extraction of intercellular wash fluid from wheat apoplast for cell wall-related enzymes analysis

RWA 侵染小麦品种中细胞壁相关酶的表征

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.

作者聚焦：在小麦-RWA 相互作用中整合 #946;-葡聚糖酶和过氧化物酶的生化功能

阐明小麦细胞 壁防御机制 的 #946;-1,3-葡聚糖酶和过氧化物酶的物理化学特性

从小麦质外体中提取细胞间洗涤液用于细胞壁相关酶分析 

extraction-intercellular-wash-fluid-from-wheat-apoplast-for-cell-wall

Research

JoVE Journal

Biochemistry

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

55 次观看. 首先，用蒸馏水冲洗小麦叶片两次，以去除切割端的任何胞质污染。将叶片插入含有提取缓冲液的厚壁玻璃管中。使用水射流泵，真空渗透 5 分钟，用提取缓冲液浸渍叶子。从玻璃管中取出叶片，用纸巾吸干。将干燥的叶片垂直插入装有多孔盘的预冷离心管中。将叶子在 500 摄氏度下以 4 G 离心 10 分钟。使用 100 微升移液器，将上清液转移?...

视频: 从小麦质外体中提取细胞间洗涤液用于细胞壁相关酶分析 

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.

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 ...