Publications by authors named "Stephen C Fry"

102 Publications

Ancient origin of fucosylated xyloglucan in charophycean green algae.

Commun Biol 2021 06 17;4(1):754. Epub 2021 Jun 17.

Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark.

The charophycean green algae (CGA or basal streptophytes) are of particular evolutionary significance because their ancestors gave rise to land plants. One outstanding feature of these algae is that their cell walls exhibit remarkable similarities to those of land plants. Xyloglucan (XyG) is a major structural component of the cell walls of most land plants and was originally thought to be absent in CGA. This study presents evidence that XyG evolved in the CGA. This is based on a) the identification of orthologs of the genetic machinery to produce XyG, b) the identification of XyG in a range of CGA and, c) the structural elucidation of XyG, including uronic acid-containing XyG, in selected CGA. Most notably, XyG fucosylation, a feature considered as a late evolutionary elaboration of the basic XyG structure and orthologs to the corresponding biosynthetic enzymes are shown to be present in Mesotaenium caldariorum.
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http://dx.doi.org/10.1038/s42003-021-02277-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8211770PMC
June 2021

Fruit softening: evidence for pectate lyase action in vivo in date (Phoenix dactylifera) and rosaceous fruit cell walls.

Ann Bot 2021 Jun 10. Epub 2021 Jun 10.

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK.

Background And Aims: The programmed softening occurring during fruit development requires scission of cell-wall polysaccharides, especially pectin. Proposed mechanisms include the action of wall enzymes or hydroxyl radicals. Enzyme activities found in fruit extracts include pectate lyase (PL) and endo-polygalacturonase (EPG), which, in vitro, cleave de-esterified homogalacturonan in mid-chain by β-elimination and hydrolysis respectively. However, the important biological question of whether PL exhibits action in vivo had not been tested.

Methods: We developed a method for specifically and sensitively detecting in-vivo PL products, based on Driselase digestion of cell-wall polysaccharides and detection of the characteristic unsaturated product of PL action.

Key Results: In model in-vitro experiments, pectic homogalacturonan that had been partially cleaved by commercial PL was digested to completion with Driselase, releasing an unsaturated disaccharide ('ΔUA-GalA'), taken as diagnostic of PL action. ΔUA-GalA was separated from saturated oligogalacturonides (EPG products) by electrophoresis, then subjected to thin-layer chromatography (TLC), resolving ΔUA-GalA from higher homologues. The ΔUA-GalA was confirmed as 4-deoxy-β-l-threo-hex-4-enopyranuronosyl-(1➝4)-d-galacturonic acid by NMR spectroscopy. Driselase digestion of cell walls from ripe fruits of date (Phoenix dactylifera), pear (Pyrus communis), rowan (Sorbus aucuparia) and apple (Malus pumila) yielded ΔUA-GalA, demonstrating that PL had been acting in vivo in these fruits prior to harvest. Date-derived ΔUA-GalA was verified by negative-mode mass spectrometry, including CID fragmentation. The ΔUA-GalA : GalA ratio from ripe dates was roughly 1:20 (mol/mol), indicating that ~5% of the bonds in endogenous homogalacturonan had been cleaved by in-vivo PL action.

Conclusions: The results provide the first demonstration that PL, previously known from studies of fruit gene expression, proteomic studies and in-vitro enzyme activity, exhibits enzyme action in the walls of soft fruits and may thus be proposed to contribute to fruit softening.
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http://dx.doi.org/10.1093/aob/mcab072DOI Listing
June 2021

Cutin:xyloglucan transacylase (CXT) activity covalently links cutin to a plant cell-wall polysaccharide.

J Plant Physiol 2021 Jul 21;262:153446. Epub 2021 May 21.

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh, EH9 3BF, UK. Electronic address:

The shoot epidermal cell wall in land-plants is associated with a polyester, cutin, which controls water loss and possibly organ expansion. Covalent bonds between cutin and its neighbouring cell-wall polysaccharides have long been proposed. However, the lack of biochemical evidence makes cutin-polysaccharide linkages largely conjectural. Here we optimised a portfolio of radiochemical assays to look for cutin-polysaccharide ester bonds in the epidermis of pea epicotyls, ice-plant leaves and tomato fruits, based on the hypothesis that a transacylase remodels cutin in a similar fashion to cutin synthase and cutin:cutin transacylase activities. Through in-situ enzyme assays and chemical degradations coupled with chromatographic analysis of the H-labelled products, we observed that among several wall-related oligosaccharides tested, only a xyloglucan oligosaccharide ([H]XXXGol) could acquire ester-bonds from endogenous cutin, suggesting a cutin:xyloglucan transacylase (CXT). CXT activity was heat-labile, time-dependent, and maximal at near-neutral pH values. In-situ CXT activity peaked in nearly fully expanded tomato fruits and ice-plant leaves. CXT activity positively correlated with organ growth rate, suggesting that it contributes to epidermal integrity during rapid expansion. This study uncovers hitherto unappreciated re-structuring processes in the plant epidermis and provides a step towards the identification of CXT and its engineering for biotechnological applications.
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http://dx.doi.org/10.1016/j.jplph.2021.153446DOI Listing
July 2021

Cutin:cutin-acid endo-transacylase (CCT), a cuticle-remodelling enzyme activity in the plant epidermis.

Biochem J 2021 02;478(4):777-798

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh EH9 3BF, U.K.

Cutin is a polyester matrix mainly composed of hydroxy-fatty acids that occurs in the cuticles of shoots and root-caps. The cuticle, of which cutin is a major component, protects the plant from biotic and abiotic stresses, and cutin has been postulated to constrain organ expansion. We propose that, to allow cutin restructuring, ester bonds in this net-like polymer can be transiently cleaved and then re-formed (transacylation). Here, using pea epicotyl epidermis as the main model, we first detected a cutin:cutin-fatty acid endo-transacylase (CCT) activity. In-situ assays used endogenous cutin as the donor substrate for endogenous enzymes; the exogenous acceptor substrate was a radiolabelled monomeric cutin-acid, 16-hydroxy-[3H]hexadecanoic acid (HHA). High-molecular-weight cutin became ester-bonded to intact [3H]HHA molecules, which thereby became unextractable except by ester-hydrolysing alkalis. In-situ CCT activity correlated with growth rate in Hylotelephium leaves and tomato fruits, suggesting a role in loosening the outer epidermal wall during organ growth. The only well-defined cutin transacylase in the apoplast, CUS1 (a tomato cutin synthase), when produced in transgenic tobacco, lacked CCT activity. This finding provides a reference for future CCT protein identification, which can adopt our sensitive enzyme assay to screen other CUS1-related enzymes.
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http://dx.doi.org/10.1042/BCJ20200835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925011PMC
February 2021

Defining natural factors that stimulate and inhibit cellulose:xyloglucan hetero-transglucosylation.

Plant J 2021 03 21;105(6):1549-1565. Epub 2021 Jan 21.

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, The University of Edinburgh, Edinburgh, EH9 3BF, UK.

Certain transglucanases can covalently graft cellulose and mixed-linkage β-glucan (MLG) as donor substrates onto xyloglucan as acceptor substrate and thus exhibit cellulose:xyloglucan endotransglucosylase (CXE) and MLG:xyloglucan endotransglucosylase (MXE) activities in vivo and in vitro. However, missing information on factors that stimulate or inhibit these hetero-transglucosylation reactions limits our insight into their biological functions. To explore factors that influence hetero-transglucosylation, we studied Equisetum fluviatile hetero-trans-β-glucanase (EfHTG), which exhibits both CXE and MXE activity, exceeding its xyloglucan:xyloglucan homo-transglucosylation (XET) activity. Enzyme assays employed radiolabelled and fluorescently labelled oligomeric acceptor substrates, and were conducted in vitro and in cell walls (in situ). With whole denatured Equisetum cell walls as donor substrate, exogenous EfHTG (extracted from Equisetum or produced in Pichia) exhibited all three activities (CXE, MXE, XET) in competition with each other. Acting on pure cellulose as donor substrate, the CXE action of Pichia-produced EfHTG was up to approximately 300% increased by addition of methanol-boiled Equisetum extracts; there was no similar effect when the same enzyme acted on soluble donors (MLG or xyloglucan). The methanol-stable factor is proposed to be expansin-like, a suggestion supported by observations of pH dependence. Screening numerous low-molecular-weight compounds for hetero-transglucanase inhibition showed that cellobiose was highly effective, inhibiting the abundant endogenous CXE and MXE (but not XET) action in Equisetum internodes. Furthermore, cellobiose retarded Equisetum stem elongation, potentially owing to its effect on hetero-transglucosylation reactions. This work provides insight and tools to further study the role of cellulose hetero-transglucosylation in planta by identifying factors that govern this reaction.
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http://dx.doi.org/10.1111/tpj.15131DOI Listing
March 2021

Enzymically attaching oligosaccharide-linked 'cargoes' to cellulose and other commercial polysaccharides via stable covalent bonds.

Int J Biol Macromol 2020 Dec 10;164:4359-4369. Epub 2020 Sep 10.

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, The University of Edinburgh, Edinburgh EH9 3BF, United Kingdom.

The Equisetum enzyme hetero-trans-β-glucanase (HTG) covalently grafts native plant cellulose (donor-substrate) to xyloglucan (acceptor-substrate), potentially offering a novel 'green' method of cellulose functionalisation. However, the range of cellulosic and non-cellulosic donor substrates that can be utilised by HTG is unknown, limiting our insight into its biotechnological potential. Here we show that HTG binds all celluloses tested (papers, tissues, hydrogels, bacterial cellulose) to radioactively- or fluorescently-labelled xyloglucan-heptasaccharide (XXXGol; acceptor-substrate). Glycol-chitin, glycol-chitosan and chitosan also acted as donor substrates but less effectively than cellulose. Cellulose-XXXGol conjugates were formed throughout the volume of a block of hydrogel, demonstrating penetration. Plant-derived celluloses (cellulose Iβ) became more effective donor-substrates after 'mercerisation' in ≥3 M NaOH; the opposite was true for bacterial cellulose Iα. Cellulose-XXXGol bonds resisted boiling 6 M NaOH, demonstrating strong glycosidic bonding. In conclusion, HTG stably grafts native and processed celluloses to xyloglucan-oligosaccharides, which may carry valuable 'cargoes', exemplified by sulphorhodamine. We thus demonstrate HTG's biotechnological potential to modify various cellulose-based substrates such as textiles, pulps, papers, packaging, sanitary products and hydrogels.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.09.039DOI Listing
December 2020

Activity and Action of Cell-Wall Transglycanases.

Methods Mol Biol 2020 ;2149:165-192

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh, UK.

Transglycanases (endotransglycosylases) are enzymes that "cut and paste" polysaccharide chains. Several transglycanase activities have been discovered which can cut (i.e., use as donor substrate) each of the major hemicelluloses [xyloglucan, mannans, xylans, and mixed-linkage β-glucan (MLG)], and, as a recent addition, cellulose. These enzymes may play interesting roles in adjusting the wall's physical properties, influencing cell expansion, stem strengthening, and fruit softening.Activities discussed include the homotransglycanases XET (xyloglucan endotransglucosylase, i.e., xyloglucan-xyloglucan endotransglycosylase), trans-β-mannanase (mannan -mannan endotransglycosylase), and trans-β-xylanase (xylan -xylan endotransglucosylase), plus the heterotransglycanases MXE (MLG -xyloglucan endotransglucosylase) and CXE (cellulose -xyloglucan endotransglucosylase).Transglycanases acting on polysaccharide donor substrates can utilize small, labeled oligosaccharides as acceptor substrates, generating easily recognizable polymeric labeled products. We present methods for extracting transglycanases from plant tissues and assaying them in vitro, either quantitatively in solution assays or by high-throughput dot-blot screens. Both radioactively and fluorescently labeled substrates are mentioned. A general procedure (glass-fiber blotting) is illustrated by which proposed novel transglycanase activities can be tested for.In addition, we describe strategies for detecting transglycanase action in vivo. These methods enable the quantification of, separately, XET and MXE action in Equisetum stems. Related methods enable the tissue distribution of transglycanase action to be visualized cytologically.
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http://dx.doi.org/10.1007/978-1-0716-0621-6_10DOI Listing
March 2021

High-Voltage Paper Electrophoresis (HVPE).

Authors:
Stephen C Fry

Methods Mol Biol 2020 ;2149:1-31

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, The University of Edinburgh, Edinburgh, UK.

HVPE is an excellent and often overlooked method for obtaining objective and meaningful information about cell-wall "building blocks" and their metabolic precursors. It provides not only a means of analysis of known compounds but also an insight into the charge and/or mass of any unfamiliar compounds that may be encountered. It can be used preparatively or analytically. It can achieve either "class separations" (e.g., delivering all hexose monophosphates into a single pool) or the resolution of different compounds within a given class (e.g., ADP-Glc from UDP-Glc; or GlcA from GalA).All information from HVPE about charge and mass can be obtained on minute traces of analytes, especially those that have been radiolabeled, for example by in-vivo feeding of a H- or C-labeled precursor. HVPE does not usually damage the substance under investigation (unless staining is used), so samples of interest can be eluted intact from the paper ready for further analysis. Although HVPE is a technique that has been available for several decades, recently it has tended to be sidelined, possible because the apparatus is not widely available. Interested scientists are invited to contact the author about the possibility of accessing the Edinburgh apparatus.
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http://dx.doi.org/10.1007/978-1-0716-0621-6_1DOI Listing
March 2021

Three highly acidic Equisetum XTHs differ from hetero-trans-β-glucanase in donor substrate specificity and are predominantly xyloglucan homo-transglucosylases.

J Plant Physiol 2020 Aug 4;251:153210. Epub 2020 Jun 4.

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK. Electronic address:

Transglycanases are enzymes that remodel the primary cell wall in plants, potentially loosening and/or strengthening it. Xyloglucan endotransglucosylase (XET; EC 2.4.1.207), ubiquitous in land plants, is a homo-transglucanase activity (donor, xyloglucan; acceptor, xyloglucan) exhibited by XTH (xyloglucan endotransglucosylase/hydrolase) proteins. By contrast, hetero-trans-β-glucanase (HTG) is the only known enzyme that is preferentially a hetero-transglucanase. Its two main hetero-transglucanase activities are MLG : xyloglucan endotransglucosylase (MXE) and cellulose : xyloglucan endotransglucosylase (CXE). HTG is highly acidic and found only in the evolutionarily isolated genus of fern-allies, Equisetum. We now report genes for three new highly acidic HTG-related XTHs in E. fluviatile (EfXTH-A, EfXTH-H and EfXTH-I). We expressed them heterologously in Pichia and tested the encoded proteins' enzymic activities to determine whether their acidity and/or their Equisetum-specific sequences might confer high hetero-transglucanase activity. Untransformed Pichia was found to secrete MLG-degrading enzyme(s), which had to be removed for reliable MXE assays. All three acidic EfXTHs exhibited very predominantly XET activity, although low but measurable hetero-transglucanase activities (MXE and CXE) were also detected in EfXTH-H and EfXTH-I. We conclude that the extremely high hetero-transglucanase activities of Equisetum HTG are not emulated by similarly acidic Equisetum XTHs that share up to 55.5% sequence identity with HTG.
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http://dx.doi.org/10.1016/j.jplph.2020.153210DOI Listing
August 2020

Hetero-trans-β-Glucanase Produces Cellulose-Xyloglucan Covalent Bonds in the Cell Walls of Structural Plant Tissues and Is Stimulated by Expansin.

Mol Plant 2020 07 4;13(7):1047-1062. Epub 2020 May 4.

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, The University of Edinburgh, Edinburgh EH9 3BF, United Kingdom.

Current cell-wall models assume no covalent bonding between cellulose and hemicelluloses such as xyloglucan or mixed-linkage β-d-glucan (MLG). However, Equisetum hetero-trans-β-glucanase (HTG) grafts cellulose onto xyloglucan oligosaccharides (XGOs) - and, we now show, xyloglucan polysaccharide - in vitro, thus exhibiting CXE (cellulose:xyloglucan endotransglucosylase) activity. In addition, HTG also catalyzes MLG-to-XGO bonding (MXE activity). In this study, we explored the CXE action of HTG in native plant cell walls and tested whether expansin exposes cellulose to HTG by disrupting hydrogen bonds. To quantify and visualize CXE and MXE action, we assayed the sequential release of HTG products from cell walls pre-labeled with substrate mimics. We demonstrated covalent cellulose-xyloglucan bonding in plant cell walls and showed that CXE and MXE action was up to 15% and 60% of total transglucanase action, respectively, and peaked in aging, strengthening tissues: CXE in xylem and cells bordering intercellular canals and MXE in sclerenchyma. Recombinant bacterial expansin (EXLX1) strongly augmented CXE activity in vitro. CXE and MXE action in living Equisetum structural tissues potentially strengthens stems, while expansin might augment the HTG-catalyzed CXE reaction, thereby allowing efficient CXE action in muro. Our methods will enable surveys for comparable reactions throughout the plant kingdom. Furthermore, engineering similar hetero-polymer formation into angiosperm crop plants may improve certain agronomic traits such as lodging tolerance.
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http://dx.doi.org/10.1016/j.molp.2020.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7339142PMC
July 2020

Characterisation of the non-oxidative degradation pathway of dehydroascorbic acid in slightly acidic aqueous solution.

Arch Biochem Biophys 2020 03 26;681:108240. Epub 2019 Dec 26.

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, The King's Buildings, Max Born Crescent, Edinburgh, EH9 3BF, UK. Electronic address:

Although l-ascorbate (vitamin C) is an important biological antioxidant, its degradation pathways in vivo remain incompletely characterised. Ascorbate is oxidised to dehydroascorbic acid, which can be either hydrolysed to diketogulonate (DKG) or further oxidised. DKG can be further degraded, oxidatively or non-oxidatively. Here we characterise DKG products formed non-enzymically and non-oxidatively at 20 °C and at a slightly acidic pH typical of the plant apoplast. High-voltage electrophoresis revealed at least five products, including two novel CPLs (epimers of 2-carboxy-l-threo-pentonolactone), which slowly interconverted with CPA (2-carboxy-l-threo-pentonate). One of the two CPLs has an exceptionally low pK. The CPL structures were supported by MS [(CHO)] and by H and C NMR spectroscopy. Xylonate and its lactone also appeared. Experiments with [1-C]DKG showed that all five products (including the 5-carbon xylonate and its lactone) retained DKG's carbon-1; therefore, most xylonate arose by decarboxylation of CPLs or CPA, one of whose -COOH groups originates from C-2 or C-3 of DKG after a 'benzilic acid rearrangement'. Since CPLs appeared before CPA, a DKG lactone is probably the main species undergoing this rearrangement. CPA and CPL also form non-enzymically in vivo, where they may be useful to researchers as 'fingerprints', or to organisms as 'signals', indicating a non-oxidative, slightly acidic biological compartment.
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http://dx.doi.org/10.1016/j.abb.2019.108240DOI Listing
March 2020

Montbresides A-D: antibacterial p-coumaroyl esters of a new sucrose-based tetrasaccharide from Crocosmia × crocosmiiflora (montbretia) flowers.

Fitoterapia 2019 Nov 19;139:104377. Epub 2019 Oct 19.

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, UK. Electronic address:

Crocosmia × crocosmiiflora (montbretia) flowers yielded four esters (montbresides A-D) of a new sucrose-based tetrasaccharide, 3-O-β-d-glucopyranosyl-4´-O-α-d-rhamnopyranosyl-sucrose [β-d-Glc-(1 → 3)-α-d-Glc-(1↔2)-β-d-Fru-(4 ← 1)-α-d-Rha]. All four possess O-p-coumaroyl residues on C-3 of fructose and C-4 of α-glucose, plus O-acetyl residues on C-2 and C-3 of rhamnose and C-6 of fructose. Montbresides A and B are additionally O-acetylated on C-1 of fructose. The p-coumaroyls are trans- in montbresides A and C and cis- in B and D. Elemental compositions were determined from MS data, and structures from 1D and 2D NMR spectra. Monosaccharide residues were identified from selective 1D TOCSY spectra and TLC, and acylation sites from 2D HMBC spectra. Enantiomers were distinguished by enzymic digestion. Montbretia flower extracts were cytotoxic against six human cancerous cell-lines, but purified montbresides lacked cytotoxicity. Each montbreside displayed antibacterial activity against Staphylococcus aureus (minimal inhibitory concentration ~6 μg/ml). Montbretia is a potential source of new cytotoxins and antibacterial agents.
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http://dx.doi.org/10.1016/j.fitote.2019.104377DOI Listing
November 2019

MUR1-mediated cell-wall fucosylation is required for freezing tolerance in Arabidopsis thaliana.

New Phytol 2019 12;224(4):1518-1531

Department of Biosciences & Durham Centre for Crop Improvement Technology, Durham University, South Road, Durham, DH1 3LE, UK.

Forward genetic screens play a key role in the identification of genes contributing to plant stress tolerance. Using a screen for freezing sensitivity, we have identified a novel freezing tolerance gene, SENSITIVE-TO-FREEZING8, in Arabidopsis thaliana. We identified SFR8 using recombination-based mapping and whole-genome sequencing. As SFR8 was predicted to have an effect on cell wall composition, we used GC-MS and polyacrylamide gel electrophoresis to measure cell-wall fucose and boron (B)-dependent dimerization of the cell-wall pectic domain rhamnogalacturonan II (RGII) in planta. After treatments to promote borate-bridging of RGII, we assessed freeze-induced damage in wild-type and sfr8 plants by measuring electrolyte leakage from freeze-thawed leaf discs. We mapped the sfr8 mutation to MUR1, a gene encoding the fucose biosynthetic enzyme GDP-d-mannose-4,6-dehydratase. sfr8 cell walls exhibited low cell-wall fucose levels and reduced RGII bridging. Freezing sensitivity of sfr8 mutants was ameliorated by B supplementation, which can restore RGII dimerization. B transport mutants with reduced RGII dimerization were also freezing-sensitive. Our research identifies a role for the structure and composition of the plant primary cell wall in determining basal plant freezing tolerance and highlights the specific importance of fucosylation, most likely through its effect on the ability of RGII pectin to dimerize.
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http://dx.doi.org/10.1111/nph.16209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899859PMC
December 2019

Higher expression of the strawberry xyloglucan endotransglucosylase/hydrolase genes FvXTH9 and FvXTH6 accelerates fruit ripening.

Plant J 2019 12 8;100(6):1237-1253. Epub 2019 Oct 8.

Biotechnology of Natural Products, Technische Universität München, Liesel-Beckmann-Str. 1, 85354, Freising, Germany.

Fruit softening in Fragaria (strawberry) is proposed to be associated with the modification of cell wall components such as xyloglucan by the action of cell wall-modifying enzymes. This study focuses on the in vitro and in vivo characterization of two recombinant xyloglucan endotransglucosylase/hydrolases (XTHs) from Fragaria vesca, FvXTH9 and FvXTH6. Mining of the publicly available F. vesca genome sequence yielded 28 putative XTH genes. FvXTH9 showed the highest expression level of all FvXTHs in a fruit transcriptome data set and was selected with the closely related FvXTH6 for further analysis. To investigate their role in fruit ripening in more detail, the coding sequences of FvXTH9 and FvXTH6 were cloned into the vector pYES2 and expressed in Saccharomyces cerevisiae. FvXTH9 and FvXTH6 displayed xyloglucan endotransglucosylase (XET) activity towards various acceptor substrates using xyloglucan as the donor substrate. Interestingly, FvXTH9 showed activity of mixed-linkage glucan:xyloglucan endotransglucosylase (MXE) and cellulose:xyloglucan endotransglucosylase (CXE). The optimum pH of both FvXTH9 and FvXTH6 was 6.5. The prediction of subcellular localization suggested localization to the secretory pathway, which was confirmed by localization studies in Nicotiana tabacum. Overexpression showed that Fragaria × ananassa fruits infiltrated with FvXTH9 and FvXTH6 ripened faster and showed decreased firmness compared with the empty vector control pBI121. Thus FvXTH9 and also FvXTH6 might promote strawberry fruit ripening by the modification of cell wall components.
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http://dx.doi.org/10.1111/tpj.14512DOI Listing
December 2019

Functional and chemical characterization of XAF: a heat-stable plant polymer that activates xyloglucan endotransglucosylase/hydrolase (XTH).

Ann Bot 2019 08;124(1):131-148

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Max Born Crescent, Edinburgh, UK.

Background And Aims: Xyloglucan endotransglucosylase/hydrolase (XTH) proteins that possess xyloglucan endotransglucosylase (XET) activity contribute to cell-wall assembly and remodelling, orchestrating plant growth and development. Little is known about in-vivo XET regulation, other than at the XTH transcriptional level. Plants contain 'cold-water-extractable, heat-stable polymers' (CHPs) which are XTH-activating factors (XAFs) that desorb and thereby activate wall-bound XTHs. Because XAFs may control cell-wall modification in vivo, we have further explored their nature.

Methods: Material was cold-water-extracted from 25 plant species; proteins were precipitated by heat-denaturation, then CHP was ethanol-precipitated. For XAF assays, CHP (or sub-fractions thereof) was applied to washed Arabidopsis thaliana cell walls, and the enzymes thus solubilized were assayed radiochemically for XET activity. In some experiments, the CHP was pre-treated with trifluoroacetic acid (TFA), alkali (NaOH) or glycanases.

Key Results: CHP specifically desorbed wall-bound XTHs, but not β-glucosidases, phosphatases or peroxidases. CHP preparations from 25 angiosperms all possessed XAF activity but had no consistent monosaccharide composition. Of 11 individual plant polymers tested, only gum arabic and tamarind xyloglucan were XAF-active, albeit less so than CHP. On gel-permeation chromatography, XAF-active cauliflower CHP eluted with a molecular weight of ~7000-140 000, although no specific sugar residue(s) co-eluted exactly with XAF activity. Cauliflower XAF activity survived cold alkali and warm dilute TFA (which break ester and glycofuranosyl linkages, respectively), but was inactivated by hot 2 m TFA (which breaks glycopyranosyl linkages). Cauliflower XAF activity was remarkably stable to diverse glycanases and glycosidases.

Conclusions: XAFs are naturally occurring heat-stable polymers that specifically desorb (thereby activating) wall-bound XTHs. Their XAF activity considerably exceeds that of gum arabic and tamarind xyloglucan, and they were not identifiable as any major plant polysaccharide. We propose that XAF is a specific, minor, plant polymer that regulates xyloglucan transglycosylation in vivo, and thus wall assembly and restructuring.
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http://dx.doi.org/10.1093/aob/mcz050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6676392PMC
August 2019

The oxidation of dehydroascorbic acid and 2,3-diketogulonate by distinct reactive oxygen species.

Biochem J 2018 11 9;475(21):3451-3470. Epub 2018 Nov 9.

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, The King's Buildings, Edinburgh EH9 3BF, U.K.

l-Ascorbate, dehydro-l-ascorbic acid (DHA), and 2,3-diketo-l-gulonate (DKG) can all quench reactive oxygen species (ROS) in plants and animals. The vitamin C oxidation products thereby formed are investigated here. DHA and DKG were incubated aerobically at pH 4.7 with peroxide (HO), 'superoxide' (a ∼50 : 50 mixture of [Formula: see text] and [Formula: see text]), hydroxyl radicals (OH, formed in Fenton mixtures), and illuminated riboflavin (generating singlet oxygen, O). Products were monitored electrophoretically. quenched HO far more effectively than superoxide, but the main products in both cases were 4--oxalyl-l-threonate (4-OxT) and smaller amounts of 3-OxT and OxA + threonate. HO, but not superoxide, also yielded cyclic-OxT. Dilute Fenton mixture almost completely oxidised a 50-fold excess of DHA, indicating that it generated oxidant(s) greatly exceeding the theoretical OH yield; it yielded oxalate, threonate, and OxT. O had no effect on DHA. was oxidatively decarboxylated by HO, Fenton mixture, and O, forming a newly characterised product, 2-oxo-l--pentonate (OTP; '2-keto-l-xylonate'). Superoxide yielded negligible OTP. Prolonged HO treatment oxidatively decarboxylated OTP to threonate. Oxidation of DKG by HO, Fenton mixture, or O also gave traces of 4-OxT but no detectable 3-OxT or cyclic-OxT. In conclusion, DHA and DKG yield different oxidation products when attacked by different ROS. DHA is more readily oxidised by HO and superoxide; DKG more readily by O The diverse products are potential signals, enabling organisms to respond appropriately to diverse stresses. Also, the reaction-product 'fingerprints' are analytically useful, indicating which ROS are acting .
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http://dx.doi.org/10.1042/BCJ20180688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6225978PMC
November 2018

A Trihelix Family Transcription Factor Is Associated with Key Genes in Mixed-Linkage Glucan Accumulation.

Plant Physiol 2018 11 17;178(3):1207-1221. Epub 2018 Sep 17.

Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824

Mixed-linkage glucan (MLG) is a polysaccharide that is highly abundant in grass endosperm cell walls and present at lower amounts in other tissues. () and genes synthesize MLG, but it is unknown if other genes participate in the production and restructuring of MLG. Using transcriptional profiling data, we identified a trihelix family transcription factor () that is highly coexpressed with the gene (), which suggests that BdTHX1 is involved in the regulation of MLG biosynthesis. To determine the genes regulated by this transcription factor, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) experiments using immature seeds and an anti-BdTHX1 polyclonal antibody. The ChIP-seq experiment identified the second intron of as one of the most enriched sequences. The binding of BdTHX1 to the intron sequence was confirmed using electrophoretic mobility shift assays (EMSA). ChIP-seq also showed that a gene encoding a grass-specific glycoside hydrolase family 16 endotransglucosylase/hydrolase () is bound by BdTHX1, and the binding was confirmed by EMSA. Radiochemical transglucanase assays showed that BdXTH8 exhibits predominantly MLG:xyloglucan endotransglucosylase activity, a hetero-transglycosylation reaction, and can thus produce MLG-xyloglucan covalent bonds; it also has a lower xyloglucan:xyloglucan endotransglucosylase activity. shoots regenerated from transformed calli overexpressing showed an abnormal arrangement of vascular tissue and seedling-lethal phenotypes. These results indicate that the transcription factor BdTHX1 likely plays an important role in MLG biosynthesis and restructuring by regulating the expression of and .
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http://dx.doi.org/10.1104/pp.18.00978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6236600PMC
November 2018

Oxalyltransferase, a plant cell-wall acyltransferase activity, transfers oxalate groups from ascorbate metabolites to carbohydrates.

Plant J 2018 Jun 8. Epub 2018 Jun 8.

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh, EH9 3BF, UK.

In the plant apoplast, ascorbate is oxidised, via dehydroascorbic acid, to O-oxalyl esters [oxalyl-l-threonate (OxT) and cyclic oxalyl-l-threonate (cOxT)]. We tested whether OxT and cOxT can donate the oxalyl group in transacylation reactions to form oxalyl-polysaccharides, potentially modifying the cell wall. [oxalyl- C]OxT was incubated with living spinach (Spinacia oleracea) and Arabidopsis cell-suspension cultures in the presence or absence of proposed acceptor substrates (carbohydrates). In addition, [ C]OxT and [ C]cOxT were incubated in vitro with cell-wall enzyme preparations plus proposed acceptor substrates. Radioactive products were monitored electrophoretically. Oxalyltransferase activity was detected. Living cells incorporated oxalate groups from OxT into cell-wall polymers via ester bonds. When sugars were added, [ C]oxalyl-sugars were formed, in competition with OxT hydrolysis. Preferred acceptor substrates were carbohydrates possessing primary alcohols e.g. glucose. A model transacylation product, [ C]oxalyl-glucose, was relatively stable in vivo (half-life >24 h), whereas [ C]OxT underwent rapid turnover (half-life ~6 h). Ionically wall-bound enzymes catalysed similar transacylation reactions in vitro with OxT or cOxT as oxalyl donor substrates and any of a range of sugars or hemicelluloses as acceptor substrates. Glucosamine was O-oxalylated, not N-oxalylated. We conclude that plants possess apoplastic acyltransferase (oxalyltransferase) activity that transfers oxalyl groups from ascorbate catabolites to carbohydrates, forming relatively long-lived O-oxalyl-carbohydrates. The findings increase the range of known metabolites whose accumulation in vivo indicates vitamin C catabolism. Possible signalling roles of the resulting oxalyl-sugars can now be investigated, as can the potential ability of polysaccharide oxalylation to modify the wall's physical properties.
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http://dx.doi.org/10.1111/tpj.13984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6099474PMC
June 2018

Active proton efflux, nutrient retention and boron-bridging of pectin are related to greater tolerance of proton toxicity in the roots of two Erica species.

Plant Physiol Biochem 2018 May 2;126:142-151. Epub 2018 Mar 2.

Department of Plant Biotechnology, IRNAS-CSIC, Av Reina Mercedes 10, 41012 Seville, Spain. Electronic address:

Background And Aims: Tolerance to soil acidity was studied in two species of Ericaceae that grow in mine-contaminated soils (S Portugal, SW Spain) to find out if there are interspecific variations in H tolerance which might be related to their particular location.

Methods: Tolerance to H toxicity was tested in nutrient solutions using seeds collected in SW Spain. Plant growth and nutrient contents in leaves, stems and roots were determined. Viability tests and proton exchange were studied in roots exposed, short-term, to acidic conditions. Membrane ATPase activity and the cell-wall pectic polysaccharide domain rhamnogalacturonan-II (RG-II) were analysed to find out interspecific differences.

Results: Variation in survival, growth and mineral composition was found between species. The H-tolerant species (Erica andevalensis) showed greater concentration of nutrients than E. australis. Very low pH (pH 2) produced a significant loss of root nutrients (K, P, Mg) in the sensitive species. Root ATPase activity was slightly higher in the tolerant species with a correspondingly greater H efflux capacity. In both species, the great majority of the RG-II domains were in their boron-bridged dimeric form. However, shifting to a medium of pH 2 caused some of the boron bridges to break in the sensitive species.

Conclusions: Variation in elements linked to the cell wall-membrane complex and the stability of their components (RG-II, H-ATPases) are crucial for acid stress tolerance. Thus, by maintaining root cell structure, active proton efflux avoided toxic H build-up in the cytoplasm and supported greater nutrient acquisition in H-tolerant species.
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http://dx.doi.org/10.1016/j.plaphy.2018.02.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902606PMC
May 2018

Potassium, not lepidimoide, is the principal 'allelochemical' of cress-seed exudate that promotes amaranth hypocotyl elongation.

Authors:
Stephen C Fry

Ann Bot 2017 10;120(4):511-520

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK.

Background And Aims: Imbibed cress ( Lepidium sativum L.) seeds exude 'allelochemicals' that promote excessive hypocotyl elongation and inhibit root growth in neighbouring competitors, e.g. amaranth ( Amaranthus caudatus L.) seedlings. The major hypocotyl promoter has recently been shown not to be the previously suggested acidic disaccharide, lepidimoic acid (LMA), a fragment of the pectic polysaccharide domain rhamnogalacturonan-I. The nature of the hypocotyl promoter has now been re-assessed.

Methods: Low-molecular weight cress-seed exudate (LCSE) was fractionated by high-voltage electrophoresis, and components with different charge:mass ratios were tested for effects on dark-grown amaranth seedlings. Further samples of LCSE were size-fractionated by gel permeation chromatography, and active fractions were analysed electrophoretically.

Key Results: The LCSE strongly promoted amaranth hypocotyl elongation. The active principle was hydrophilic and, unlike LMA, stable to hot acid. After electrophoresis at pH 6·5, the only fractions that strongly promoted hypocotyl elongation were those with a very high positive charge:mass ratio, migrating towards the cathode 3-4 times faster than glucosamine. Among numerous naturally occurring cations tested, the only one with such a high mobility was potassium. K + was present in LCSE at approx. 4 m m , and pure KCl (1-10 m m ) strongly promoted amaranth hypocotyl elongation. No other cation tested (including Na + , spermidine and putrescine) had this effect. The peak of bioactivity from a gel permeation chromatography column exactly coincided with the peak of K + .

Conclusions: The major 'allelopathic' substance present in cress-seed exudate that stimulates hypocotyl elongation in neighbouring seedlings is the inorganic cation, K + , not the oligosaccharin LMA.
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http://dx.doi.org/10.1093/aob/mcx081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737857PMC
October 2017

Xyloglucan endotransglucosylase/hydrolases (XTHs) are inactivated by binding to glass and cellulosic surfaces, and released in active form by a heat-stable polymer from cauliflower florets.

J Plant Physiol 2017 Nov 5;218:135-143. Epub 2017 Aug 5.

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK. Electronic address:

Xyloglucan endotransglucosylase (XET) activity, which cuts and re-joins hemicellulose chains in the plant cell wall, contributing to wall assembly and growth regulation, is the major activity of XTH proteins. During purification, XTHs often lose XET activity which, however, is restored by treatment with certain cold-water-extractable, heat-stable polymers (CHPs), e.g. from cauliflower florets. It was not known whether the XTH-activating factor (XAF) present in CHPs works by promoting (e.g. allosterically) XET activity or by re-solubilising sequestered XTH proteins. We now show that XTHs in dilute solution bind to diverse surfaces (e.g. glass and cellulose), and that CHPs can re-solubilise the bound enzyme, re-activating it. Cell walls prepared from cauliflower florets, mung bean shoots and Arabidopsis cell-suspension cultures each contained endogenous, tightly bound, inactive XTHs, which were likewise rapidly solubilised (within 0.5h) and thus activated by cauliflower XAF. We present a convenient quantitative assay for XAF acting on the native sequestered XTHs of Arabidopsis cell walls; using this assay, we show that CHPs from all plants tested possess XAF activity. The XAF activity of diverse CHPs does not correlate with their conductivity, showing that this activity is not a simple ionic effect. The XAF action of cauliflower CHPs was augmented by NaCl, although NaCl alone was much less effective than a CHP solution of similar conductivity, confirming that the cauliflower polymers did not simply exert a salt effect. We suggest that XAF is an endogenous regulator of XET action, modulating cell-wall loosening and/or assembly in vivo.
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http://dx.doi.org/10.1016/j.jplph.2017.07.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5669584PMC
November 2017

Novel insights into ascorbate retention and degradation during the washing and post-harvest storage of spinach and other salad leaves.

Food Chem 2017 Oct 17;233:237-246. Epub 2017 Apr 17.

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK. Electronic address:

Post-harvest treatments of pre-packaged salad leaves potentially cause l-ascorbate loss, but the mechanisms of ascorbate degradation remain incompletely understood, especially in planta. We explored the extent and pathways of ascorbate loss in variously washed and stored salad leaves. Ascorbate was assayed by 2,6-dichlorophenolindophenol titration, and pathways were monitored by C-radiolabelling followed by high-voltage electrophoresis. All leaves tested showed ascorbate loss during storage: lettuce showed the greatest percentage loss, wild rocket the least. Spinach leaves were particularly prone to losing ascorbate during washing, especially with simultaneous mechanical agitation; however, washing in the presence of hypochlorite did not significantly increase ascorbate loss. In spinach, [C]oxalate was the major product of [C]ascorbate degradation, suggesting that commercial washing causes oxidative stress. This study highlights that ascorbate/dehydroascorbic acid are lost via the oxidative pathway during washing and post-harvest storage of salad leaves. Thus changes to washing procedures could potentially increase the post-harvest retention of ascorbate.
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http://dx.doi.org/10.1016/j.foodchem.2017.04.082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5441274PMC
October 2017

Phenolic metabolism and molecular mass distribution of polysaccharides in cellulose-deficient maize cells.

J Integr Plant Biol 2017 Jul 21;59(7):475-495. Epub 2017 Jun 21.

Área de Fisiología Vegetal. Dpto. Ingeniería y Ciencias Agrarias. Facultad de Biología y Ciencias Ambientales, Universidad de León, Leon E-24071, Spain.

As a consequence of the habituation to low levels of dichlobenil (DCB), cultured maize cells presented an altered hemicellulose cell fate with a lower proportion of strongly wall-bound hemicelluloses and an increase in soluble extracellular polymers released into the culture medium. The aim of this study was to investigate the relative molecular mass distributions of polysaccharides as well as phenolic metabolism in cells habituated to low levels of DCB (1.5 μM). Generally, cell wall bound hemicelluloses and sloughed polymers from habituated cells were more homogeneously sized and had a lower weight-average relative molecular mass. In addition, polysaccharides underwent massive cross-linking after being secreted into the cell wall, but this cross-linking was less pronounced in habituated cells than in non-habituated ones. However, when relativized, ferulic acid and p-coumaric acid contents were higher in this habituated cell line. Feasibly, cells habituated to low levels of DCB synthesized molecules with a lower weight-average relative molecular mass, although cross-linked, as a part of their strategy to compensate for the lack of cellulose.
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http://dx.doi.org/10.1111/jipb.12549DOI Listing
July 2017

Metabolites of 2,3-diketogulonate delay peroxidase action and induce non-enzymic HO generation: Potential roles in the plant cell wall.

Arch Biochem Biophys 2017 04 14;620:12-22. Epub 2017 Mar 14.

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh EH9 3BF, UK.

A proportion of the plant's l-ascorbate (vitamin C) occurs in the apoplast, where it and its metabolites may act as pro-oxidants and anti-oxidants. One ascorbate metabolite is 2,3-diketogulonate (DKG), preparations of which can non-enzymically generate HO and delay peroxidase action on aromatic substrates. As DKG itself generates several by-products, we characterised these and their ability to generate HO and delay peroxidase action. DKG preparations rapidly produced a by-product, compound (1), with λ 271 and 251 nm at neutral and acidic pH respectively. On HPLC, (1) co-eluted with the major HO-generating and peroxidase-delaying principle. Compound (1) was slowly destroyed by ascorbate oxidase, and was less stable at pH 6 than at pH 1. Electrophoresis of an HPLC-enriched preparation of (1) suggested a strongly acidic (pK ≈ 2.3) compound. Mass spectrometry suggested that un-ionised (1) has the formula CHO, i.e. it is a reduction product of DKG (CHO). In conclusion, compound (1) is the major HO-generating, peroxidase-delaying principle formed non-enzymically from DKG in the pathway ascorbate → dehydroascorbic acid → DKG → (1). We hypothesise that (1) generates apoplastic HO (and consequently hydroxyl radicals) and delays cell-wall crosslinking - both these effects favouring wall loosening, and possibly playing a role in pathogen defence.
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http://dx.doi.org/10.1016/j.abb.2017.03.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5398285PMC
April 2017

Bonds broken and formed during the mixed-linkage glucan : xyloglucan endotransglucosylase reaction catalysed by hetero-trans-β-glucanase.

Biochem J 2017 03 8;474(7):1055-1070. Epub 2017 Mar 8.

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, The King's Buildings, Edinburgh EH9 3BF, U.K.

Mixed-linkage glucan∶xyloglucan endotransglucosylase (MXE) is one of the three activities of the recently characterised hetero-trans-β-glucanase (HTG), which among land plants is known only from species. The biochemical details of the MXE reaction were incompletely understood - details that would promote understanding of MXE's role and enable its full technological exploitation. We investigated HTG's site of attack on one of its donor substrates, mixed-linkage (1→3),(1→4)-β-d-glucan (MLG), with radioactive oligosaccharides of xyloglucan as the acceptor substrate. Comparing three different MLG preparations, we showed that the enzyme favours those with a high content of cellotetraose blocks. The reaction products were analysed by enzymic digestion, thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC) and gel-permeation chromatography (GPC). HTG consistently cleaved the MLG at the third consecutive β-(1→4)-bond following (towards the reducing terminus) a β-(1→3)-bond. It then formed a β-(1→4)-bond between the MLG and the non-reducing terminal glucose residue of the xyloglucan oligosaccharide, consistent with its xyloglucan endotransglucosylase/hydrolase subfamily membership. Using size-homogeneous barley MLG as the donor substrate, we showed that HTG does not favour any particular region of the MLG chain relative to the polysaccharide's reducing and non-reducing termini; rather, it selects its target cellotetraosyl unit stochastically along the MLG molecule. This work improves our understanding of how enzymes can exhibit promiscuous substrate specificities and provides the foundations to explore strategies for engineering novel substrate specificities into transglycanases.
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http://dx.doi.org/10.1042/BCJ20160935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341106PMC
March 2017

Drought and Heat Differentially Affect XTH Expression and XET Activity and Action in 3-Day-Old Seedlings of Durum Wheat Cultivars with Different Stress Susceptibility.

Front Plant Sci 2016 10;7:1686. Epub 2016 Nov 10.

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento Lecce, Italy.

Heat and drought stress have emerged as major constraints for durum wheat production. In the Mediterranean area, their negative effect on crop productivity is expected to be exacerbated by the occurring climate change. Xyloglucan endotransglucosylase/hydrolases (XTHs) are chief enzymes in cell wall remodeling, whose relevance in cell expansion and morphogenesis suggests a central role in stress responses. In this work the potential role of XTHs in abiotic stress tolerance was investigated in durum wheat. The separate effects of dehydration and heat exposure on XTH expression and its endotransglucosylase (XET) activity and action have been monitored, up to 24 h, in the apical and sub-apical root regions and shoots excised from 3-day-old seedlings of durum wheat cultivars differing in stress susceptibility/tolerance. Dehydration and heat stress differentially influence the XTH expression profiles and the activity and action of XET in the wheat seedlings, depending on the degree of susceptibility/tolerance of the cultivars, the organ, the topological region of the root and, within the root, on the gradient of cell differentiation. The root apical region was the zone mainly affected by both treatments in all assayed cultivars, while no change in XET activity was observed at shoot level, irrespective of susceptibility/tolerance, confirming the pivotal role of the root in stress perception, signaling, and response. Conflicting effects were observed depending on stress type: dehydration evoked an overall increase, at least in the apical region of the root, of XET activity and action, while a significant inhibition was caused by heat treatment in most cultivars. The data suggest that differential changes in XET action in defined portions of the root of young durum wheat seedlings may have a role as a response to drought and heat stress, thus contributing to seedling survival and crop establishment. A thorough understanding of the mechanisms underlying these variations could represent the theoretical basis for implementing breeding strategies to develop new highly productive hybrids adapted to future climate scenarios.
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http://dx.doi.org/10.3389/fpls.2016.01686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5102909PMC
November 2016

Ascorbate degradation in tomato leads to accumulation of oxalate, threonate and oxalyl threonate.

Plant J 2017 Mar 9;89(5):996-1008. Epub 2017 Feb 9.

INRA, UR-1115, Plantes et Systèmes de culture Horticoles, CS40509, 84914 Avignon Cedex 9, France.

Ascorbate content in plants is controlled by its synthesis from carbohydrates, recycling of the oxidized forms and degradation. Of these pathways, ascorbate degradation is the least studied and represents a lack of knowledge that could impair improvement of ascorbate content in fruits and vegetables as degradation is non-reversible and leads to a depletion of the ascorbate pool. The present study revealed the nature of degradation products using [ C]ascorbate labelling in tomato, a model plant for fleshy fruits; oxalate and threonate are accumulated in leaves, as is oxalyl threonate. Carboxypentonates coming from diketogulonate degradation were detected in relatively insoluble (cell wall-rich) leaf material. No [ C]tartaric acid was found in tomato leaves. Ascorbate degradation was stimulated by darkness, and the degradation rate was evaluated at 63% of the ascorbate pool per day, a percentage that was constant and independent of the initial ascorbate or dehydroascorbic acid concentration over periods of 24 h or more. Furthermore, degradation could be partially affected by the ascorbate recycling pathway, as lines under-expressing monodehydroascorbate reductase showed a slight decrease in degradation product accumulation.
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http://dx.doi.org/10.1111/tpj.13439DOI Listing
March 2017

The pectic disaccharides lepidimoic acid and β-d-xylopyranosyl-(1→3)-d-galacturonic acid occur in cress-seed exudate but lack allelochemical activity.

Ann Bot 2016 Apr 8;117(4):607-23. Epub 2016 Mar 8.

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, The King's Buildings, Edinburgh EH9 3BF, UK and

Background And Aims: Cress-seed (Lepidium sativum) exudate exerts an allelochemical effect, promoting excessive hypocotyl elongation and inhibiting root growth in neighbouring Amaranthus caudatus seedlings. We investigated acidic disaccharides present in cress-seed exudate, testing the proposal that the allelochemical is an oligosaccharin-lepidimoic acid (LMA; 4-deoxy-β-l-threo-hex-4-enopyranuronosyl-(1→2)-l-rhamnose).

Methods: Cress-seed exudate was variously treated [heating, ethanolic precipitation, solvent partitioning, high-voltage paper electrophoresis and gel-permeation chromatography (GPC)], and the products were bioassayed for effects on dark-grown Amaranthus seedlings. Two acidic disaccharides, including LMA, were isolated and characterized by electrophoresis, thin-layer chromatography (TLC) and nuclear magnetic resonance (NMR) spectroscopy, and then bioassayed.

Key Results: Cress-seed exudate contained low-Mr, hydrophilic, heat-stable material that strongly promoted Amaranthus hypocotyl elongation and inhibited root growth, but that separated from LMA on electrophoresis and GPC. Cress-seed exudate contained ∼250 µmLMA, whose TLC and electrophoretic mobilities, susceptibility to mild acid hydrolysis and NMR spectra are reported. A second acidic disaccharide, present at ∼120 µm, was similarly characterized, and shown to be β-d-xylopyranosyl-(1→3)-d-galacturonic acid (Xyl→GalA), a repeat unit of xylogalacturonan. Purified LMA and Xyl→GalA when applied at 360 and 740 µm, respectively, only slightly promoted Amaranthus hypocotyl growth, but equally promoted root growth and thus had no effect on the hypocotyl:root ratio, unlike total cress-seed exudate.

Conclusions: LMA is present in cress seeds, probably formed by rhamnogalacturonan lyase action on rhamnogalacturonan-I during seed development. Our results contradict the hypothesis that LMA is a cress allelochemical that appreciably perturbs the growth of potentially competing seedlings. Since LMA and Xyl→GalA slightly promoted both hypocotyl and root elongation, their effect could be nutritional. We conclude that rhamnogalacturonan-I and xylogalacturonan (pectin domains) are not sources of oligosaccharins with allelochemical activity, and the biological roles (if any) of the disaccharides derived from them are unknown. The main allelochemical principle in cress-seed exudate remains to be identified.
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http://dx.doi.org/10.1093/aob/mcw008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4817500PMC
April 2016

Pectic polysaccharides are attacked by hydroxyl radicals in ripening fruit: evidence from a fluorescent fingerprinting method.

Ann Bot 2016 Mar 9;117(3):441-55. Epub 2016 Feb 9.

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK

Background And Aims: Many fruits soften during ripening, which is important commercially and in rendering the fruit attractive to seed-dispersing animals. Cell-wall polysaccharide hydrolases may contribute to softening, but sometimes appear to be absent. An alternative hypothesis is that hydroxyl radicals ((•)OH) non-enzymically cleave wall polysaccharides. We evaluated this hypothesis by using a new fluorescent labelling procedure to 'fingerprint' (•)OH-attacked polysaccharides.

Methods: We tagged fruit polysaccharides with 2-(isopropylamino)-acridone (pAMAC) groups to detect (a) any mid-chain glycosulose residues formed in vivo during (•)OH action and (b) the conventional reducing termini. The pAMAC-labelled pectins were digested with Driselase, and the products resolved by high-voltage electrophoresis and high-pressure liquid chromatography.

Key Results: Strawberry, pear, mango, banana, apple, avocado, Arbutus unedo, plum and nectarine pectins all yielded several pAMAC-labelled products. GalA-pAMAC (monomeric galacturonate, labelled with pAMAC at carbon-1) was produced in all species, usually increasing during fruit softening. The six true fruits also gave pAMAC·UA-GalA disaccharides (where pAMAC·UA is an unspecified uronate, labelled at a position other than carbon-1), with yields increasing during softening. Among false fruits, apple and strawberry gave little pAMAC·UA-GalA; pear produced it transiently.

Conclusions: GalA-pAMAC arises from pectic reducing termini, formed by any of three proposed chain-cleaving agents ((•)OH, endopolygalacturonase and pectate lyase), any of which could cause its ripening-related increase. In contrast, pAMAC·UA-GalA conjugates are diagnostic of mid-chain oxidation of pectins by (•)OH. The evidence shows that (•)OH radicals do indeed attack fruit cell wall polysaccharides non-enzymically during softening in vivo. This applies much more prominently to drupes and berries (true fruits) than to false fruits (swollen receptacles). (•)OH radical attack on polysaccharides is thus predominantly a feature of ovary-wall tissue.
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http://dx.doi.org/10.1093/aob/mcv192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4765547PMC
March 2016

Boron bridging of rhamnogalacturonan-II is promoted in vitro by cationic chaperones, including polyhistidine and wall glycoproteins.

New Phytol 2016 Jan 24;209(1):241-51. Epub 2015 Aug 24.

The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, The University of Edinburgh, The King's Buildings, Mayfield Road, Edinburgh, EH9 3JH, UK.

Dimerization of rhamnogalacturonan-II (RG-II) via boron cross-links contributes to the assembly and biophysical properties of the cell wall. Pure RG-II is efficiently dimerized by boric acid (B(OH)3 ) in vitro only if nonbiological agents for example Pb(2+) are added. By contrast, newly synthesized RG-II domains dimerize very rapidly in vivo. We investigated biological agents that might enable this. We tested for three such agents: novel enzymes, borate-transferring ligands and cationic 'chaperones' that facilitate the close approach of two polyanionic RG-II molecules. Dimerization was monitored electrophoretically. Parsley shoot cell-wall enzymes did not affect RG-II dimerization in vitro. Borate-binding ligands (apiose, dehydroascorbic acid, alditols) and small organic cations (including polyamines) also lacked consistent effects. Polylysine bound permanently to RG-II, precluding electrophoretic analysis. However, another polycation, polyhistidine, strongly promoted RG-II dimerization by B(OH)3 without irreversible polyhistidine-RG-II complexation. Likewise, partially purified spinach extensins (histidine/lysine-rich cationic glycoproteins), strongly promoted RG-II dimerization by B(OH)3 in vitro. Thus certain polycations, including polyhistidine and wall glycoproteins, can chaperone RG-II, manoeuvring this polyanionic polysaccharide domain such that boron-bridging is favoured. These chaperones dissociate from RG-II after facilitating its dimerization, indicating that they act catalytically rather than stoichiometrically. We propose a natural role for extensin-RG-II interaction in steering cell-wall assembly.
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http://dx.doi.org/10.1111/nph.13596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4973674PMC
January 2016
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