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

אפיון אנזימים הקשורים לדופן התא בזני חיטה שורצי RWA

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.

מחבר זרקור: שילוב פונקציות ביוכימיות של #946;-גלוקנאזות ואנזימי פרוקסידז באינטראקציה חיטה-RWA

הבהרת #946;-1,3-גלוקנאז ופרוקסידז תכונות פיסיקוכימיות של מנגנון ההגנה על דופן תא החיטה מפני התפשטות Diuraphis noxia

With this research, we try to elucidate the biochemical functions of peroxidase and beta 1, 3-glucanase that are induced during wheat, Russian wheat aphid interaction. Enzymes that I mentioned form the recent interesting developments in our research because they modify the cell wall, the plant cell wall during Russian wheat aphid interaction. Because of that, they deter feeding in the resistant culture.And this study is part of the pre-breeding program, so it shows that the biochemistry has a role to play in plant effects. So the challenges with production currently is the evolution of Russian wheat aphid biotypes. Currently we have Russian wheat aphid biotype one to biotype five.Biotype five is the most virulent because it has overcome all the resistance conferred by the Diuraphis noxia genes in commercial wheat cultivars. Therefore, it means we need to develop strategies that incorporate the biochemistry, the breeding, and the insecticides to protect wheat. We found this enzymes in peroxidase.It was significant for us to extract the IWF from the cell wall matrix and then we could see that beta 1, 3-glucanase and peroxidase are linked to the cell wall or they are bound to the cell wall. And their function on the cell is as follows. Peroxidase perform cross-linking of the lipin to the carbohydrates, while Beta 1, 3-glucanase regulate callose deposition making the plant more resistant.The method developed in our lab can be used to extract Beta 1, 3-glucanase as well as peroxidase from the cell wall as well as the apoplastic region. This can be used in the fields that are involved in plant growth and development, as well as plant pest interaction. Additionally, IWF can be studying further to find new types of defectants that make the plant susceptible.

מיצוי נוזל שטיפה בין-תאי מאפופלסט חיטה לניתוח אנזימים הקשורים לדופן התא 

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 

53 Views. כדי להתחיל, שטפו את חתיכות עלי החיטה פעמיים במים מזוקקים כדי להסיר כל זיהום ציטוסולי מהקצוות החתוכים. הכניסו את חתיכות העלה לצינור זכוכית עבה דופן המכיל חיץ חילוץ. באמצעות משאבת סילון מים, יש להפעיל חדירת ואקום למשך חמש דקות כדי לספוג עלים עם חיץ השאיבה....

Video: מיצוי נוזל שטיפה בין-תאי מאפופלסט חיטה לניתוח אנזימים הקשורים לדופן התא 

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 ...

To begin, rinse the wheat leaf pieces twice in distilled water to remove any cytosolic contamination from the cut ends. Insert the leaf pieces into a thick-walled glass tube containing an extraction buffer. Using a water jet pump, apply vacuum infiltration for five minutes to impregnate leaves with the extraction buffer.Remove the leaf pieces from the glass tube and blot dry them with a paper towel. Insert the dried leaf pieces vertically into a pre-cooled centrifuge tube fitted with a perforated disc. Centrifuge the leaves at 500G for 10 minutes at 4 degrees Celsius.Using a 100-microliter pipette, transfer the supernatant into a pre-cooled 1.5-milliliter micro-centrifuge tube. Store the collected supernatant at minus 20 degrees Celsius until use.