Plant carbohydrate binding module enhances activity of hybrid microbial cellulase enzyme

Extent: 8 p.A synthetic, highly active cellulase enzyme suitable for in planta production may be a valuable tool for biotechnological approaches to develop transgenic biofuel crops with improved digestibility. Here, we demonstrate that the addition of a plant derived carbo-hydrate binding module (CBM) to a synthetic glycosyl hydrolase improved the activity of the hydrolase in releasing sugar from plant biomass. A CEL-HYB1-CBM enzyme was generated by fusing a hybrid microbial cellulase, CEL-HYB1, with the CBM of the tomato (Solanum lycopersicum) SlCel9C1 cellulase. CEL-HYB1 and CEL-HYB1-CBM enzymes were produced in vitro using Pichia pastoris and the activity of these enzymes was tested using carboxymethylcellulose, MUC, and native crystalline cellulose assays. The presence of the CBM substantially improved the endoglucanase activity of CEL-HYB1, especially against the native crystalline cellulose encountered in Sorghum bicolor plant cell walls. These results indicate that addition of an endogenous plant derived CBM to cellulase enzymes may enhance hydrolytic activity.Caitlin S. Byrt, Ricky Cahyanegara and Christopher P.L. Gro

Major genes for Na(+) exclusion, Nax1 and Nax2 (wheat HKT1;4 and HKT1;5), decrease Na(+) accumulation in bread wheat leaves under saline and waterlogged conditions

Two major genes for Na+ exclusion in durum wheat, Nax1 and Nax2, that were previously identified as the Na+ transporters TmHKT1;4-A2 and TmHKT1;5-A, were transferred into bread wheat in order to increase its capacity to restrict the accumulation of Na+ in leaves. The genes were crossed from tetraploid durum wheat (Triticum turgidum ssp. durum) into hexaploid bread wheat (Triticum aestivum) by interspecific crossing and marker-assisted selection for hexaploid plants containing one or both genes. Nax1 decreased the leaf blade Na+ concentration by 50%, Nax2 decreased it by 30%, and both genes together decreased it by 60%. The signature phenotype of Nax1, the retention of Na+ in leaf sheaths resulting in a high Na+ sheath:blade ratio, was found in the Nax1 lines. This conferred an extra advantage under a combination of waterlogged and saline conditions. The effect of Nax2 on lowering the Na+ concentration in bread wheat was surprising as this gene is very similar to the TaHKT1;5-D Na+ transporter already present in bread wheat, putatively at the Kna1 locus. The results indicate that both Nax genes have the potential to improve the salt tolerance of bread wheat.Richard A. James, Carol Blake, Caitlin S. Byrt, and Rana Munn

Root cell wall solutions for crop plants in saline soils

Available online 11 January 2018The root growth of most crop plants is inhibited by soil salinity. Roots respond by modulating metabolism, gene expression and protein activity, which results in changes in cell wall composition, transport processes, cell size and shape, and root architecture. Here, we focus on the effects of salt stress on cell wall modifying enzymes, cellulose microfibril orientation and non-cellulosic polysaccharide deposition in root elongation zones, as important determinants of inhibition of root elongation, and highlight cell wall changes linked to tolerance to salt stressed and water limited roots. Salt stress induces changes in the wall composition of specific root cell types, including the increased deposition of lignin and suberin in endodermal and exodermal cells. These changes can benefit the plant by preventing water loss and altering ion transport pathways. We suggest that binding of Na⁺ ions to cell wall components might influence the passage of Na⁺ and that Na⁺ can influence the binding of other ions and hinder the function of pectin during cell growth. Naturally occurring differences in cell wall structure may provide new resources for breeding crops that are more salt tolerant.Caitlin S. Byrt, Rana Munns, Rachel A. Burton, Matthew Gilliham, Stefanie Weg

A holistic high-throughput screening framework for biofuel feedstock assessment that characterises variations in soluble sugars and cell wall composition in Sorghum bicolor

Background: A major hindrance to the development of high yielding biofuel feedstocks is the ability to rapidly assess large populations for fermentable sugar yields. Whilst recent advances have outlined methods for the rapid assessment of biomass saccharification efficiency, none take into account the total biomass, or the soluble sugar fraction of the plant. Here we present a holistic high-throughput methodology for assessing sweet Sorghum bicolor feedstocks at 10 days post-anthesis for total fermentable sugar yields including stalk biomass, soluble sugar concentrations, and cell wall saccharification efficiency. Results: A mathematical method for assessing whole S. bicolor stalks using the fourth internode from the base of the plant proved to be an effective high-throughput strategy for assessing stalk biomass, soluble sugar concentrations, and cell wall composition and allowed calculation of total stalk fermentable sugars. A high-throughput method for measuring soluble sucrose, glucose, and fructose using partial least squares (PLS) modelling of juice Fourier transform infrared (FTIR) spectra was developed. The PLS prediction was shown to be highly accurate with each sugar attaining a coefficient of determination (R2) of 0.99 with a root mean squared error of prediction (RMSEP) of 11.93, 5.52, and 3.23 mM for sucrose, glucose, and fructose, respectively, which constitutes an error of <4% in each case. The sugar PLS model correlated well with gas chromatography–mass spectrometry (GC-MS) and brix measures. Similarly, a high-throughput method for predicting enzymatic cell wall digestibility using PLS modelling of FTIR spectra obtained from S. bicolor bagasse was developed. The PLS prediction was shown to be accurate with an R2 of 0.94 and RMSEP of 0.64 μg.mgDW-1.h-1. Conclusions: This methodology has been demonstrated as an efficient and effective way to screen large biofuel feedstock populations for biomass, soluble sugar concentrations, and cell wall digestibility simultaneously allowing a total fermentable yield calculation. It unifies and simplifies previous screening methodologies to produce a holistic assessment of biofuel feedstock potential.Antony P Martin, William M Palmer, Caitlin S Byrt, Robert T Furbank and Christopher PL Gro

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

Drift or shift? Continuity, change, and international variation in knowledge production in OR/MS

With the aim of contributing to the debate around OR/MS as a discipline, this study provides a historical comparative investigation of publicly available knowledge production in the field. The empirical investigation is based on a content analysis of 300 randomly selected articles from six major journals in the field. We have found: (1) since the late 1950s to the present day there has been no significant change in the types of published research in OR/MS in North America; (2) from the late 1950s to the present day, there have been significant differences in types of published research in OR/MS internationally. The imputed imbalance between theory and applications in published work had already occurred in the early stages of the development of OR/MS in North America and has since remained very much the same. Furthermore, research in the United Kingdom has been distinctly different from that dominant in North America and elsewhere. There are also indications that outside North America and the United Kingdom there is an emerging turn towards applications-oriented research. Over the last two or three decades there has been a significant increase overall in the share of articles published by academic authors

Structural variations in wheat HKT1;5 underpin differences in Na+ transport capacity

An important trait associated with the salt tolerance of wheat is the exclusion of sodium ions ( Na⁺) from the shoot. We have previously shown that the sodium transporters TmHKT1;5-A and TaHKT1;5-D, from Triticum monoccocum (Tm) and Triticum aestivum (Ta), are encoded by genes underlying the major shoot Na⁺- exclusion loci Nax1 and Kna1, respectively. Here, using heterologous expression, we show that the affinity (Km) for the Na⁺ transport of TmHKT1;5-A, at 2.66 mM, is higher than that of TaHKT1;5-D at 7.50 mM. Through 3D structural modelling, we identify residues D⁴⁷¹/a gap and D⁴⁷⁴/ G⁴⁷³ that contribute to this property. We identify four additional mutations in amino acid residues that inhibit the transport activity of TmHKT1;5-A, which are predicted to be the result of an occlusion of the pore. We propose that the underlying transport properties of TmHKT1;5-A and TaHKT1;5-D contribute to their unique ability to improve Na⁺ exclusion in wheat that leads to an improved salinity tolerance in the field.Bo Xu, Shane Waters, Caitlin S. Byrt, Darren Plett, Stephen D. Tyerman, Mark Tester, Rana Munns, Maria Hrmova, Matthew Gilliha

Prospecting for Energy-Rich Renewable Raw Materials: \u3cem\u3eAgave\u3c/em\u3e 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

Optimal strategy to identify incidence of diagnostic of diabetes using administrative data

<p>Abstract</p> <p>Background</p> <p>Accurate estimates of incidence and prevalence of the disease is a vital step toward appropriate interventions for chronic disease like diabetes. A growing body of scientific literature is now available on producing accurate information from administrative data. Advantages of use of administrative data to determine disease incidence include feasibility, accessibility and low cost, but straightforward use of administrative data can produce biased information on incident cases of chronic disease like diabetes. The present study aimed to compare criteria for the selection of diabetes incident cases in a medical administrative database.</p> <p>Methods</p> <p>An exhaustive retrospective cohort of diabetes cases was constructed for 2002 using the Canadian National Diabetes Surveillance System case definition (one hospitalization or two physician claims with a diagnosis of diabetes over a 2-year period) with the Quebec health service database. To identify previous occurrence of diabetes in the database, a five-year observation period was evaluated using retrograde survival function and kappa agreement. The use of NDSS case definition to identify incident cases was compared to a single occurrence of an ICD-9 code 250 in the records using the McNemar test.</p> <p>Results</p> <p>Retrograde survival function showed that the probability of being a true incident case after a 5-year diabetes-free observation period was almost constant and near 0.14. Agreement between 10 years (maximum period) and 5 years and more diabetes-free observation periods were excellent (kappa > 0.9). Respectively 41,261 and 37,473 incident cases were identified using a 5-year diabetes-free observation period with NDSS definition and using a single ICD-9 code 250.</p> <p>Conclusion</p> <p>A 5-year diabetes-free observation period was a conservative time to identify incident cases in an administrative database using one ICD-9 code 250 record.</p

Barley sodium content is regulated by natural variants of the Na+ transporter HvHKT1;5

During plant growth, sodium (Na+) in the soil is transported via the xylem from the root to the shoot. While excess Na+ is toxic to most plants, non-toxic concentrations have been shown to improve crop yields under certain conditions, such as when soil K+ is low. We quantified grain Na+ across a barley genome-wide association study panel grown under non-saline conditions and identified variants of a Class 1 HIGH-AFFINITY-POTASSIUM-TRANSPORTER (HvHKT1;5)-encoding gene responsible for Na+ content variation under these conditions. A leucine to proline substitution at position 189 (L189P) in HvHKT1;5 disturbs its characteristic plasma membrane localisation and disrupts Na+ transport. Under low and moderate soil Na+, genotypes containing HvHKT1:5P189 accumulate high concentrations of Na+ but exhibit no evidence of toxicity. As the frequency of HvHKT1:5P189 increases significantly in cultivated European germplasm, we cautiously speculate that this non-functional variant may enhance yield potential in non-saline environments, possibly by offsetting limitations of low available K+