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Plant roots secret a lot of peroxidases to counteract environmental influences. This protocol describes a way to extract root apoplastic wall fluid from Arabidopsis plants and to determine the peroxidase activity using guaiacol as substrate.

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[Bio101] Extraction of Root Apoplastic Wall Fluid for Apoplastic Peroxidase Activity Assay
[Bio101] 根壁外非原质体液的提取及非原质体过氧化物酶活性分析

植物科学 > 植物细胞生物学 > 细胞结构
作者: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
9/5/2011, 7751 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.127

[Abstract] Plant roots secret a lot of peroxidases to counteract environmental influences. This protocol describes a way to extract root apoplastic wall fluid from Arabidopsis plants and to determine the peroxidase activity using guaiacol as substrate.
Keywords: Peroxidase(过氧化物酶), Root(根), Guaiacol(愈创木酚), Apoplastic fluid(质外体液流), Enzyme activity(酶活)

[Abstract] 植物的根中有很多过氧化物酶可以应对外界环境的影响。本实验提供了一种从拟南芥植物根中提取壁外非原质体液并使用邻甲氧基苯酚作为底物检测其过氧化物酶活性的方法。

Materials and Reagents

  1. 10 mM Na+-PO4 (pH 6.0)
  2. Guaiacol
  3. Agar
  4. MES-K
  5. 1/4x modified Hoagland medium (see general growth protocol for recipes)
  6. Solid medium (see Recipes)

Equipment

  1. UV-vis Spectrometer
  2. Arabidopsis growth facility
  3. 15 ml Falcon tube

Procedure

  1. Plant growth
    Surface-sterilized Arabidopsis seeds are planted on ? x modified Hoagland medium (1 % agar, 5 mM MES-K, pH 6.0) solidified on 1 ml pipette tip box insert (the part with holes). After germination for 2-3 days under light, the boxes with young seedlings are floated in ? modified Hoagland liquid medium with 5 mM MES-K at pH 6.0, aerated by fish tank water pumps.
  2. Extraction of apoplastic wall fluid (AWF)
    1. Roots of Col seedlings (2.5 weeks old) are rinsed in de-ionized water and cut into 10 mM Na+-PO4 (pH 6.0).
    2. Vacuum for 5 min on ice, followed by slow release for infiltration.
    3. The roots are then dabbed onto filter paper to dry, measured on a balance and recorded as fresh weight.
    4. The roots are then put to the plunge barrel of a 5 ml syringe. The syringe barrel is placed in a 15 ml Falcon tube and centrifuged at 3,000 x g for 15 min at 4 °C. 
    5. The AWF is rescued from the tube bottom. Total volume is measured by pipetting. AWF is kept on ice.
  3. Total protein quantification
    Protein concentration measurement (Bradford micro assay) is done according to Bio-Rad reagent instruction.
    To set up a reaction, mix the following:
    Chemical Volume FW Stock conc.
    Guaiacol 880 μl 124.14; D=1.112 g/ml, 20 mM in 10 mM Na-PO4 pH 6.0
    AWF x μl
    Add 20-x μl 10 mM Na-PO4 (pH 6.0).
    Start the reaction by adding:
    H2O2 100 μl 34.01; 31.4% 0.3% (v/v)
    Total volume is adjusted to 1 ml.
    Reactions are followed spectrophotometrically at 470 nm for 2 min at room temperature. Extinction coefficient = 26.6/Mm/cm

    Figure 1.

方法与试剂

 

1.       1/4 改良的Hoagland培养基 (配方与普通生长配方一致)

配制固体培养基,加入1%的琼脂粉,使用5mM MES-K缓冲液将pH调至6.0 

2.       10 mM Na+-PO4 pH 6.0

3.       Guaiacol邻甲氧基苯酚

 

仪器

 

1.       紫外可见分光光度计

2.       拟南生长培养箱

 

步骤

 

1.       植物生长

1mL的套管(管底有格洞)中制备1/4 改良的Hoagland固体培养基(1%的琼脂粉,使用5mM MES-K缓冲液将pH调至6.0),将表面消毒后的拟南种子点种到固体培养基的格洞上。在光照下萌发2-3天后,将培养拟南幼苗的套管底部浸泡到含有1/4 改良的Hoagland液体培养基(1%的琼脂粉,使用5mM MES-K缓冲液将pH调至6.0)中,使用鱼缸水泵进行通气处理。

2.       根壁外非原质体液的提取 (AWF)

1)          先用去离子水清洗2.5周的Col生态型的植株,然后将其根切下浸泡到pH6.010 mM Na+-PO4溶液中。

2)          冰浴抽真空5分钟,缓慢通气使溶液充分浸透到材料里。

3)          将抽真空后的根放到滤纸上除去表面溶液,然后称量其鲜重。

4)          将称重后的根放到5mL注射器的注射筒中,然后将注射筒放到15mL的尖头离心管中,4 oC 3000g离心15分钟。

5)          离心管底部的液体就是根壁外非原质体液体,使用移液器对非原质体液进行体积定量,将提取出来的非原质体液放置到冰浴

3.       总蛋白定量

蛋白浓度测定 (Bradford 微量分析)是根据伯乐公司的试剂使用方法来进行检测的。

4.       非原质体过氧化物酶(APX)活性检测

根据下列反应体系进行反应,对其活性进行检测:

试剂及反应液             体积                        分子量及母液浓度.

邻甲氧基苯酚              880 ul           124.14; D=1.112 g/ml       20mM溶于pH6.010 mM Na-PO4溶液

壁外非原质体液(AWF  x ul

加入下列试剂开始反应:

H2O2      100 ul           34.01; 31.4 %                                    0.3 % (v/v)

总体积补充至1mL

室温反应2分钟后,于470nm下使用分光光度计读数。消光系数 = 26.6 mM-1 cm-1

 

参考文献

 

1.        Li X., Chanroj S., Wu Z., Romanowsky S.M., Harper J.F., Sze H. (2008). A distinct endosomal Ca2+/Mn2+ pump affects root growth through the secretory process. Plant Physiology 147(4): 1675-89. 

 

 

 

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How to cite this protocol: Li, X. (2011). Extraction of Root Apoplastic Wall Fluid for Apoplastic Peroxidase Activity Assay. Bio-protocol Bio101: e127. DOI: 10.21769/BioProtoc.127; Full Text



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