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Featured researches published by Jackie Freeman.


Plant Physiology | 2010

Down-Regulation of the CSLF6 Gene Results in Decreased (1,3;1,4)-β-d-Glucan in Endosperm of Wheat

Csilla Nemeth; Jackie Freeman; Huw D. Jones; Caroline A. Sparks; Till K. Pellny; Mark D. Wilkinson; Jim M. Dunwell; Annica A.M. Andersson; Per Åman; Fabienne Guillon; Luc Saulnier; Rowan A. C. Mitchell; Peter R. Shewry

(1,3;1,4)-β-d-Glucan (β-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative β-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total β-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable β-glucan was also reduced by about 50% in the transgenic lines, and the Mr distribution of the fraction was decreased from an average of 79 to 85 × 104 g/mol in the controls and 36 to 57 × 104 g/mol in the transgenics. Immunolocalization of β-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a β-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.


Plant Physiology | 2012

Cell Walls of Developing Wheat Starchy Endosperm: Comparison of Composition and RNA-Seq Transcriptome

Till K. Pellny; Alison Lovegrove; Jackie Freeman; Paola Tosi; C. G. Love; J. P. Knox; Peter R. Shewry; Rowan A. C. Mitchell

The transcriptome of the developing starchy endosperm of hexaploid wheat (Triticum aestivum) was determined using RNA-Seq isolated at five stages during grain fill. This resource represents an excellent way to identify candidate genes responsible for the starchy endosperm cell wall, which is dominated by arabinoxylan (AX), accounting for 70% of the cell wall polysaccharides, with 20% (1,3;1,4)-β-d-glucan, 7% glucomannan, and 4% cellulose. A complete inventory of transcripts of 124 glycosyltransferase (GT) and 72 glycosylhydrolase (GH) genes associated with cell walls is presented. The most highly expressed GT transcript (excluding those known to be involved in starch synthesis) was a GT47 family transcript similar to Arabidopsis (Arabidopsis thaliana) IRX10 involved in xylan extension, and the second most abundant was a GT61. Profiles for GT43 IRX9 and IRX14 putative orthologs were consistent with roles in AX synthesis. Low abundances were found for transcripts from genes in the acyl-coA transferase BAHD family, for which a role in AX feruloylation has been postulated. The relative expression of these was much greater in whole grain compared with starchy endosperm, correlating with the levels of bound ferulate. Transcripts associated with callose (GSL), cellulose (CESA), pectin (GAUT), and glucomannan (CSLA) synthesis were also abundant in starchy endosperm, while the corresponding cell wall polysaccharides were confirmed as low abundance (glucomannan and callose) or undetectable (pectin) in these samples. Abundant transcripts from GH families associated with the hydrolysis of these polysaccharides were also present, suggesting that they may be rapidly turned over. Abundant transcripts in the GT31 family may be responsible for the addition of Gal residues to arabinogalactan peptide.


Journal of Aerosol Science | 2002

Detection of airborne fungal spores sampled by rotating-arm and Hirst-type spore traps using polymerase chain reaction assays

Carmen Calderon; Elaine Ward; Jackie Freeman; Alastair McCartney

AbstractConventional methods for detectingairborne fungal spores rely on either optical identication orculturingand can be time consumingor unreliable. A method for purifyingDNA from conventionalsporesamplersanddetectingitusingpolymerasechainreaction(PCR)assaysisdescribed.Experimentswere done using Penicillium roqueforti. As few as 10 spores could be detected in the PCR andP.roquefortisporesweredetectedinabackgroundofsporesofsixotherunrelatedspecies.Themethodsuccessfully detected P. roqueforti spores collected by rotatingarm and Hirst-type spore traps in windtunnel tests. The tests suggested that the detection limit was about 10 spores or less in the PCR. Fungalspores were also detected in air samples collected in Mexico City usingfungal consensus primers, witha detection limit of about 200 spores in the PCR. The potential for usingPCR-assays in conjunctionwith impactor samplers is discussed. c 2001 Elsevier Science Ltd. All rights reserved. 1. IntroductionBioaerosolsmaycausearangeofhealtheectsinhumansandanimals,dependingonthepre-dominant components present and their concentrations. Such eects include mucous membraneirritation, chronic bronchitis, allergic rhinitis and asthma, extrinsic allergic alveolitis (hyper-sensitivity pneumonitis), inhalation fever, humidier fever or organic dust toxic syndrome and


Frontiers in Plant Science | 2013

Grass cell wall feruloylation: distribution of bound ferulate and candidate gene expression in Brachypodium distachyon

Till K. Pellny; Jackie Freeman; Peter R. Shewry; Rowan A. C. Mitchell

The cell walls of grasses such as wheat, maize, rice, and sugar cane, contain large amounts of ferulate that is ester-linked to the cell wall polysaccharide glucuronoarabinoxylan (GAX). This ferulate is considered to limit the digestibility of polysaccharide in grass biomass as it forms covalent linkages between polysaccharide and lignin components. Candidate genes within a grass-specific clade of the BAHD acyl-coA transferase superfamily have been identified as being responsible for the ester linkage of ferulate to GAX. Manipulation of these BAHD genes may therefore be a biotechnological target for increasing efficiency of conversion of grass biomass into biofuel. Here, we describe the expression of these candidate genes and amounts of bound ferulate from various tissues and developmental stages of the model grass Brachypodium distachyon. BAHD candidate transcripts and significant amounts of bound ferulate were present in every tissue and developmental stage. We hypothesize that BAHD candidate genes similar to the recently described Oryza sativa p-coumarate monolignol transferase (OsPMT) gene (PMT sub-clade) are principally responsible for the bound para-coumaric acid (pCA), and that other BAHD candidates (non-PMT sub-clade) are responsible for bound ferulic acid (FA). There were some similarities with between the ratio of expression non-PMT/PMT genes and the ratio of bound FA/pCA between tissue types, compatible with this hypothesis. However, much further work to modify BAHD genes in grasses and to characterize the heterologously expressed proteins is required to demonstrate their function.


Planta | 2010

Temporal and spatial changes in cell wall composition in developing grains of wheat cv. Hereward

Geraldine A. Toole; G. Le Gall; Ian J. Colquhoun; C. Nemeth; Luc Saulnier; Alison Lovegrove; Till K. Pellny; Mark D. Wilkinson; Jackie Freeman; Rowan A. C. Mitchell; E. N. C. Mills; Peter R. Shewry

A combination of enzyme mapping, FT-IR microscopy and NMR spectroscopy was used to study temporal and spatial aspects of endosperm cell wall synthesis and deposition in developing grain of bread wheat cv. Hereward. This confirmed previous reports that changes in the proportions of the two major groups of cell wall polysaccharides occur, with β-glucan accumulating earlier in development than arabinoxylan. Changes in the structure of the arabinoxylan occurred, with decreased proportions of disubstituted xylose residues and increased proportions of monosubstituted xylose residues. These are likely to result, at least in part, from arabinoxylan restructuring catalysed by enzymes such as arabinoxylan arabinofurano hydrolase and lead to changes in cell wall mechanical properties which may be required to withstand stresses during grain maturation and desiccation.


Plant Physiology | 2013

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

Alison Lovegrove; Mark D. Wilkinson; Jackie Freeman; Till K. Pellny; Paola Tosi; Luc Saulnier; Peter R. Shewry; Rowan A. C. Mitchell

Suppression of either of two wheat genes decreases the amount of arabinoxylan, the major cell wall polymer in wheat flour, by 50%. 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.


New Phytologist | 2018

Suppression of a single BAHD gene in Setaria viridis causes large, stable decreases in cell wall feruloylation and increases biomass digestibility

Wagner Rodrigo de Souza; Polyana Kelly Martins; Jackie Freeman; Till K. Pellny; Louise V. Michaelson; Bruno L. Sampaio; Felipe Vinecky; Ana Paula Ribeiro; Bárbara Andrade Dias Brito da Cunha; Adilson Kenji Kobayashi; Patrícia Abrão de Oliveira; Raquel Bombarda Campanha; Thályta Fraga Pacheco; Danielly C. I. Martarello; Rogério Marchiosi; Osvaldo Ferrarese-Filho; Wanderley Dantas dos Santos; Robson Tramontina; Fabio M. Squina; Danilo da Cruz Centeno; Marília Gaspar; Marcia R. Braga; Marco Aurélio Silva Tiné; John Ralph; Rowan A. C. Mitchell

Summary Feruloylation of arabinoxylan (AX) in grass cell walls is a key determinant of recalcitrance to enzyme attack, making it a target for improvement of grass crops, and of interest in grass evolution. Definitive evidence on the genes responsible is lacking so we studied a candidate gene that we identified within the BAHD acyl‐CoA transferase family. We used RNA interference (RNAi) silencing of orthologs in the model grasses Setaria viridis (SvBAHD01) and Brachypodium distachyon (BdBAHD01) and determined effects on AX feruloylation. Silencing of SvBAHD01 in Setaria resulted in a c. 60% decrease in AX feruloylation in stems consistently across four generations. Silencing of BdBAHD01 in Brachypodium stems decreased feruloylation much less, possibly due to higher expression of functionally redundant genes. Setaria SvBAHD01 RNAi plants showed: no decrease in total lignin, approximately doubled arabinose acylated by p‐coumarate, changes in two‐dimensional NMR spectra of unfractionated cell walls consistent with biochemical estimates, no effect on total biomass production and an increase in biomass saccharification efficiency of 40–60%. We provide the first strong evidence for a key role of the BAHD01 gene in AX feruloylation and demonstrate that it is a promising target for improvement of grass crops for biofuel, biorefining and animal nutrition applications.


Archive | 2010

Chapter 2:Challenges and Opportunities for Using Wheat for Biofuel Production

Peter R. Shewry; Jackie Freeman; Mark D. Wilkinson; Till K. Pellny; Rowan Mitchell

Wheat is an attractive raw material for biofuel production, with over 600 million tonnes grain being harvested annually, and a potentially similar amount of straw available as a bi-product. Wheat whole grain has a starch content of about 70% dry weight, but about 10% consists of cell wall polysaccharides which are not currently saccharified for fermentation and limit the quality of the residual grain for livestock feed. The major cell wall polysaccharides in wheat grain are arabinoxylans, with smaller amounts of β-glucans. However, in outer grain tissues (bran) and straw the dominant polysaccharides are cellulose and glucuronoarabinoxylan, with large quantities of lignin. We discuss our current knowledge of arabinoxylan synthesis in wheat and how this can be exploited by manipulating the expression of key biosynthetic enzymes to change the structures and interactions of the cell wall xylans to improve the yield of biofuels from whole grain and straw.


Plant Biotechnology Journal | 2016

Effect of suppression of arabinoxylan synthetic genes in wheat endosperm on chain length of arabinoxylan and extract viscosity

Jackie Freeman; Alison Lovegrove; Mark D. Wilkinson; Luc Saulnier; Peter R. Shewry; Rowan A. C. Mitchell

Summary Arabinoxylan (AX) is the dominant component within wheat (Triticum aestivum L.) endosperm cell walls, accounting for 70% of the polysaccharide. The viscosity of aqueous extracts from wheat grain is a key trait influencing the processing for various end uses, and this is largely determined by the properties of endosperm AX. We have previously shown dramatic effects on endosperm AX in transgenic wheat by down‐regulating either TaGT43_2 or TaGT47_2 genes (orthologues to IRX9 and IRX10 in Arabidopsis, respectively) implicated in AX chain extension and the TaXAT1 gene responsible for monosubstitution by 3‐linked arabinose. Here, we use these transgenic lines to investigate the relationship between amounts of AX in soluble and insoluble fractions, the chain‐length distribution of these measured by intrinsic viscosity and the overall effect on extract viscosity. In transgenic lines expressing either the TaGT43_2 or TaGT47_2 RNAi transgenes, the intrinsic viscosities of water‐extractable (WE‐AX) and of a water‐insoluble alkaline‐extracted fraction (AE‐AX) were decreased by between 10% and 50% compared to control lines. In TaXAT1 RNAi lines, there was a 15% decrease in intrinsic viscosity of WE‐AX but no consistent effect on that of AE‐AX. All transgenic lines showed decreases in extract viscosity with larger effects in TaGT43_2 and TaGT47_2 RNAi lines (by up to sixfold) than in TaXAT1 RNAi lines (by twofold). These effects were explained by the decreases in amount and chain length of WE‐AX, with decreases in amount having the greater influence. Extract viscosity from wheat grain can therefore be greatly decreased by suppression of single gene targets.


Plant Biotechnology Journal | 2017

Feruloylation and structure of arabinoxylan in wheat endosperm cell walls from RNAi lines with suppression of genes responsible for backbone synthesis and decoration

Jackie Freeman; Jane L. Ward; Ondrej Kosik; Alison Lovegrove; Mark D. Wilkinson; Peter R. Shewry; Rowan A. C. Mitchell

Summary Arabinoxylan (AX) is the major component of the cell walls of wheat grain (70% in starchy endosperm), is an important determinant of end‐use qualities affecting food processing, use for animal feed and distilling and is a major source of dietary fibre in the human diet. AX is a heterogeneous polysaccharide composed of fractions which can be sequentially extracted by water (WE‐AX), then xylanase action (XE‐AX) leaving an unextractable (XU‐AX) fraction. We determined arabinosylation and feruloylation of AX in these fractions in both wild‐type wheat and RNAi lines with decreased AX content (TaGT43_2 RNAi, TaGT47_2 RNAi) or decreased arabinose 3‐linked to mono‐substituted xylose (TaXAT1 RNAi). We show that these fractions are characterized by the degree of feruloylation of AX, <5, 5–7 and 13–19 mg bound ferulate (g−1 AX), and their content of diferulates (diFA), <0.3, 1–1.7 and 4–5 mg (g−1 AX), for the WE, XE and XU fractions, respectively, in all RNAi lines and their control lines. The amount of AX and its degree of arabinosylation and feruloylation were less affected by RNAi transgenes in the XE‐AX fraction than in the WE‐AX fraction and largely unaffected in the XU‐AX fraction. As the majority of diFA is associated with the XU‐AX fraction, there was only a small effect (TaGT43_2 RNAi, TaGT47_2 RNAi) or no effect (TaXAT1 RNAi) on total diFA content. Our results are compatible with a model where, to maintain cell wall function, diFA is maintained at stable levels when other AX properties are altered.

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Mark D. Wilkinson

Technical University of Madrid

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Luc Saulnier

Institut national de la recherche agronomique

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