M. Mafa received funding from the NRF-Thuthuka (Reference Number: TTK2204102938). S.N. Zondo received the National Research Foundation Postgraduate Scholarship for his MSc degree. The authors are grateful to the Agricultural Research Council - Small Grain (ARC-SG) Institute for providing the seeds used in this study. Any opinion, findings, and recommendations expressed in this material are those of the author(s), and therefore, the funders do not accept any liability in regard thereto.

Characterization of Cell Wall-Related Enzymes in RWA-Infested Wheat Cultivars

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reactive+oxygen+species+are+involved+in+resistance+responses+of+wheat+to+the+Russian+wheat+aphid.">The reactive oxygen species are involved in resistance responses of wheat to the Russian wheat aphid.</a> J Plant Physiol. 163 (11), 1118-1125 (2005).</li><li>Manghwar, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+analysis+of+defense-related+genes+in+wheat+and+maize+against+Bipolaris+sorokiniana.">Expression analysis of defense-related genes in wheat and maize against Bipolaris sorokiniana.</a> Physiol Mol Plant Pathol. 103, 36-46 (2018).</li><li>Botha, C. E., Matsiliza, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+in+transport+in+wheat+(Triticum+aestivum)+is+caused+by+sustained+phloem+feeding+by+the+Russian+wheat+aphid+(Diuraphis+noxia).">Reduction in transport in wheat (Triticum aestivum) is caused by sustained phloem feeding by the Russian wheat aphid (Diuraphis noxia).</a> S Afr J Bot. 70 (2), 249-254 (2004).</li><li>Mafa, M. S., Rufetu, E., Alexander, O., Kemp, G., Mohase, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-wall+structural+carbohydrates+reinforcements+are+part+of+the+defense+mechanisms+of+wheat+against+Russian+wheat+aphid+(Diuraphis+noxia)+infestation.">Cell-wall structural carbohydrates reinforcements are part of the defense mechanisms of wheat against Russian wheat aphid (Diuraphis noxia) infestation.</a> Plant Physiol Biochem. 179, 168-178 (2022).</li><li>Walker, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sieve+element+occlusion:+Interaction+with+phloem+sap-feeding+insects+-+A+review.">Sieve element occlusion: Interaction with phloem sap-feeding insects - A review.</a> J Plant Physiol. 269, 153582 (2022).</li><li>Botha, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+developing+Russian+wheat+aphid+biotypes+remains+an+unsolved+enigma.">Fast developing Russian wheat aphid biotypes remains an unsolved enigma.</a> Curr Opin Insect Sci. 45, 1-11 (2020).</li><li>Saheed, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stronger+induction+of+callose+deposition+in+barley+by+Russian+wheat+aphid+than+bird+cherry-oat+aphid+is+not+associated+with+differences+in+callose+synthase+or+&#946;-1,3-glucanase+transcript+abundance.">Stronger induction of callose deposition in barley by Russian wheat aphid than bird cherry-oat aphid is not associated with differences in callose synthase or &#946;-1,3-glucanase transcript abundance.</a> Physiol Plant. 135 (2), 150-161 (2009).</li><li>Zondo, S. N. 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The authors declare no conflict of interest involved in this work.

Wheat and barley are cereal crops frequently infested by aphid species, including Russian wheat aphids (Diuraphis noxia)7,24. Resistant wheat plants induce the upregulation of POD and &#946;-1,3-glucanase activities as defense responses throughout the infestation period to modify the cell wall by regulating callose and lignin accumulation6,25,26,<sup ...

Russian wheat aphids (RWA) infest wheat and barley, causing significant yield loss or grain quality reduction. Wheat responds to infestation by inducing several defense responses, including increasing the &#946;-1,3-glucanase and peroxidase activity levels in the resistant cultivars, while susceptible cultivars reduce the activity of these enzymes at early infestation period1,2,3,4. The key functions of &#946;-1,3-glucanase and POD in the wheat plant included regulating callose accumulation in the resistant cultivar and reactive oxygen species (ROS) quenching at the cell wall and apoplastic regions during RWA infestation1,3,5,6,7. Mafa et al.6 demonstrated that there was a strong correlation between the increased POD activity and increased lignin content in the resistant wheat cultivar upon RWASA2 infestations. In addition, increased lignin content indicated that the cell wall of the infested resistant wheat cultivar was reinforced, leading to reduced RWA feeding.
Most researcher groups extracted and studied apoplastic &#946;-1,3-glucanase and POD during the wheat/barley-RWA interaction; in addition, most of these studies claimed that these enzymes influence the cell wall of the wheat plant infested with RWA without measuring the enzyme presence in the cell wall region. Only a few studies have used microscopic techniques to show that &#946;-1,3-glucanase activity levels were linked to callose regulation7,8,9 or extracted major cell wall components to demonstrate the correlation between POD activities and cell wall modification in the resistant6,10. The lack of probing the &#946;-1,3-glucanase and POD association to the cell wall indicates a need to develop methods that allow researchers to measure the cell wall-bound enzymes directly.
The current method proposes that removing the apoplastic fluid from the leaf tissue before extracting the cell wall-bound enzymes is necessary. The extraction procedure of apoplastic fluid must be performed twice from the leaf tissue, which is used for extracting the cell wall-bound enzymes. This process reduces contamination and confusion of the apoplastic enzymes with those found in the cell wall regions. Thus, in this study, we extracted cell wall-bound POD, &#946;-1,3-glucanase, and MLG-specific &#946;-glucanase and performed their biochemical characterization.

<table><tbody><tr><td>10 kDa Centrifuge concentrating membrane device</td><td>Sigma-Aldrich</td><td>R1NB84206</td><td>For research use only. Not for use in Diagnostic procedures. For concentration and purification of biological solutions.</td></tr><tr><td>2 g Laminaribiose</td><td>Megazyme (Wicklow, Ireland)</td><td>O-LAM2</td><td>High purity laminaribiose for use in research, biochemical enzyme assays and in vitro diagnostic analysis.</td></tr><tr><td>3 g Laminaritriose</td><td>Megazyme (Wicklow, Ireland)</td><td>O-LAM3</td><td>High purity laminaritriose for use in research, biochemical enzyme assays and in vitro diagnostic analysis.</td></tr><tr><td>3,5 Dinitro salicylic acid</td><td>Sigma-Aldrich</td><td>D0550</td><td>Used in colorimetric determination of reducing sugars</td></tr><tr><td>4 g Laminaritetraose&amp;nbsp;</td><td>Megazyme (Wicklow, Ireland)</td><td>O-LAM4</td><td>High purity laminaritetraose for use in research, biochemical enzyme assays and in vitro diagnostic analysis.</td></tr><tr><td>5 g Laminaripentaose</td><td>Megazyme (Wicklow, Ireland)</td><td>O-LAM5</td><td>High purity laminaripentaose for use in research, biochemical enzyme assays and in vitro diagnostic analysis.</td></tr><tr><td>95% Absolute ethanol</td><td>Sigma-Aldrich</td><td>107017</td><td>Ethanol absolute for analysis</td></tr><tr><td>acetic acid</td><td>Sigma-Aldrich</td><td>B00063</td><td>Acetc acid glacial 100% for analysis (contains acetic acid)</td></tr><tr><td>Azo-CM-Cellulose</td><td>Megazyme (Wicklow, Ireland)</td><td>S-ACMC</td><td>The polysaccharide is dyed with Remazolbrilliant Blue R to an extent of approx. one dye molecule per 20 sugar residues.</td></tr><tr><td>Beta glucan (barley)&amp;nbsp;</td><td>Megazyme (Wicklow, Ireland)</td><td>G6513</td><td>A powdered substrate, less soluble in water. Used in determining &amp;beta;-1,3-glucanase activity.</td></tr><tr><td>Bio-Rad Protein Assay Dye</td><td>Bio-Rad Laboratories, South africa</td><td>500-0006</td><td>Colorimetric assay dye, concentrate, for use with Bio-Rad Protein Assay Kits I and II&amp;nbsp;</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Gibco Europe</td><td>810-1018</td><td>For Laboratory use only</td></tr><tr><td>Citrate acid</td><td>Sigma-Aldrich</td><td>C0759</td><td>For Life Science research only. Not for use in diagnostic procedures.</td></tr><tr><td>CM-curdlan&amp;nbsp;</td><td>Megazyme (Wicklow, Ireland)</td><td>P-CMCUR</td><td>Powdered substrate for determining &amp;beta;-1,3-glucanase activity. Insoluble in water.</td></tr><tr><td>D-Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td>For Life Science research only. Not for use in diagnostic procedures.</td></tr><tr><td>Guaiacol</td><td>Sigma-Aldrich</td><td>G5502</td><td>Oxidation indicator. Used for determining peroxidase activity.</td></tr><tr><td>Hydrogen peroxide</td><td>BDH Laboratory Supplies, England</td><td>10366</td><td>Powerful oxidising agent.</td></tr><tr><td>Mikskaar Professional Substarte</td><td>Mikskaar (Estonia)</td><td>NI</td><td>Peat moss-based seedling substrate.</td></tr><tr><td>Multifeed fertiliser (5.2.4 (43))</td><td>Multifeed Classic</td><td>B1908248</td><td>A water soluble fertiliser for young developing plants and seedlings with a high phosphorus (P) requirement to ensure optimum root development.</td></tr><tr><td>Naphthol</td><td>Merck, Germany</td><td>N2780</td><td>Undergoes hydrogenations in the presence of a catalyst.</td></tr><tr><td>Phenol</td><td>Sigma-Aldrich</td><td>33517</td><td>Light sensitive. For R&amp;amp;D use only. Not for drug, household, or other uses. SDS available</td></tr><tr><td>Potassium sodium tartrate tetrahydrate (Rochelle salt)</td><td>Sigma-Aldrich</td><td>S2377</td><td>used in the preparation of 3,5-dinitrosalicylic acid solution used in the determination of the reducing sugar.</td></tr><tr><td>Silica plate (TLC Silica gel 60 F&lt;sub&gt;254&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>60778-25EA</td><td>Silica gel matrix, with fluorescent indicator 254 nm</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>S8045</td><td>For R&amp;amp;D use only. Not for drug, household, or other uses.</td></tr><tr><td>Sodium metabisulfite</td><td>Sigma-Aldrich</td><td>31448</td><td>Added as an antioxidant during the preparation of 3,5-dinitrosalicylic acid solutions.</td></tr><tr><td>Sodium phosphate dibasic heptahydrate</td><td>Sigma-Aldrich</td><td>S9390</td><td>Used as a buffer solution in biological research to keep the pH constant.</td></tr><tr><td>Sodium phosphate monobasic heptahydrate</td><td>Sigma-Aldrich</td><td>71500</td><td>An inorganic compound, which is soluble in water. Used as a reagent in the development of silicate-based grouts.</td></tr><tr><td>Statistical analysis software</td><td>TIBCO Statistica</td><td>version 13.1</td><td></td></tr><tr><td>Sulfuric acid</td><td>Merck, Darmstadt, Germany</td><td>30743</td><td>Sulfuric acid 95-97% for analysis of Hg, ACS reagent.</td></tr><tr><td>Tris-HCl</td><td>Sigma-Aldrich</td><td>10812846001</td><td>Buffering agent in incubation mixtures.&amp;nbsp;It has also been used as a component of lysis and TE (Tris-EDTA) buffer. For life science research only. Not for use in diagnostic procedures.</td></tr><tr><td>UV&amp;ndash;Visible Spectrophotometer</td><td>GENESYS 120&amp;nbsp;</td><td></td><td></td></tr><tr><td>&amp;nbsp;NI = not identified.</td><td></td><td></td><td></td></tr></tbody></table>

elucidating &#946;-1,3-glucanase and peroxidase physicochemical properties of wheat cell wall defense mechanism against diuraphis noxia infestation

Wheat plants infested by Russian wheat aphids (RWA) induce a cascade of defense responses, including the hypersensitive responses (HR) and induction of pathogenesis-related (PR) proteins, such as &#946;-1,3-glucanase and peroxidase (POD). This study aims to characterize the physicochemical properties of cell wall-associated POD and &#946;-1,3-glucanase and determine their synergism on the cell wall modification during RWASA2-wheat interaction. The susceptible Tugela, moderately resistant Tugela-Dn1, and resistant Tugela-Dn5 cultivars were pregerminated and planted under greenhouse conditions, fertilized 14 days after planting, and irrigated every 3 days. The plants were infested with 20 parthenogenetic individuals of the same RWASA2 clone at the 3-leaf stage, and leaves were harvested at 1 to 14 days post-infestation. The Intercellular wash fluid (IWF) was extracted using vacuum filtration and stored at -20 &#176;C. Leaf residues were crushed into powder and used for cell wall components. POD activity and characterization were determined using 5 mM guaiacol substrate and H2O2, monitoring change in absorbance at 470 nm. &#946;-1,3-glucanase activity, pH, and temperature optimum conditions were demonstrated by measuring the total reducing sugars in the hydrolysate with DNS reagent using &#946;-1,3-glucan and &#946;-1,3-1,4-glucan substrates, measuring the absorbance at 540 nm, and using glucose standard curve. The pH optimum was determined between pH 4 to 9, temperature optimum between 25 and 50 &#176;C, and thermal stability between 30 &#176;C and 70 &#176;C. &#946;-1,3-glucanase substrate specificity was determined at 25 &#176;C and 40 &#176;C using curdlan and barley &#946;-1,3-1,4-glucan substrates. Additionally, the &#946;-1,3-glucanase mode of action was determined using laminaribiose to laminaripentaose. The oligosaccharide hydrolysis product patterns were qualitatively analyzed with thin-layer chromatography (TLC) and quantitatively analyzed with HPLC. The method presented in this study demonstrates a robust approach for infesting wheat with RWA, extracting peroxidase and &#946;-1,3-glucanase from the cell wall region and their comprehensive biochemical characterization.

Four biological replicates of wheat cultivars (Tugela, Tugela-Dn1, and Tugela-Dn5) were infested with RWASA2 at the 3-leaf growth stage. After infestation, the leaves were harvested at 1-, 2-, 3-, 7-, and 14 dpi. The control treatments were not infested with RWASA2 to make the experiment results comparable to wheat plants not exposed to stress. The experiments were conducted in quadruplicates, and the results were presented as the mean values.
The protein concentrations of bo...

Author Spotlight: Integrating Biochemical Functions of &#946;-Glucanases and Peroxidase Enzymes in Wheat-RWA Interaction

The present protocol describes procedures used to study and characterize cell wall-related enzymes, mainly &#946;-1,3-glucanase and peroxidase, in wheat plants. Their activity levels increase during wheat-RWA interaction and are involved in the plant defense response through cell wall reinforcement, which deters aphid feeding.

Elucidating &#946;-1,3-Glucanase and Peroxidase Physicochemical Properties of Wheat Cell Wall Defense Mechanism Against Diuraphis noxia Infestation

Wheat plants infested by Russian wheat aphids (RWA) induce a cascade of defense responses, including the hypersensitive responses (HR) and induction of ...

elucidating-13-glucanase-peroxidase-physicochemical-properties-wheat

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JoVE Journal

Biochemistry

599 Views.  University of the Free State. Met dit onderzoek proberen we de biochemische functies van peroxidase en bèta 1, 3-glucanase op te helderen die worden geïnduceerd tijdens de interactie tussen tarwe en Russische tarwebladluis. Enzymen die ik noemde komen voort uit de recente interessante ontwikkelingen in ons onderzoek omdat ze de celwand wijzigen, de celwand van planten tijdens de interactie tussen Russische tarweluis. Daarom ontmoedigen ze het voeden in de resistente cultuur.En de...

Video: Auteur Spotlight: Integratie van biochemische functies van #946;-glucanasen en peroxidase-enzymen in tarwe-RWA-interactie

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts of purified material. Here, we describe a simple method for gaining detailed polysaccharide structural information, including resolution of structural isomers. For polysaccharide analysis by gel electrophoresis (PACE), plant cell wall material is hydrolyzed with glycosyl hydrolases specific to the polysaccharide of interest (e.g., mannanases for mannan). Large format polyacrylamide gels are then used to separate the released oligosaccharides, which have been fluorescently labeled. Gels can be visualized with a modified gel imaging system (see Table of Materials). The resulting oligosaccharide fingerprint can either be compared qualitatively or, with replication, quantitatively. Linkage and branching information can be established using additional glycosyl hydrolases (e.g., mannosidases and galactosidases). Whilst this protocol describes a method for analyzing glucomannan structure, it can be applied to any polysaccharide for which characterized glycosyl hydrolases exist. Alternatively, it can be used to characterize novel glycosyl hydrolases using defined polysaccharide substrates.

A protocol for the structural analysis of polysaccharides by gel electrophoresis (PACE), using mannan as an example, is described.

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Chemistry

Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR and sensitivity-enhanced DNP experiments. The sample processing procedure of plant biomass is also detailed. This method allows the measurement of a series of 1D and 2D 13C-13C/15N correlations spectra, which enables high-resolution structural elucidation of complex biomaterials in their native state, with minimal perturbation. The isotope-labeling can be examined by quantifying the intensity in 1D spectra and the polarization transfer efficiency in 2D correlation spectra. The success of dynamic nuclear polarization (DNP) sample preparation can be evaluated by the sensitivity enhancement factor. Further experiments examining the structural aspects of the polysaccharides and proteins will lead to a model of the three-dimensional architecture. These methods can be modified and adapted to investigate a wide range of carbohydrate-rich materials, including the natural cell walls of plants, fungi, algae and bacteria, as well as synthesized or designed carbohydrate polymers and their complex with other molecules.

A protocol for preparing 13C,15N-labeled fungal and plant samples for multidimensional solid-state NMR spectroscopy and dynamic nuclear polarization (DNP) investigations is presented.

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR ...

Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital structural and protective role in the development and life of higher plants. Although the intricate mechanisms regulating lignification processes in vivo strongly impact the industrial valorization of many plant-derived products, the scientific community still has a long way to go to decipher them. In a simple three-step workflow, the dual labeling protocol presented herein enables bioimaging studies of actively lignifying zones of plant tissues. The first step consists in the metabolic incorporation of two independent chemical reporters, surrogates of the two native monolignols that give rise to lignin H- and G-units. After incorporation into growing lignin polymers, each reporter is then specifically labeled with its own fluorescent probe via a sequential combination of bioorthogonal SPAAC/CuAAC click reactions. Combined with lignin autofluorescence, this approach leads to the generation of three-color localization maps of lignin within plant cell walls by confocal fluorescence microscopy and provides precise spatial information on the presence or absence of active lignification machinery at the scale of plant tissues, cells and different cell wall layers.

BLISS, a dual labeling protocol for studying lignification dynamics, was developed. Using synthetic monolignol reporters and a sequential combination of SPAAC and CuAAC bioorthogonal click reactions, this methodology paves the way to in-depth analysis of the factors that regulate the biogenesis of lignins in planta.

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital ...

Determination of the Absorption, Translocation, and Distribution of Imidacloprid in Wheat

Neonicotinoids, a class of insecticides, are widely used because of their novel modes of action, high insecticidal activity, and strong root uptake. Imidacloprid, the most widely used insecticide worldwide, is a representative first-generation neonicotinoid and is used in pest control for crops, vegetables, and fruit trees. With such a broad application of imidacloprid, its residue in crops has attracted increasing scrutiny. In the present study, 15 wheat seedlings were placed in a culture medium containing 0.5 mg/L or 5 mg/L imidacloprid for hydroculture. The content of imidacloprid in the wheat roots and leaves was determined after 1 day, 2 days, and 3 days of hydroculture to explore the migration and distribution of imidacloprid in wheat. The results showed that imidacloprid was detected both in the roots and leaves of the wheat plant, and the content of imidacloprid in the roots was higher than that in the leaves. Furthermore, the imidacloprid concentration in the wheat increased with increasing exposure time. After 3 days of exposure, the roots and leaves of the wheat in the 0.5 mg/L treatment group contained 4.55 mg/kg &plusmn; 1.45 mg/kg and 1.30 mg/kg &plusmn; 0.08 mg/kg imidacloprid, respectively, while the roots and leaves of the 5 mg/L treatment group contained 42.5 mg/kg &plusmn; 0.62 mg/kg and 8.71 mg/kg &plusmn; 0.14 mg/kg imidacloprid, respectively. The results from the present study allow for a better understanding of pesticide residues in crops and provide a data reference for the environmental risk assessment of pesticides.

Presented here is a protocol for the determination of the absorption, translocation, and distribution of imidacloprid in wheat under hydroponic conditions using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The results showed that imidacloprid can be absorbed by wheat, and imidacloprid was detected in both the wheat roots and leaves.

Neonicotinoids, a class of insecticides, are widely used because of their novel modes of action, high insecticidal activity, and strong root uptake. ...

Environment

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Biology

Glycan Profiling of Plant Cell Wall Polymers using Microarrays 

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins

Small cysteine-rich defensins are one of the largest groups of host defense peptides present in all plants. Many plant defensins exhibit potent in vitro antifungal activity against a broad-spectrum of fungal pathogens and therefore have the potential to be used as antifungal agents in transgenic crops. In order to harness the full potential of plant defensins for diseases control, it is crucial to elucidate their mechanisms of action (MOA). With the advent of advanced microscopy techniques, live-cell imaging has become a powerful tool for understanding the dynamics of the antifungal MOA of plant defensins. Here, a confocal microscopy based live-cell imaging method is described using two fluorescently labeled plant defensins (MtDef4 and MtDef5) in combination with vital fluorescent dyes. This technique enables real-time visualization and analysis of the dynamic events of MtDef4 and MtDef5 internalization into fungal cells. Importantly, this assay generates a wealth of information including internalization kinetics, mode of entry and subcellular localization of these peptides. Along with other cell biological tools, these methods have provided critical insights into the dynamics and complexity of the MOA of these peptides. These tools can also be used to compare the MOA of these peptides against different fungi.

Plant defensins play an important role in plant defense against pathogens. For effective use of these antifungal peptides as antifungal agents, understanding their modes of action (MOA) is critical. Here, a live-cell imaging method is described to study critical aspects of the MOA of these peptides.

Small cysteine-rich defensins are one of the largest groups of host defense peptides present in all plants. Many plant defensins exhibit potent in vitro ...

Double-Staining Method to Detect Pectin in Plant-Fungus Interaction

Plant cells use different structural mechanisms, either constitutive or inducible, to defend themselves from fungal infection. Encapsulation is an efficient inducible mechanism to isolate the fungal haustoria from the plant cell protoplast. Conversely, pectin, one of the polymeric components of the cell wall, is a target of several pectolytic enzymes in necrotrophic interactions. Here, a protocol to detect pectin and fungal hyphae through optical microscopy is presented. The pectin-rich encapsulation in the cells of coffee leaves infected by the rust fungus Hemileia vastatrix and the mesophyll cell wall modification induced by Cercospora coffeicola are investigated. Lesioned leaf samples were fixed with the Karnovsky solution, dehydrated, and embedded in glycol methacrylate for 2-4 days. All steps were followed by vacuum-pumping to remove air in the intercellular spaces and improve the embedding process. The embedded blocks were sectioned into 5-7 &#181;m thick sections, which were deposited on a glass slide covered with water and subsequently heated at 40 &#176;C for 30 min. Next, the slides were double-stained with 5% cotton blue in lactophenol to detect the fungus and 0.05% ruthenium red in water to detect pectin (acidic groups of polyuronic acids of pectin). Fungal haustoria of Hemileia vastatrix were found to be encapsulated by pectin. In coffee cercosporiosis, mesophyll cells exhibited dissolution of cell walls, and intercellular hyphae and conidiophores were observed. The method presented here is effective to detect a pectin-associated response in the plant-fungi interaction.

This protocol describes a microscopical method to detect pectin in coffee-fungus interaction.

Elucidating β-1,3-Glucanase and Peroxidase Physicochemical Properties of Wheat Cell Wall Defense Mechanism Against Diuraphis noxia Infestation

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