Carbohydrate microarrays and their use for the identification of molecular markers for plant cell wall composition.

Genetic improvement of the plant cell wall has enormous potential to increase the quality of food, fibers, and fuels. However, the identification and characterization of genes involved in plant cell wall synthesis is far from complete. Association mapping is one of the few techniques that can help identify candidate genes without relying on our currently incomplete knowledge of cell wall synthesis. However, few cell wall phenotyping methodologies have proven sufficiently precise, robust, or scalable for association mapping to be conducted for specific cell wall polymers. Here, we created high-density carbohydrate microarrays containing chemically extracted cell wall polysaccharides collected from 331 genetically diverse Brassica napus cultivars and used them to obtain detailed, quantitative information describing the relative abundance of selected noncellulosic polysaccharide linkages and primary structures. We undertook genome-wide association analysis of data collected from 57 carbohydrate microarrays and identified molecular markers reflecting a diversity of specific xylan, xyloglucan, pectin, and arabinogalactan moieties. These datasets provide a detailed insight into the natural variations in cell wall carbohydrate moieties between B. napus genotypes and identify associated markers that could be exploited by marker-assisted breeding. The identified markers also have value beyond B. napus for functional genomics, facilitated by the close genetic relatedness to the model plant Arabidopsis Together, our findings provide a unique dissection of the genetic architecture that underpins plant cell wall biosynthesis and restructuring.

Durum wheat particle size affects starch and protein digestion in vitro.

PURPOSE: The term bioaccessibility refers to the proportion of a nutrient released from a complex food matrix during digestion and, therefore, becoming potentially available for absorption in the gastrointestinal tract. In the present study, we assessed the starch and protein bioaccessibility from a range of wheat endosperm products differing in particle size. METHODS: Five porridge meals (size A, flour, mean particle size 0.11 mm, size B, small, mean particle size 0.38 mm, size C, semolina, mean particle size 1.01 mm, size D, medium, mean particle size 1.44 mm, size E, large, mean particle size 1.95 mm) with theoretically different postprandial glycaemic responses were subjected to oral processing in vitro, followed by simulated gastric and duodenal digestion. RESULTS: A significant increase (P < 0.001) in starch degradation was observed in size A (52%) compared with size E (25%). Both sizes C and D gave less, although not significantly, digestible starch (32 and 28%, respectively). The glucose release significantly decreased as the particle size of the meal increased (92.16% detected for size A vs 47.39% for size E). In agreement with starch degradation and glucose release, size A gave the most digestible protein. CONCLUSIONS: This data provide further evidence that, by decreasing the size of wheat endosperm, starch release and glycaemic response are enhanced. We also showed that protein bioaccessibility followed a similar trend as for starch digestion. Finally, these results support the hypothesis that different degrees of starch encapsulation elicit different blood glucose responses.

Feedstock selection for polymer and chemical production: feedstock-specific recalcitrance.

Plant cell wall materials derived from a range of waste biomass sources have great potential as a source of sustainable alternatives to petrochemicals. Perhaps the most straightforward way of realising this potential would be to hydrolyse the most efficiently fermentable polymers into their constituent sugars and use yeast to ferment these into useful chemicals. However, it also makes sense to pre-extract components which have a greater value in polymeric form. This is particularly true for non-cellulosic polymers, which are rich in poorly-fermentable pentose sugars. Liquid hot water (LHW) pretreatment can be used to extract non-cellulosic carbohydrates in a cost-effective manner, leaving a cellulose-rich substrate which is easier to hydrolyse using commercial cellulases. However, inherent differences in the plant cell wall structure and composition mean that some biomass sources may be more suitable for exploitation than others. Here, we examine eight different feedstocks (two each from hardwood, softwood, cereal straws and dicotyledonous crops), expose them to 26 different LHW pretreatment conditions and hydrolyse the entire pretreated slurry with a commercial cellulase. This enables side-by-side comparisons, in terms of saccharification yield, of the feedstocks. The results clearly demonstrate considerable differences in suitability between the feedstocks, in relation to the quantity of products released and the processes needed to obtain them.

Elucidation of the genetic basis of variation for stem strength characteristics in bread wheat by Associative Transcriptomics.

BACKGROUND: The current approach to reducing the tendency for wheat grown under high fertilizer conditions to collapse (lodge) under the weight of its grain is based on reducing stem height via the introduction of Rht genes. However, these reduce the yield of straw (itself an important commodity) and introduce other undesirable characteristics. Identification of alternative height-control loci is therefore of key interest. In addition, the improvement of stem mechanical strength provides a further way through which lodging can be reduced. RESULTS: To investigate the prospects for genetic alternatives to Rht, we assessed variation for plant height and stem strength properties in a training genetic diversity panel of 100 wheat accessions fixed for Rht. Using mRNAseq data derived from RNA purified from leaves, functional genotypes were developed for the panel comprising 42,066 Single Nucleotide Polymorphism (SNP) markers and 94,060 Gene Expression Markers (GEMs). In the first application in wheat of the recently-developed method of Associative Transcriptomics, we identified associations between trait variation and both SNPs and GEMs. Analysis of marker-trait associations revealed candidates for the causative genes underlying the trait variation, implicating xylan acetylation and the COP9 signalosome as contributing to stem strength and auxin in the control of the observed variation for plant height. Predictive capabilities of key markers for stem strength were validated using a test genetic diversity panel of 30 further wheat accessions. CONCLUSIONS: This work illustrates the power of Associative Transcriptomics for the exploration of complex traits of high agronomic importance in wheat. The careful selection of genotypes included in the analysis, allowed for high resolution mapping of novel trait-controlling loci in this staple crop. The use of Gene Expression markers coupled with the more traditional sequence-based markers, provides the power required to understand the biological context of the marker-trait associations observed. This not only adds to the wealth of knowledge that we strive to accumulate regarding gene function and plant adaptation, but also provides breeders with the information required to make more informed decisions regarding the potential consequences of incorporating the use of particular markers into future breeding programmes.

Antisense down-regulation of the strawberry ß-galactosidase gene FaßGal4 increases cell wall galactose levels and reduces fruit softening.

Strawberry softening is characterized by an increase in the solubilization and depolymerization of pectins from cell walls. Galactose release from pectin side chains by ß-galactosidase enzymes has been proposed as one reason for the increase in soluble pectins. A putative ß-galactosidase gene, FaßGal4, has been identified using a custom-made oligonucleotide-based strawberry microarray platform. FaßGal4 was expressed mainly in the receptacle during fruit ripening, and was positively regulated by abscisic acid and negatively regulated by auxins. To ascertain the role of FaßGal4 in strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the CaMV35S promoter were generated. Phenotypic analyses were carried out in transgenic plants during three consecutive growing seasons, using non-transformed plants as control. Two out of nine independent transgenic lines yielded fruits that were 30% firmer than control at the ripe stage. FaßGal4 mRNA levels were reduced by 70% in ripe fruits from these selected transgenic lines, but they also showed significant silencing of FaßGal1, although the genes did not share significant similarity. These two transgenic lines also showed an increase in pectin covalently bound to the cell wall, extracted using Na2CO3. The amount of galactose in cell walls from transgenic fruits was 30% higher than in control; notably, the galactose increase was larger in the 1 M KOH fraction, which is enriched in hemicellulose. These results suggest that FaßGal4 participates in the solubilization of covalently bound pectins during ripening, reducing strawberry fruit firmness.

The effects of processing and mastication on almond lipid bioaccessibility using novel methods of in vitro digestion modelling and micro-structural analysis.

A number of studies have demonstrated that consuming almonds increases satiety but does not result in weight gain, despite their high energy and lipid content. To understand the mechanism of almond digestion, in the present study, we investigated the bioaccessibility of lipids from masticated almonds during in vitro simulated human digestion, and determined the associated changes in cell-wall composition and cellular microstructure. The influence of processing on lipid release was assessed by using natural raw almonds (NA) and roasted almonds (RA). Masticated samples from four healthy adults (two females, two males) were exposed to a dynamic gastric model of digestion followed by simulated duodenal digestion. Between 7·8 and 11·1 % of the total lipid was released as a result of mastication, with no significant differences between the NA and RA samples. Significant digestion occurred during the in vitro gastric phase (16·4 and 15·9 %) and the in vitro duodenal phase (32·2 and 32·7 %) for the NA and RA samples, respectively. Roasting produced a smaller average particle size distribution post-mastication; however, this was not significant in terms of lipid release. Light microscopy showed major changes that occurred in the distribution of lipid in all cells after the roasting process. Further changes were observed in the surface cells of almond fragments and in fractured cells after exposure to the duodenal environment. Almond cell walls prevented lipid release from intact cells, providing a mechanism for incomplete nutrient absorption in the gut. The composition of almond cell walls was not affected by processing or simulated digestion.

Expression of a bacterial, phenylpropanoid-metabolizing enzyme in tobacco reveals essential roles of phenolic precursors in normal leaf development and growth.

Tobacco plants (Nicotiana tabacum cv XHFD 8) were genetically modified to express a bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) enzyme which is active with intermediates of the phenylpropanoid pathway. We have previously shown that HCHL expression in tobacco stem resulted in various pleiotropic effects, indicative of a reduction in the carbon flux through the phenylpropanoid pathway, accompanied by an abnormal phenotype. Here, we report that in addition to the reduction in lignin and phenolic biosynthesis, HCHL expression also resulted in several gross morphological changes in poorly lignified tissue, such as abnormal mesophyll and palisade. The effect of HCHL expression was also noted in lignin-free single cells, with suspension cultures displaying an altered shape and different growth patterns. Poorly/non-lignified cell walls also exhibited a greater ease of alkaline extractability of simple phenolics and increased levels of incorporation of vanillin and vanillic acid. However, HCHL expression had no significant effect on the cell wall carbohydrate chemistry of these tissues. Evidence from this study suggests that changes in the transgenic lines result from a reduction in phenolic intermediates which have an essential role in maintaining structural integrity of low-lignin or lignin-deprived cell walls. These results emphasize the importance of the intermediates and products of phenylpropanoid pathway in modulating aspects of normal growth and development of tobacco. Analysis of these transgenic plants also shows the plasticity of the lignification process and reveals the potential to bioengineer plants with reduced phenolics (without deleterious effects) which could enhance the bioconversion of lignocellulose for industrial applications.

Anticholinesterase Activities of Different Solvent Extracts of Brewer's Spent Grain.

Cholinesterases, involved in acetylcholine catabolism in the central and peripheral nervous system, have been strongly linked with neurodegenerative diseases. Current therapeutic approaches using synthetic drugs present several side effects. Hence, there is an increasing research interest in naturally-occurring dietary polyphenols, which are also considered efficacious. Food processing by-products such as brewer's spent grain (BSG) would be a potential bio-source of polyphenols. In this study, polyphenol-rich BSG extracts using 60% acetone and 0.75% NaOH solutions were generated, which were further subjected to liquid-liquid partitioning using various organic solvents. The water-partitioned fractions of the saponified extracts had the highest total polyphenol content (6.2 ± 2.8 mgGAE/g dw) as determined by Folin-Ciocalteu reagent, while the LC-MS/MS showed ethyl acetate fraction with the highest phenolics (2.9 ± 0.3 mg/g BSG dw). The best inhibitions of acetyl- (37.9 ± 2.9%) and butyryl- (53.6 ± 7.7%) cholinesterases were shown by the diethyl ether fraction of the saponified extract. This fraction contained the highest sum of quantified phenolics (99 ± 21.2 µg/mg of extract), and with significant (p < 0.01) inhibitory contribution of decarboxylated-diferulic acid. Amongst the standards, caffeic acid presented the highest inhibition for both cholinesterases, 25.5 ± 0.2% for acetyl- and 52.3 ± 0.8% for butyryl-cholinesterase, respectively, whilst the blends insignificantly inhibited both cholinesterases. The results showed that polyphenol-rich BSG fractions have potentials as natural anti-cholinesterase agents.

Recovery of Polyphenols from Brewer's Spent Grains.

The recovery of antioxidant polyphenols from light, dark and mix brewer's spent grain (BSG) using conventional maceration, microwave and ultrasound assisted extraction was investigated. Total polyphenols were measured in the crude (60% acetone), liquor extracts (saponified with 0.75% NaOH) and in their acidified ethyl acetate (EtOAc) partitioned fractions both by spectrophotometry involving Folin-Ciocalteu reagent and liquid-chromatography-tandem mass spectrometry (LC-MS/MS) methods. Irrespective of the extraction methods used, saponification of BSG yielded higher polyphenols than in the crude extracts. The EtOAc fractionations yielded the highest total phenolic content (TPC) ranging from 3.01 ± 0.19 to 4.71 ± 0.28 mg gallic acid equivalent per g of BSG dry weight. The corresponding total polyphenols quantified by LC-MS/MS ranged from 549.9 ± 41.5 to 2741.1 ± 5.2 µg/g of BSG dry weight. Microwave and ultrasound with the parameters and equipment used did not improve the total polyphenol yield when compared to the conventional maceration method. Furthermore, the spectrophotometric quantification of the liquors overestimated the TPC, while the LC-MS/MS quantification gave a closer representation of the total polyphenols in all the extracts. The total polyphenols were in the following order in the EtOAc fractions: BSG light > BSG Mix > BSG dark, and thus suggested BSG light as a sustainable, low cost source of natural antioxidants that may be tapped for applications in food and phytopharmaceutical industries.

Peroxidase-mediated oxidative cross-linking and its potential to modify mechanical properties in water-soluble polysaccharide extracts and cereal grain residues.

Analysis of wheat bran and spent grain shows that concentrations of ferulate and diferulates offer considerable scope to modify the cross-linking of feruloylated polysaccharides and hence the mechanical properties of these residues. In solution ferulic acid (FA) can be readily polymerized by horseradish peroxidase, but when esterified to a polysaccharide, the profile of diferulates becomes restricted. This situation also exists in muro and suggests structural constraints may limit the availability of FA for cross-linking. At relatively low polysaccharide concentration, (approximately 3%), steric restrictions were apparent in gels prepared using isolated polysaccharides. Mechanical properties such as swelling also appear to be fixed at these relatively low polysaccharide concentrations. This limits the potential to modify mechanical properties in muro through oxidoreductase activity. To modify mechanical properties such treatments will need to focus on increasing the "flexibility" of the cell wall matrix and the accessibility of enzymes to the cell wall matrix.

Release of protein, lipid, and vitamin E from almond seeds during digestion.

The evaluation of the bioaccessibility of almond nutrients is incomplete. However, it may have implications for the prevention and management of obesity and cardiovascular disease. This study quantified the release of lipid, protein, and vitamin E from almonds during digestion and determined the role played by cell walls in the bioaccessibility of intracellular nutrients. Natural almonds (NA), blanched almonds (BA), finely ground almonds (FG), and defatted finely ground almonds (DG) were digested in vitro under simulated gastric and gastric followed by duodenal conditions. FG were the most digestible with 39, 45, and 44% of lipid, vitamin E, and protein released after duodenal digestion, respectively. Consistent with longer residence time in the gut, preliminary in vivo studies showed higher percentages of nutrient release, and microscopic examination of digested almond tissue demonstrated cell wall swelling. Bioaccessibility is improved by increased residence time in the gut and is regulated by almond cell walls.

Environmental impacts of food waste in Europe.

Approximately 88 Million tonnes (Mt) of food is wasted in the European Union each year and the environmental impacts of these losses throughout the food supply chain are widely recognised. This study illustrates the impacts of food waste in relation to the total food utilised, including the impact from food waste management based on available data at the European level. The impacts are calculated for the Global Warming Potential, the Acidification Potential and the Eutrophication Potential using a bottom-up approach using more than 134 existing LCA studies on nine representative products (apple, tomato, potato, bread, milk, beef, pork, chicken, white fish). Results show that 186 Mt CO2-eq, 1.7 Mt SO2-eq. and 0.7 Mt PO4-eq can be attributed to food waste in Europe. This is 15 to 16% of the total impact of the entire food supply chain. In general, the study confirmed that most of the environmental impacts are derived from the primary production step of the chain. That is why animal-containing food shows most of the food waste related impacts when it is extrapolated to total food waste even if cereals are higher in mass. Nearly three quarters of all food waste-related impacts for Global Warming originate from greenhouse gas emissions during the production step. Emissions by food processing activities contribute 6%, retail and distribution 7%, food consumption, 8% and food disposal, 6% to food waste related impacts. Even though the results are subject to certain data and scenario uncertainties, the study serves as a baseline assessment, based on current food waste data, and can be expanded as more knowledge on the type and amount of food waste becomes available. Nevertheless, the importance of food waste prevention is underlined by the results of this study, as most of the impacts originate from the production step. Through food waste prevention, those impacts can be avoided as less food needs to be produced.

Dissecting the complex regulation of lodging resistance in Brassica napus.

Lodging continues to be a major cause of yield loss in important crop species such as Brassica napus. Understanding the genetic regulation of lodging resistance is therefore of key interest to breeders worldwide. Current strategies aimed at minimising lodging risk involve the incorporation of dwarfing genes or the application of plant growth regulators. However, despite these efforts, lodging continues to be a persistent problem and it is therefore of high interest that novel, complimentary strategies for lodging control are implemented. One approach would be to focus on understanding the genetic properties underlying stem mechanical strength. With this in mind, we screened a training genetic diversity panel of B. napus accession for variation in stem mechanical strength and related traits. Using Associative Transcriptomics, we identified molecular markers for a suite of valuable traits. Using an independent test genetic diversity panel, we show that the methods employed are robust for identification of predictive markers. Furthermore, based on conserved synteny with Arabidopsis thaliana, we are able to provide a biological context to the marker associations detected and provide evidence for a role in pectin methylesterification in contributing to stem mechanical strength in Brassicaceae.

Release of cell wall phenolic esters during hydrothermal pretreatment of rice husk and rice straw.

BACKGROUND: Rice husk and rice straw represent promising sources of biomass for production of renewable fuels and chemicals. For efficient utilisation, lignocellulosic components must first be pretreated to enable efficient enzymatic saccharification and subsequent fermentation. Existing pretreatments create breakdown products such as sugar-derived furans, and lignin-derived phenolics that inhibit enzymes and fermenting organisms. Alkali pretreatments have also been shown to release significant levels of simple, free phenolics such as ferulic acid that are normally esterified to cell wall polysaccharides in the intact plant. These phenolics have recently been found to have considerable inhibitory properties. The aim of this research has been to establish the extent to which such free phenolic acids are also released during hydrothermal pretreatment of rice straw (RS) and rice husk (RH). RESULTS: RS and RH were subjected to hydrothermal pretreatments over a wide range of severities (1.57-5.45). FTIR analysis showed that the pretreatments hydrolysed and solubilised hemicellulosic moieties, leading to an enrichment of lignin and crystalline cellulose in the insoluble residue. The residues also lost the capacity for UV autofluorescence at pH 7 or pH 10, indicating the breakdown or release of cell wall phenolics. Saponification of raw RS and RH enabled identification and quantification of substantial levels of simple phenolics including ferulic acid (tFA), coumaric acid (pCA) and several diferulic acids (DiFAs) including 8-O-4'-DiFA, 8,5'-DiFA and 5,5'-DiFA. RH had higher levels of pCA and lower levels of tFA and DiFAs compared with RS. Assessment of the pretreatment liquors revealed that pretreatment-liberated phenolics present were not free but remained as phenolic esters (at mM concentrations) that could be readily freed by saponification. Many were lost, presumably through degradation, at the higher severities. CONCLUSION: Differences in lignin, tFA, DiFAs and pCA between RS and RH reflect differences in cell wall physiology, and probably contribute to the higher recalcitrance of RH compared with RS. Hydrothermal pretreatments, unlike alkali pretreatments, release cinnamic acid components as esters. The potential for pretreatment-liberated phenolic esters to be inhibitory to fermenting microorganisms is not known. However, the present study shows that they are found at concentrations that could be significantly inhibitory if released as free forms by enzyme activity.

Optimising conditions for bioethanol production from rice husk and rice straw: effects of pre-treatment on liquor composition and fermentation inhibitors.

BACKGROUND: Rice straw and husk are globally significant sources of cellulose-rich biomass and there is great interest in converting them to bioethanol. However, rice husk is reportedly much more recalcitrant than rice straw and produces larger quantities of fermentation inhibitors. The aim of this study was to explore the underlying differences between rice straw and rice husk with reference to the composition of the pre-treatment liquors and their impacts on saccharification and fermentation. This has been carried out by developing quantitative NMR screening methods. RESULTS: Air-dried rice husk and rice straw from the same cultivar were used as substrates. Carbohydrate compositions were similar, whereas lignin contents differed significantly (husk: 35.3% w/w of raw material; straw 22.1% w/w of raw material). Substrates were hydrothermally pre-treated with high-pressure microwave processing across a wide range of severities. 25 compounds were identified from the liquors of both pre-treated rice husk and rice straw. However, the quantities of compounds differed between the two substrates. Fermentation inhibitors such as 5-HMF and 2-FA were highest in husk liquors, and formic acid was higher in straw liquors. At a pre-treatment severity of 3.65, twice as much ethanol was produced from rice straw (14.22% dry weight of substrate) compared with the yield from rice husk (7.55% dry weight of substrate). Above severities of 5, fermentation was inhibited in both straw and husk. In addition to inhibitors, high levels of cellulase-inhibiting xylo-oligomers and xylose were found and at much higher concentrations in rice husk liquor. At low severities, organic acids and related intracellular metabolites were released into the liquor. CONCLUSIONS: Rice husk recalcitrance to saccharification is probably due to the much higher levels of lignin and, from other studies, likely high levels of silica. Therefore, if highly polluting chemical pre-treatments and multi-step biorefining processes are to be avoided, rice husk may need to be improved through selective breeding strategies, although more careful control of pre-treatment may be sufficient to reduce the levels of fermentation inhibitors, e.g. through steam explosion-induced volatilisation. For rice straw, pre-treating at severities of between 3.65 and 4.25 would give a glucose yield of between 37.5 and 40% (w/DW, dry weight of the substrate) close to the theoretical yield of 44.1% w/DW, and an insignificant yield of total inhibitors.

Effects of partial enzymic degradation of sugar beet pectin on oxidative coupling of pectin-linked ferulates in vitro.

Pectins were extracted from roots and petioles of sugar beet, and treated with alpha-arabinosidase, 1,4-beta-galactanase or polygalacturonase. They were then cross-linked using hydrogen peroxide and peroxidase. The effects on pectin molecular size were monitored by size-exclusion chromatography and viscometry. A decrease in apparent molecular size was observed after alpha-arabinosidase and polygalacturonase treatment, and all three enzymes caused a decrease in viscosity. The pectins were then cross-linked using hydrogen peroxide and peroxidase, and the effects on dehydrodiferulate formation were monitored by HPLC. Pretreatment with polygalacturonase caused no significant change in subsequent dehydrodiferulate cross-linking, while pretreatment with alpha-arabinosidase caused a slight change in the ratios of the different dehydrodiferulates formed. Pretreatment with 1,4-beta-d-galactanase caused a more significant change in the ratios of the different dehydrodiferulates formed, and also greatly increased the overall recovery of total ferulates (monomers plus dehydrodiferulates), both in root pectin and petiole pectin. The possible effects of polysaccharide microstructure on the dimerisation and further polymerisation of pectin-linked ferulates are discussed.