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

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

Molecular membrane separation: plants inspire new technologies

Plants draw up their surrounding soil solution to gain water and nutrients required for growth, development and reproduction. Obtaining adequate water and nutrients involves taking up both desired and undesired elements from the soil solution and separating resources from waste. Desirable and undesirable elements in the soil solution can share similar chemical properties, such as size and charge. Plants use membrane separation mechanisms to distinguish between different molecules that have similar chemical properties. Membrane separation enables distribution or retention of resources and efflux or compartmentation of waste. Plants use specialised membrane separation mechanisms to adapt to challenging soil solution compositions and distinguish between resources and waste. Coordination and regulation of these mechanisms between different tissues, cell types and subcellular membranes supports plant nutrition, environmental stress tolerance and energy management. This review considers membrane separation mechanisms in plants that contribute to specialised separation processes and highlights mechanisms of interest for engineering plants with enhanced performance in challenging conditions and for inspiring the development of novel industrial membrane separation technologies. Knowledge gained from studying plant membrane separation mechanisms can be applied to developing precision separation technologies. Separation technologies are needed for harvesting resources from industrial wastes and transitioning to a circular green economy.Annamaria De Rosa, Samantha McGaughey, Isobel Magrath and Caitlin Byr

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

Perception of risk of HIV and sexual risk behaviours among students in the United States, Turkey and South Africa

The aim of this study was to examine HIV sexual risk behaviours and perception of HIV risk among 1 095 students from the United States of America (US), Turkey and South Africa. Randomly selected students who were enrolled in general education courses completed a structured questionnaire. Results revealed statistically significant differences in specific HIV-related sexual behaviours among students from the three countries and among male and female students in each country. A higher percentage of US and South African students engage in HIV risky sexual behaviours compared with their Turkish counterparts, and a higher percentage of female students in the US and South Africa engage in HIV sexual risk behaviours compared with their male colleagues. A higher proportion of Turkish male students engaged in sexual risk behaviours compared with their female counterparts. The perception of HIV risk was low among US and Turkish students, and high among South African students. There was no agreement between engaging in risky sexual behaviour and self-perception of HIV risk among South African female students, while agreement was poor for US male and female students, Turkish male and female students, and South African male students. The observed optimistic bias needs to be considered in the design and implementation of HIV prevention programmes for these populations.Department of HE and Training approved lis

Divalent cations regulate the ion conductance properties of diverse classes of aquaporins

Aquaporins (AQPs) are known to facilitate water and solute fluxes across barrier membranes. An increasing number of AQPs are being found to serve as ion channels. Ion and water permeability of selected plant and animal AQPs (plant Arabidopsis thaliana AtPIP2;1, AtPIP2;2, AtPIP2;7, human Homo sapiens HsAQP1, rat Rattus norvegicus RnAQP4, RnAQP5, and fly Drosophila melanogaster DmBIB) were expressed in Xenopus oocytes and examined in chelator-buffered salines to evaluate the effects of divalent cations (Ca2+, Mg2+, Ba2+ and Cd2+) on ionic conductances. AtPIP2;1, AtPIP2;2, HsAQP1 and DmBIB expressing oocytes had ionic conductances, and showed differential sensitivity to block by external Ca2+. The order of potency of inhibition by Ca2+ was AtPIP2;2 > AtPIP2;1 > DmBIB > HsAQP1. Blockage of the AQP cation channels by Ba2+ and Cd2+ caused voltage-sensitive outward rectification. The channels with the highest sensitivity to Ca2+ (AtPIP2;1 and AtPIP2;2) showed a distinctive relief of the Ca2+ block by co-application of excess Ba2+, suggesting that divalent ions act at the same site. Recognizing the regulatory role of divalent cations may enable the discovery of other classes of AQP ion channels, and facilitate the development of tools for modulating AQP ion channels. Modulators of AQPs have potential value for diverse applications including improving salinity tolerance in plants, controlling vector-borne diseases, and intervening in serious clinical conditions involving AQPs, such as cancer metastasis, cardiovascular or renal dysfunction.Mohamad Kourghi, Saeed Nourmohammadi, Jinxin V. Pei, Jiaen Qiu, Samantha McGaughey, Stephen D. Tyerman, Caitlin S. Byrt and Andrea J. Yoo

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

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

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