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. N., Mohase, L., Tolmay, V., Mafa, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+cell+wall-associated+&#946;-1,3-glucanase+and+peroxidase+induced+during+wheat-Diuraphis+noxia+interactions.">Functional characterization of cell wall-associated &#946;-1,3-glucanase and peroxidase induced during wheat-Diuraphis noxia interactions.</a> Research Square. , (2024).</li><li>Jimoh, M. A., Saheed, S. A., Botha, C. E. 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Clipper.</a> Master thesis in Molecular Cell Biology. , (2008).</li><li>Rahar, S., Swami, G., Nagpal, N., Nagpal, M. A., Singh, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+characterization,+and+biological+properties+of+&#946;-glucans.">Preparation, characterization, and biological properties of &#946;-glucans.</a> J Adv Pharm Technol Res. 2 (2), 94 (2011).</li><li>Mafa, M. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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)&#160;</td>
			<td>Megazyme (Wicklow, Ireland)</td>
			<td>G6513</td>
			<td>A powdered substrate, less soluble in water. Used in determining &#946;-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&#160;</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&#160;</td>
			<td>Megazyme (Wicklow, Ireland)</td>
			<td>P-CMCUR</td>
			<td>Powdered substrate for determining &#946;-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&#38;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 F254)</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&#38;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.&#160;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&#8211;Visible Spectrophotometer</td>
			<td>GENESYS 120&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;NI = not identified.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
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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.

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

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

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...

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.

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

Research

JoVE Journal

Biochemistry

Extraction of Intercellular Wash Fluid from Wheat Apoplast for Cell Wall-Related Enzymes Analysis

Extraction and Characterization of Cell Wall-Associated &#946;-1,3-Glucanase and Peroxidase in Wheat Cultivars

407 Views.  University of the Free State.  The present protocol describes procedures used to study and characterize cell wall-related enzymes, mainly β-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.

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

Summary