multiple-case study uses the Environmental Science Agency framework to interpret learning evidence of 33 young CCS volunteers (aged 10-13 years) in post-participation surveys, semi-structured interviews, and in ethnographic field notes for the field-based participants. Across the cases, we found particular features of the CCS projects and the scientific framings that may have encouraged aspects of ESA. Design features such as access to new knowledge, training, and scientific tools provided by the CCS projects encouraged youth to learn rich and varied understandings of disciplinary content, scientific skills and practices. An increased sense of confidence and competence in youth around the scientific practices of the projects were stimulated by scientific framing of CSS and ongoing participation. Overall, these aspects also supported small manifestations of youth agency with science

A novel (1,4)-beta-linked glucoxylan is synthesized by members of the cellulose synthase-like F gene family in land plants

As a significant component of monocot cell walls, (1,3;1,4)-β-glucan has conclusively been shown to be synthesized by the cellulose synthase-like F6 protein. In this study, we investigated the synthetic activity of other members of the barley (Hordeum vulgare) CslF gene family using heterologous expression. As expected, the majority of the genes encode proteins that are capable of synthesizing detectable levels of (1,3;1,4)-β-glucan. However, overexpression of HvCslF3 and HvCslF10 genes resulted in the synthesis of a novel linear glucoxylan that consists of (1,4)-β-linked glucose and xylose residues. To demonstrate that this product was not an aberration of the heterologous system, the characteristic (1,4)-β-linkage between glucose and xylose was confirmed to be present in wild type barley tissues known to contain HvCslF3 and HvCslF10 transcripts. This polysaccharide linkage has also been reported in species of Ulva, a marine green alga, and has significant implications for defining the specificity of the cell wall content of many crop species. This finding supports previous observations that members of a single CSL family may not possess the same carbohydrate synthetic activity, with the CSLF family now associated with the formation of not only (1,3)- and (1,4)-β-glucosidic linkages, but also (1,4)-β-glucosidic and (1,4)-β-xylosidic linkages.Alan Little, Jelle Lahnstein, David W. Jeffery, Shi F. Khor, Julian G. Schwerdt, Neil J. Shirley, Michelle Hooi, Xiaohui Xing, Rachel A. Burton, and Vincent Bulon

Prospecting for energy-rich renewable raw materials: agave leaf case study

Plant biomass from different species is heterogeneous, and this diversity in composition can be mined to identify materials of value to fuel and chemical industries. Agave produces high yields of energy-rich biomass, and the sugar-rich stem tissue has traditionally been used to make alcoholic beverages. Here, the compositions of Agave americana and Agave tequilana leaves are determined, particularly in the context of bioethanol production. Agave leaf cell wall polysaccharide content was characterized by linkage analysis, non-cellulosic polysaccharides such as pectins were observed by immuno-microscopy, and leaf juice composition was determined by liquid chromatography. Agave leaves are fruit-like-rich in moisture, soluble sugars and pectin. The dry leaf fiber was composed of crystalline cellulose (47-50% w/w) and non-cellulosic polysaccharides (16-22% w/w), and whole leaves were low in lignin (9-13% w/w). Of the dry mass of whole Agave leaves, 85-95% consisted of soluble sugars, cellulose, non-cellulosic polysaccharides, lignin, acetate, protein and minerals. Juice pressed from the Agave leaves accounted for 69% of the fresh weight and was rich in glucose and fructose. Hydrolysis of the fructan oligosaccharides doubled the amount of fermentable fructose in A. tequilana leaf juice samples and the concentration of fermentable hexose sugars was 41-48 g/L. In agricultural production systems such as the tequila making, Agave leaves are discarded as waste. Theoretically, up to 4000 L/ha/yr of bioethanol could be produced from juice extracted from waste Agave leaves. Using standard Saccharomyces cerevisiae strains to ferment Agave juice, we observed ethanol yields that were 66% of the theoretical yields. These data indicate that Agave could rival currently used bioethanol feedstocks, particularly if the fermentation organisms and conditions were adapted to suit Agave leaf composition.Kendall R. Corbin, Caitlin S. Byrt, Stefan Bauer, Seth DeBolt, Don Chambers, Joseph A. M. Holtum, Ghazwan Karem, Marilyn Henderson, Jelle Lahnstein, Cherie T. Beahan, Antony Bacic, Geoffrey B. Fincher, Natalie S. Betts, Rachel A. Burto

Affective processes as network hubs

The practical problems of designing and coding a web-based flight simulator for teachers has led to a ‘three-tier plus environment’ model (COVE model) for a software agent’s cognition (C), psychologicsal (O), physical (V) processes and responses to tasks and interpersonal relationships within a learning environment (E). The purpose of this article is to introduce how some of the COVE model layers represent preconscious processing hubs in an AI human-agent’s representation of learning in a serious game, and how an application of the Five Factor Model of psychology in the O layer determines the scope of dimensions for a practical computational model of affective processes. The article illustrates the model with the classroom-learning context of the simSchool application (www.simschool.org); presents details of the COVE model of an agent’s reactions to academic tasks; discusses the theoretical foundations; and outlines the research-based real world impacts from external validation studies as well as new testable hypotheses of simSchool