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Analysis of Malondialdehyde, Chlorophyll Proline, Soluble Sugar, and Glutathione Content in Arabidopsis seedling
拟南芥中丙二醛、叶绿素脯氨酸、可溶性糖和谷胱甘肽含量的分析   

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Abstract

The protocol has four sub-protocols, which are about the measurement of malondialdehyde, chlorophyll proline, soluble sugar, and glutathione content, respectively, in Arabidopsis seedling by using spectrophotometer. These methods are simple, effective and reproducible, which will help the researchers who are not familiar with these approaches, quickly get reliable results.

Keywords: Malondialdehyde(丙二醛), Proline(脯氨酸), Sugar(糖), Glutathione(谷胱甘肽), Arabidopsis(拟南芥)

I.   Measurement of Malondialdehyde

Materials and Reagents

  1. Thiobarbituric acid (TBA) (Sigma-Aldrich, catalog number: T5500 )
  2. Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: T9159 )
  3. Malondialdehyde (MDA) (BOC Sciences, catalog number: 542-78-9 )

Equipment

  1. Centrifuge
  2. Spectrophotometer

Procedure

Note: The experiment is done at room temperature (RT) except of specific indication.

  1. 0.1 g leaf tissue (with similar age, and young expanded leaf may be better) is ground into powder with liquid nitrogen, and then put the powder into a tube containing 1 ml 0.1% (w/v) TCA and mix by inverting the tube to homogenize the leaf tissue.
  2. Centrifuge homogenized samples at 10,000 x g for 10 min, and then transfer supernatant to a new tube.
  3. 4 ml of 20% TCA containing 0.5% TBA was added to the supernatant and mixed well.
  4. The mixture is boiled at 95 °C for 15 min and quickly cooled on ice (TBA can interact with MDA and results into red compound in acidic buffer, so the content of MDA can be calculated by measuring the density of the resulting red compound with spectrophotometer at 532 nm. The high temperature can accelerate the reaction and low temperature can inhibit it).
  5. Centrifuge the mixture at 10,000 x g for 5 min, and then transfer supernatant to a new tube.
  6. To generate a standard curve, a serial concentration of MDA is made: 1 μM, 2 μM, 5 μM, 10 μM, 20 μM and 50 μM (the volume of each dilution depends on the size of the cuvette of spectrophotometer).
  7. Measure the optical density of standard samples from step 6 at 532 nm by spectrophotometer and make the standard curve to get the extinction coefficient (Figure 1).


    Figure 1. The standard curve of MDA

  8. Measure the optical density of plant samples from step 5 at 532 nm and calculate the content of MDA according to the standard curve (Madhava Rao and Sresty, 2000; Baryla et al., 2000).

II.  Measurement of chlorophyll

Materials and Reagents

  1. Dimethyl formamide (DMF) (Sigma-Aldrich, catalog number: D4551 )

Equipment

  1. Centrifuge
  2. Spectrophotometer

Procedure

Note: The experiment is done at room temperature.

  1. 0.1 g leaf tissue is ground into powder with liquid nitrogen, and then homogenized with 1 ml 100% DMF.
  2. Centrifuge homogenized samples at 10,000 x g for 10 min, and then gather the supernatant.
  3. Measure the optical density of the supernatant at 664 nm and 647 nm, respectively.
  4. Calculate the content of chlorophyll a and chlorophyll b by the following formulas (Sibley et al.,1996; Inskeep and Bloom, 1985; Aono et al., 1993):
    [chlorophyll a] = 12.7 x A664 - 2.79 x A647
    [chlorophyll b] = 20.7 x A647 - 4.62 x A664
    [chlorophyll a + chlorophyll b] = 17.90 x A647 + 8.08 x A664


III. Measurement of Proline

Materials and Reagents

  1. Sulphosalicylic acid (DingGuo, catalog number: DS094 )
  2. Proline (Sigma-Aldrich, catalog number: 858919 )
  3. Ninhydrin (Sigma-Aldrich, catalog number: 151173 )
  4. Acetic acid (DingGuo, catalog number: DS002 )
  5. Orthophosphate (Sigma-Aldrich, catalog number: P2023 )
  6. Toluene (Sigma-Aldrich, catalog number: 650579 )
  7. Ninhydrin reagent (see Recipes)

Equipment

  1. Centrifuge
  2. Spectrophotometer

Procedure

Note: The experiment is done at room temperature (RT) except of specific indication.

  1. To generate a standard curve, a serial concentration of Proline is made in 3% sulphosalicylic acid: 1 μM, 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, 300 μM, 1 ml for each dilution.
  2. Each 500 μl standard solution is added with 500 μl acetic acid and 500 μl ninhydrin reagent in 5 ml tube and boil for 45 min, and then cooled in ice for 30 min.
  3. Add equal volume toluene to each sample and vibrate for 1 min, and then centrifuge at 1,000 x g for 5 min.
  4. Measure the optical density of toluene solution at 520 nm by spectrophotometer and make the standard curve (Figure 2).


    Figure 2. The standard curve of proline

  5. 0.5 g plant sample is ground into powder with liquid nitrogen, and then homogenized with 2 ml of 3% sulphosalicylic acid in tube.
  6. Centrifuge homogenized samples at 5,000 x g for 5 min, and then collect the supernatant
  7. The supernatant is treated as steps 2 and 3, and measure the optical density of samples as step 4, and then calculate the content of praline using the standard curve from step 3 (Bates et al., 1973; Lattanzioa et al., 2009).

Recipes

  1. Ninhydrin reagent
    2.5 g ninhydrin is successively added to 60 ml Glacial Acetic acid and 40 ml 6 M orthophosphate, and then dissolved at 70 °C. After cool down, the reagent can be stored in brown bottle at 4 °C for less than 24 h.


IV.  Measurement of Soluble Sugar

Materials and Reagents

  1. Ethanol (DingGuo, catalog number: DS023 )
  2. Glucose (DingGuo, catalog number: DS063 )
  3. Anthrone (SCRC, catalog number: 30015014 )
  4. H2SO4 (Sigma-Aldrich, catalog number: 339741 )
  5. Thiourea (Amresco, catalog number: M222 )
  6. Chloroform (Sigma-Aldrich, catalog number: C2432 )
  7. Anthrone reagent (see Recipes)

Equipment

  1. Centrifuge
  2. Spectrophotometer
  3. Shaker

Procedure

Note: The experiment is done at room temperature (RT) except of specific indication.

  1. To generate a standard curve, a serial concentration of glucose is made: 1 μM, 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, 300 μM, 5 ml for each concentration of glucose solution.
  2. 50 μl of each diluted glucose solution is mixed with 4.95 ml anthrone reagent and then boiled for 15 min.
  3. Measure the optical density of glucose standards at 620 nm by spectrophotometer to generate a standard curve.
  4. 0.1 g dried sample is ground into powder with liquid nitrogen, and then homogenized with 2 ml 80% ethanol in shaker at 200 rpm in 50 ml tube for 1 h.
  5. Centrifuge at 6,000 x g for 10 min, and then transfer as much supernatant as possible into a new 5 ml tube.
  6. Add equal volume of chloroform, completely mix, and then centrifuge at 12,000 x g for 10 min.
  7. The aqueous part is transferred to a new tube and repeat steps 2 and 3 to measure the optical density of the sample. The content of soluble sugar is calculated according the standard curve made at step 3 (Mandre et al., 2002; Jin et al., 2007).

Recipes

  1. Anthrone reagent
    72% H2SO4
    500 mg/L anthrone
    10 g/L thiourea


V.   Measurement of Glutathione

Materials and Reagents

  1. Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: T9159 )
  2. Polyvinylpolypyrrolidone (PVPP) (Sigma-Aldrich, catalog number: P6755 )
  3. 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS) (DingGuo, catalog number: DH350 )
  4. 5,50-dithio-bis (2-nitrobenzoic acid) (DTNB) (DingGuo, catalog number: DH499 )
  5. Glutathione reductase (GR) (Merck KGaA, catalog number: 359960 )
  6. Glutathiol (GSSG) (Solarbio, catalog number: G8690 )
  7. Reaction solution (see Recipes)

Equipment

  1. Centrifuge
  2. Spectrophotometer

Procedure

Note: The experiment is done at room temperature (RT) except of specific indication.

  1. To generate a standard curve, a serial concentration of GSSG is made: 0.5, 1, 2, 5, 10, 20 μM, 2 ml for each dilution of GSSG.
  2. 100 μl of each GSSG standard made at step 1 is added to 3 ml of Reaction solution and incubated for 15 min. Then add 100 mM DTNB to a final concentration of 10 mM and incubate at 25 °C for 15 min.
  3. Measure the optical density of each sample at 412 nm by spectrophotometer, and make standard curve with a function of the concentration of GSSG standard and the optical density of each GSSG standard.
  4. 0.5 g of Arabidopsis leaves is ground in liquid nitrogen.
  5. The samples are homogenized with 1 ml extract solution and mixed completely by inverting the tube.
  6. The mixture is centrifuged at 10,000 x g at 4 °C for 10 min, and then the supernatant is transferred to a new tube.
  7. 100 μl supernatant is treated as described at step 2, and the optical density of the supernatant is measured at 412 nm as described at step 3, and calculate the GSSG content of the sample according the standard curve.
  8. 100 μl supernatant is mixed with 3 ml 500 mM TRIS–HCl (pH 8.0) buffer containing 10 mM DTNB and incubated at 25 °C for 15 min. The optical density is then measured at 412 nm. The Glutathione (GSH) content is determined by the same standard curve as described at step 3 with the following formula: [GSH] = 2 x [standard curve].
  9. The total glutathione content = [GSH] + [GSSG] (Huang et al., 2005; Chen et al., 2011)

Recipes

  1. Reaction solution
    500 mM TRIS–HCl (pH 8.0) buffer
    GR (1 U for each 3 ml reaction solution)
    1 mM EDTA
    3 mM MgCl2
    150 μM NADPH
  2. Extract solution
    0.1% TCA (pH 2.8)
    1 mM EDTA
    1% (w/v) PVPP

Acknowledgments

This protocol is adapted from Huang et al. (2005) as well as other works mentioned in the reference list.

References

  1. Aono, M., Kubo, A., Saji, H., Tanaka, K. and Kondo, N. (1993). Enhanced tolerance to photooxidative stress of transgenic Nicotiana tabacum with high chloroplastic glutathione reductase activity. Plant Cell Physiol 34(1): 129-135.
  2. Baryla, A., Laborde, C., Montillet, J. L., Triantaphylides, C. and Chagvardieff, P. (2000). Evaluation of lipid peroxidation as a toxicity bioassay for plants exposed to copper. Environ Pollut 109(1): 131-135.
  3. Bates, L., Waldren, R. and Teare, I. (1973). Rapid determination of free proline for water-stress studies. Plant Soil 39(1): 205-207.
  4. Chen, L., Han, Y., Jiang, H., Korpelainen, H. and Li, C. (2011). Nitrogen nutrient status induces sexual differences in responses to cadmium in Populus yunnanensis. J Exp Bot 62(14): 5037-5050.
  5. Huang, C., He, W., Guo, J., Chang, X., Su, P. and Zhang, L. (2005). Increased sensitivity to salt stress in an ascorbate-deficient Arabidopsis mutant. J Exp Bot 56(422): 3041-3049.
  6. Inskeep, W. P. and Bloom, P. R. (1985). Extinction coefficients of chlorophyll a and B in n,n-dimethylformamide and 80% acetone. Plant Physiol 77(2): 483-485.
  7. Jin, Z. M., Wang, C. H., Liu, Z. P. and Gong, W. J. (2007). Physiological and ecological characters studies on Aloe vera under soil salinity and seawater irrigation. Process Biochem 42(4): 710-714.
  8. Lattanzio, V., Cardinali, A., Ruta, C., Fortunato, I. M., Lattanzio, V. M., Linsalata, V. and Cicco, N. (2009). Relationship of secondary metabolism to growth in oregano (Origanum vulgare L.) shoot cultures under nutritional stress. Environ Exp Bot 65(1): 54-62.
  9. Madhava Rao, K. V. and Sresty, T. V. (2000). Antioxidative parameters in the seedlings of pigeonpea (Cajanus cajan (L.) Millspaugh) in response to Zn and Ni stresses. Plant Sci 157(1): 113-128.
  10. Mandre, M., Tullus, H. and Kloseiko, J. (2002). Partitioning of carbohydrates and biomass of needles in Scots pine canopy. Z Naturforsch C 57(3-4): 296-302.
  11. Sibley, J. L., Eakes, D. J., Gilliam, C. H., Keever, G. J., Dozier, W. A. and Himelrick, D.G. (1996). Foliar SPAD-502 meter values, nitrogen levels, and extractable chlorophyll for red maple selections. Hort Sci 31(3): 468-470.

简介

该方案具有四个子协议,其分别是使用分光光度计在拟南芥苗中测量丙二醛,叶绿素脯氨酸,可溶性糖和谷胱甘肽含量。 这些方法简单,有效和可重复,这将有助于不熟悉这些方法的研究人员快速获得可靠的结果。

关键字:丙二醛, 脯氨酸, 糖, 谷胱甘肽, 拟南芥

I.    测量丙二醛

材料和试剂

  1. 硫代巴比妥酸(TBA)(Sigma-Aldrich,目录号:T5500)
  2. 三氯乙酸(TCA)(Sigma-Aldrich,目录号:T9159)
  3. 丙二醛(MDA)(BOC Sciences,目录号:542-78-9)

设备

  1. 离心机
  2. 分光光度计

程序

注意:实验在室温(RT)下进行,除了具体的指示。

  1. 将0.1g叶组织(具有相似的年龄,年轻的扩张叶可能更好)用液氮研磨成粉末,然后将粉末放入含有1ml 0.1%(w/v)TCA的管中,通过翻转管混合 以均匀化叶组织
  2. 将匀浆样品以10,000×g离心10分钟,然后将上清液转移至新管中。
  3. 向上清液中加入4ml含0.5%TBA的20%TCA,充分混合
  4. 将混合物在95℃下煮沸15分钟,并在冰上快速冷却(TBA可与MDA相互作用,并在酸性缓冲液中形成红色化合物,因此MDA的含量可通过用分光光度计测量所得红色化合物的密度来计算高温可加速反应,低温可抑制反应)。
  5. 将混合物以10,000×g离心5分钟,然后将上清液转移到新管中。
  6. 为了产生标准曲线,制备MDA的连续浓度:1μM,2μM,5μM,10μM,20μM和50μM(每种稀释的体积取决于分光光度计的比色杯的尺寸)。
  7. 用分光光度计测量步骤6的标准样品在532nm的光密度,并使标准曲线得到消光系数(图1)。


    图1. MDA
    的标准曲线
  8. 测量来自步骤5的植物样品在532nm处的光密度,并根据标准曲线计算MDA的含量(Madhava Rao和Sresty,2000; Baryla等人,2000)。br/>

II。 叶绿素的测量

材料和试剂

  1. 二甲基甲酰胺(DMF)(Sigma-Aldrich ,目录号:D4551)

设备

  1. 离心机
  2. 分光光度计

程序

注意:实验在室温下进行。

  1. 将0.1g叶组织用液氮研磨成粉末,然后用1ml 100%DMF匀化
  2. 将匀浆样品在10,000×g离心10分钟,然后收集上清液。
  3. 分别在664nm和647nm处测量上清液的光密度。
  4. 通过下式计算叶绿素a和叶绿素b的含量(Sibley等人,1996; Inskeep和Bloom,1985; Aono等人, em。,1993):
    [叶绿素a] = 12.7×A 664 - 2.79×A 647
    [叶绿素b] = 20.7×A 647+ 4.62×A 664
    [叶绿素a +叶绿素b] = 17.90×A 647+ + 8.08×A 664 <


III。 测量脯氨酸

材料和试剂

  1. 磺基水杨酸(DingGuo,目录号:DS094)
  2. 脯氨酸(Sigma-Aldrich,目录号:858919)
  3. 茚三酮(Sigma-Aldrich,目录号:151173)
  4. 乙酸(DingGuo,目录号:DS002)
  5. 正磷酸盐(Sigma-Aldrich,目录号:P2023)
  6. 甲苯(Sigma-Aldrich,目录号:650579)
  7. 水合试剂(见配方)

设备

  1. 离心机
  2. 分光光度计

程序

注意:实验在室温(RT)下进行,除了具体的指示。

  1. 为了产生标准曲线,在3%磺基水杨酸中制备脯氨酸的连续浓度:1μM,10μM,50μM,100μM,150μM,200μM,300μM,每种稀释1ml。
  2. 每个500μl标准溶液在5ml管中加入500μl乙酸和500μl茚三酮试剂并煮沸45分钟,然后在冰中冷却30分钟。
  3. 向每个样品中加入等体积甲苯,振动1分钟,然后在1,000×g离心5分钟。
  4. 用分光光度计测量甲苯溶液在520nm处的光密度,得到标准曲线(图2)

    图2.脯氨酸
    的标准曲线
  5. 将0.5g植物样品用液氮研磨成粉末,然后用管中的2ml 3%磺基水杨酸匀化。
  6. 在5,000xg离心均质样品5分钟,然后收集上清液
  7. 将上清液作为步骤2和3处理,并测量作为步骤4的样品的光密度,然后使用来自步骤3的标准曲线计算脯氨酸的含量(Bates等人,1973; Lattanzioa et al 。,2009)。

食谱

  1. 水合试剂
    将2.5g茚三酮连续加入到60ml冰乙酸和40ml 6M正磷酸盐中,然后在70℃下溶解。 冷却后,试剂可以在4℃下储存在棕色瓶中不超过24小时


IV。 测量可溶性糖

材料和试剂

  1. 乙醇(DingGuo,目录号:DS023)
  2. 葡萄糖(DingGuo,目录号:DS063)
  3. 蒽酮(SCRC,目录号:30015014)
  4. H 2 SO 4(Sigma-Aldrich,目录号:339741)
  5. 硫脲(Amresco,目录号:M222)
  6. 氯仿(Sigma-Aldrich,目录号:C2432)
  7. 蒽酮试剂(见配方)

设备

  1. 离心机
  2. 分光光度计
  3. 振动器

程序

注意:实验在室温(RT)下进行,除了具体的指示。

  1. 为了产生标准曲线,对于每一浓度的葡萄糖溶液,使葡萄糖的系列浓度为1μM,10μM,50μM,100μM,150μM,200μM,300μM,5ml。
  2. 将50μl每种稀释的葡萄糖溶液与4.95ml蒽酮试剂混合,然后煮沸15分钟。
  3. 通过分光光度计测量620nm处葡萄糖标准品的光密度,以产生标准曲线
  4. 将0.1g干燥的样品用液氮研磨成粉末,然后在50ml管中以200rpm在振荡器中用2ml 80%乙醇匀化1小时。
  5. 以6000xg离心10分钟,然后将尽可能多的上清液转移到新的5ml管中。
  6. 加入等体积的氯仿,完全混合,然后以12,000×g离心10分钟。
  7. 将含水部分转移到新管中并重复步骤2和3以测量样品的光密度。 可溶性糖的含量根据步骤3中制备的标准曲线计算(Mandre等人,2002; Jin等人,2007)。

食谱

  1. 蒽酮试剂
    72%H <2> SO 4
    500 mg/L蒽酮
    10 g/L硫脲


V 。   测量谷胱甘肽

材料和试剂

  1. 三氯乙酸(TCA)(Sigma-Aldrich,目录号:T9159)
  2. 聚乙烯聚吡咯烷酮(PVPP)(Sigma-Aldrich,目录号:P6755)
  3. 2-氨基-2-(羟甲基)-1,3-丙二醇(TRIS)(DingGuo,目录号:DH350)
  4. 5,5-二硫代 - 双(2-硝基苯甲酸)(DTNB)(DingGuo,目录号:DH499)
  5. 谷胱甘肽还原酶(GR)(Merck KGaA,目录号:359960)
  6. 谷胱甘肽(GSSG)(Solarbio,目录号:G8690)
  7. 反应溶液(参见配方)

设备

  1. 离心机
  2. 分光光度计

程序

注意:实验在室温(RT)下进行,除了具体的指示。

  1. 为了产生标准曲线,GSSG的连续浓度为:对于GSSG的每个稀释,0.5,1,2,5,10,20μM,2ml。
  2. 将100μl在步骤1制备的每种GSSG标准品加入到3ml反应溶液中并孵育15分钟。 然后加入100mM DTNB至终浓度为10mM,并在25℃下孵育15分钟。
  3. 通过分光光度计测量每个样品在412nm处的光密度,并制作具有GSSG标准品浓度和每个GSSG标准品的光密度的函数的标准曲线。
  4. 将0.5g拟南芥叶在液氮中研磨
  5. 将样品与1ml提取物溶液匀浆,通过倒置管而完全混合。
  6. 将混合物在4℃以10,000×g离心10分钟,然后将上清液转移到新管中。
  7. 如步骤2所述处理100μl上清液,如步骤3所述在412nm处测量上清液的光密度,并根据标准曲线计算样品的GSSG含量。
  8. 将100μl上清液与含有10mM DTNB的3ml 500mM TRIS-HCl(pH 8.0)缓冲液混合,并在25℃温育15分钟。然后在412nm测量光密度。谷胱甘肽(GSH)含量通过与步骤3中描述的相同的标准曲线用下式测定:[GSH] = 2x [标准曲线]。
  9. 总谷胱甘肽含量= [GSH] + [GSSG](Huang等人,2005; Chen等人,2011)

食谱

  1. 反应溶液
    500mM TRIS-HCl(pH 8.0)缓冲液
    GR(每3ml反应溶液1U) 1mM EDTA
    3mM MgCl 2/
    150μMNADPH
  2. 提取解决方案
    0.1%TCA(pH 2.8)
    1mM EDTA
    1%(w/v)PVPP

致谢

该协议改编自Huang等人(2005)以及参考列表中提及的其他作品。

参考文献

  1. Aono,M.,Kubo,A.,Saji,H.,Tanaka,K。和Kondo,N。(1993)。 增强对转基因烟草的光氧化胁迫的耐受性 谷胱甘肽还原酶活性。植物细胞生理学 34(1):129-135。
  2. Baryla,A.,Laborde,C.,Montillet,J.L.,Triantaphylides,C.and Chagvardieff,P。(2000)。 评估脂质过氧化作为暴露于铜的植物的毒性生物测定。 Environ Pollut 109(1):131-135。
  3. Bates,L.,Waldren,R。和Teare,I.(1973)。 快速测定水分胁迫研究中的游离脯氨酸。植物土壤 39(1):205-207。
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引用:Zhang, Z. and Huang, R. (2013). Analysis of Malondialdehyde, Chlorophyll Proline, Soluble Sugar, and Glutathione Content in Arabidopsis seedling. Bio-protocol 3(14): e817. DOI: 10.21769/BioProtoc.817.
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