Stephen C Fry

• BSc, PhD
• Professor at University of Edinburgh

Defects in cell wall integrity (CWI) profoundly affect plant growth, although, underlying mechanisms are not well understood. We show that in Arabidopsis mur1 mutant, CWI defects from compromising dimerization of RG-II pectin, a key component of cell wall, attenuate the expression of auxin response factors ARF7-ARF19 . As a result, polar auxin tran...

Background and aims The cell walls of charophytic algae both resemble and differ from those of land plants. Cell walls in early-diverging charophytes (e.g. Klebsormidiophyceae) are particularly distinctive, in ways that may enable survival in environments that are incompatible with land-plant polymers. This study therefore investigates the structur...

Cellulosic microfibrils in plant cell walls are largely ensheathed and probably tethered by hydrogen-bonded hemicelluloses. Ensheathing may vary developmentally as hemicelluloses are peeled to enable cell expansion. We characterised a simple method to quantify ensheathed versus naked cellulosic surfaces based on the ability to adsorb a radiolabelle...

A role for l-ascorbate as the precursor of several plant compounds adds to its already broad metabolic utility. There are many examples of plant species in which oxalate and l-threonate are formed from l-ascorbate breakdown, and a number of roles have been proposed for this: structural, physiological, and biochemical. On the other hand, the synthes...

Background and aims The softening of ripening fruit involves partial depolymerisation of cell-wall pectin by three types of reaction: enzymic hydrolysis, enzymic elimination (lyase-catalysed) and non-enzymic oxidative scission. Two known lyase activities are pectate lyase and rhamnogalacturonan lyase (RGL), potentially causing mid-chain cleavage of...

Land‐plant transglycosylases ‘cut‐and‐paste’ cell‐wall polysaccharides by endo‐transglycosylation (transglycanases) and exo‐transglycosylation (transglycosidases). Such enzymes may remodel the wall, adjusting extensibility and adhesion. Charophytes have cell‐wall polysaccharides that broadly resemble, but appreciably differ from land‐plants'. We in...

Background and aims Cress seeds release allelochemicals that overstimulate the elongation of neighbouring (potentially competing) seedlings’ hypocotyls and inhibit their root growth. The hypocotyl promoter is potassium, but the root inhibitor was unidentified; its nature is investigated here. Methods Low-molecular-weight cress-seed exudate (LCSE)...

Most pectic rhamnogalacturonan-II (RG-II) domains in plant cell walls are borate-bridged dimers. However, the sub-cellular locations, pH dependence, reversibility and biocatalyst involvement in borate bridging remain uncertain. Experiments discussed here explored these questions, utilising suspension-cultured plant cells. In-vivo pulse radiolabelli...

All land‐plant cell walls possess hemicelluloses, cellulose and anionic pectin. The walls of their cousins, the charophytic algae, exhibit some similarities to land plants’ but also major differences. Charophyte ‘pectins’ are extractable by conventional land‐plant methods, although they differ significantly in composition. Here, we explore ‘pectins...

We established model systems for exploring the roles of symplastic and apoplastic ascorbate in heavy-metal-polluted dicot and monocot cells. Cell-suspension cultures of Arabidopsis and maize were treated with copper, cadmium or nickel; growth and ascorbate metabolism were measured. Growth was halved by ∼80 µM Cu²⁺, 90 µM Cd²⁺ or 1200 µM Ni²⁺ in Ara...

Cross‐linking of the cell‐wall pectin domain rhamnogalacturonan‐II (RG‐II) via boron‐bridges between apiose residues is essential for normal plant growth and development, but little is known about its mechanism or reversibility. We characterised the making and breaking of boron‐bridges in vivo and in vitro at ‘apoplastic’ pH. RG‐II (13‐26μM) was in...

Background and aims: Rhamnogalacturonan-II (RG-II) is a domain of primary cell wall pectin. Pairs of RG-II domains are covalently cross-linked via borate diester bridges, necessary for normal cell growth. Interpreting the precise mechanism and roles of boron bridging is difficult because there are conflicting hypotheses as to whether bridging occu...

Rhamnogalacturonan-II (RG-II) is a complex pectic domain in plant primary cell walls. In vivo, most RG-II domains are covalently dimerised via borate diester bridges, essential for correct cell-wall assembly, but the dimerisation of pure RG-II monomers by boric acid in vitro is extremely slow. Cationic ‘chaperones’ can promote dimerisation, probabl...

We report that cell wall polymers in the chlorophyte algae may be modified by O-acetylation. The importance of cell wall modifications in the green algae is not well understood, although similar modifications play key roles in land plants by modulating the properties of cell wall carbohydrate polymers. Using a combination of biophysical (Fourier-tr...

Transglycanases remodel cell‐wall polymers, having critical impact on many physiological processes. Unlike xyloglucan endotransglucosylase (XET) activity, widely studied in land‐plants, very little is known about charophyte wall‐modifying enzymes — information that would promote our understanding of the ‘primordial’ wall, revealing how the wall mat...

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 la...

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, clea...

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 op...

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...

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 in...

The Equisetum enzyme hetero-trans-β-glucanase (HTG) covalently grafts native plant cellulose (donor-substrate) to xyloglucan (acceptor-substrates), 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 insi...

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...

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 preparat...

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...

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...

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...

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...

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 pre...

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...

Fruit softening in 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 in vitro and in vivo characterization of two recombinant xyloglucan endotransglucosylase/hydrolases (XTHs) from Fragaria vesca, FvXTH9 and FvXTH6. Mining of t...

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...

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 (H2O2), ‘superoxide’ (a ∼50 : 50 mixture of O2⋅− and HO2⋅), hydroxyl rad...

Mixed-linkage glucan (MLG) is a polysaccharide that is highly abundant in grass endosperm cell walls and present at lower amounts in other tissues. Cellulose synthase-like F (CSLF) and cellulose synthase-like H (CSLH) genes synthesize MLG, but it is unknown if other genes participate in the production and restructuring of MLG. Using Brachypodium di...

Figure S1. [14C]Dehydroascorbic acid labels the polymeric fraction (AIR) of live Arabidopsis, rose and spinach cell cultures. Figure S2. Effect of boiling or freeze–thawing on the ability of live spinach cells to transfer oxalate residues from oxalyl‐threonate into wall polymers. Figure S3. Time‐course for transfer of oxalate residues from oxalyl...

The expression of two genes encoding xyloglucan endotransglucosylase/hydrolases (XTHs), Cs-XTH1 and Cs-XTH3, was upregulated during the onset of cucumber somatic embryogenesis. As a means of characterising the developmental regulation of these genes, the activity of the respective upstream regulatory regions was investigated in seedlings and somati...

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‐¹⁴C]OxT was incubated with liv...

Genetics now potentially lets us modify the production, crosslinking and degradation of cell wall polysaccharides. There remains, however, the need to test experimentally whether intended modifications of polysaccharide metabolism have successfully been effected in vivo . Simple methods for this are described, including in‐vivo radiolabelling, enzy...

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 coll...

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 p...

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, lepi...

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 ma...

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,...

The cell wall is an important factor in the use of plant biomass to generate liquid biofuels. It needs to be broken down in order for its components to be available for fermentation and other biofuel-generating pathways. This chapter provides a comprehensive account of the cell wall in living plants, covering biological functions, chemical composit...

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 which could impair improvement of ascorbate content in fruits and vegetables as degradation is non-reversible and leads to a...

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 H2O2 and delay peroxidase action on aromatic substrates. As DKG itself generates several by-produ...

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 Equisetum species. The biochemical details of the MXE reaction were incompletely understood - details that would promote understanding of MXE's role in v...

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...

The plant cell is surrounded by a cell wall, mainly composed of polysaccharides (cellulose, pectins, and hemicelluloses) and also containing glycoproteins and sometimes phenolic compounds, polyesters (cutin, suberin), or silica. The various polymers are assembled into a coherent wall by covalent and noncovalent cross-linking. Wall polysaccharides a...

Ripening in 'climacteric' fruits (most commercial species) is induced by a burst of endogenous ethylene. What triggers this burst remains uncertain. The 'climacteric' is a sudden increase in ethylene and CO2 output. Ripening makes fruits attractive to animals. Chlorophylls are degraded and nongreen pigments (carotenoids and/or anthocyanins) are syn...

Plant tissues possess discrete cells, a construction conferring strength, tolerance of localized injury, division of labor, and (via intercellular air spaces) gas exchange. The cell is composed of a protoplast (nucleus plus cytoplasm) and a cell wall. Three organelle types contain genes - the nucleus, plastids, and mitochondria, each bounded by a d...

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-lepidimo...

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 polysa...

Bioassay-guided isolation and purification of phytochemicals with anti-parasitic properties from Cissus ruspolii - Volume 75 Issue OCE2 - K. Tolossa, S.C. Fry, S. Athanasiadou, G.J. Loake, J.G.M. Houdijk

Fig. S1 Some poly(amino acids) affect RG‐II dimerization.

Small molecules (xenobiotics) that inhibit cell-wall-localised enzymes are valuable for elucidating the enzymes' biological roles. We applied a high-throughput fluorescent dot-blot screen to search for inhibitors of Petroselinum xyloglucan endotransglucosylase (XET) activity in vitro. Of 4216 xenobiotics tested, with cellulose-bound xyloglucan as d...

Vitamin C (ascorbate and dehydroascorbic acid) is vital for plants and found throughout the plant cell including in the apoplast. The structure of ascorbate was determined eighty years ago; however, many of its degradation pathways remain unclear. Numerous degradation products of ascorbate have been reported to occur in the apoplast but many still...

Figure S1. Fractionation of native Equisetum HTG.

Table S3. Modelled interactions between xyloglucan and the active site in Populus XTH (PttXET) (Johansson et al., 2004), Tropaeolum XTH (TmNXG1) (Mark et al., 2009) and Equisetum HTG (present work).

Figure S6. Effect of BSA on the activity of Pichia‐produced HTG with soluble and insoluble donor substrates.

Table S1. Radiochemical characterization of cellulose–[3H]XXXGol generated by HTG extracted from E. fluviatile stems.

Table S2. A four‐step strategy for purification of native Equisetum HTG.

Methods S1. Mass spectrometry, gene amplification, cloning, and heterologous protein production.

Figure S3. Deduced sequence of E. fluviatile HTG, compared with several other family GH16b proteins.

Figure S4. Rooted cladogram showing the relationship of HTG to other GH16b sub‐family members.

Figure S2. Mass spectra of tryptic peptides of E. fluviatile HTG.

Figure S5. Inability of Pichia‐produced HTG to utilize cellohexaose as donor substrate.

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 in...

Cell walls are metabolically active components of plant cells. They contain diverse enzymes, including transglycanases (endotransglycosylases) — enzymes that ‘cut and paste’ certain structural polysaccharide molecules and thus potentially remodel the wall during growth and development. Known transglycanase activities can modify several cell-wall po...

During evolution, plants have acquired and/or lost diverse sugar residues as cell-wall constituents. Of particular interest are primordial cell-wall features that existed, and in some cases abruptly changed, during the momentous step whereby land-plants arose from charophytic algal ancestors. Polysaccharides were extracted from four charophyte orde...

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is a highly specific and sensitive technique for measuring metabolites. However, co-eluting components in tissue extracts can mutually interfere with ionization at the interface of the LC and MS/MS phases, potentially causing under- or over-estimation of metabolite concentrations....

Figure S1. View of the UV irradiation equipment showing Q‐Panel UV‐313 fluorescent lamps, four chambers with quartz windows and water cooling, gas syringes for sampling, UV and PAR sensors. Table S1. Net UV‐induced gaseous emissions of CH4, CO, CO2, C2H4, C2H6 and C3H8 from plant leaves, expressed per unit leaf dry weight and per unit leaf area, w...

Transglycanases (endotransglycosylases) cleave a polysaccharide (donor-substrate) in mid-chain, then transfer a portion onto another poly- or oligosaccharide (acceptor-substrate). Such enzymes contribute to plant cell-wall assembly and/or re-structuring. We sought a general method for revealing novel homo- and hetero-transglycanases, applicable to...

Transglycanases (endotransglycosylases) cleave a polysaccharide (donor-substrate) in mid-chain, then transfer a portion onto another poly- or oligosaccharide (acceptor-substrate). Such enzymes contribute to plant cell-wall assembly and/or re-structuring. We sought a general method for revealing novel homo- and hetero-transglycanases, applicable to...

Cell-suspension cultures habituated to 2,6-dichlorobenzonitrile (DCB) survive with reduced cellulose owing to hemicellulose network modification. We aimed to define the hemicellulose metabolism modifications in DCB-habituated maize cells showing a mild reduction in cellulose at different stages in the culture cycle. Using pulse-chase radiolabelling...

The original report that plants emit methane (CH4 ) under aerobic conditions caused much debate and controversy. Critics questioned experimental techniques, possible mechanisms for CH4 production and the nature of estimating global emissions. Several studies have now confirmed that aerobic CH4 emissions can be detected from plant foliage but the ex...

Hydroxyl radicals (•OH) cause non-enzymic scission of polysaccharides in diverse biological systems. Such reactions can be detrimental (e.g. causing rheumatic and arthritic diseases in mammals) or beneficial (e.g. promoting the softening of ripening fruit, and biomass saccharification). Here we present a method for documenting •OH action, based on...