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

Siphephelo N.N. Zondo; Lintle Mohase; Vicki Tolmay; Mpho S. Mafa

doi:10.3791/66903

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Method Article

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

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DOI:

10.3791/66903

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July 26th, 2024

Siphephelo N.N. Zondo¹ , Lintle Mohase² , Vicki Tolmay³ , Mpho S. Mafa¹

¹Carbohydrates and Enzymology Laboratory (CHEM-LAB), Department of Plant Sciences, University of the Free State, ²Faculty of Natural and Agricultural Sciences, Department of Plant Science, Botany, Plant Biochemistry and Physiology, University of the Free State, ³Agricultural Research Council - Small Grain Institute

Summary

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.

Abstract

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 β-1,3-glucanase and peroxidase (POD). This study aims to characterize the physicochemical properties of cell wall-associated POD and β-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 °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. β-1,3-glucanase activity, pH, and temperature optimum conditions were demonstrated by measuring the total reducing sugars in the hydrolysate with DNS reagent using β-1,3-glucan and β-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 °C, and thermal stability between 30 °C and 70 °C. β-1,3-glucanase substrate specificity was determined at 25 °C and 40 °C using curdlan and barley β-1,3-1,4-glucan substrates. Additionally, the β-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 β-1,3-glucanase from the cell wall region and their comprehensive biochemical characterization.

Introduction

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 β-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 β-1,3-glucanase and POD in the wheat plant included regulating callose accumulation in the resistant cultivar and reactive oxygen speci....