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Analysis of Sugar Component of a Hot Water Extract from Arabidopsis thaliana Pollen Tubes Using GC-EI-MS
采用 GC-EI-MS分析拟南芥花粉管中热水提取物的糖组分   

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Abstract

Extraction with hot water is the oldest and simplest method used to recover pectin from an alcohol insoluble residue extract, although this method has not been widely used for the cell wall analysis of pollen tube, a model used to study cell wall. This protocol described this method applied for pectin extraction from 6 h-old Arabidopsis pollen tubes followed by a sugar composition analysis by gas chromatography mass spectrometry.

Keywords: Pollen tube(花粉管), Cell wall(细胞壁), Monosaccharide composition(单糖组成), GC-MS(气相色谱-质谱联用)

Materials and Reagents

  1. Arabidopsis day-0 flowers according to Boavida and McCormick (2007)
  2. Absolute ethanol (EtOH) for analysis (Merck Millipore, catalog number: 107017 )
  3. Trifluoroacetic acid 12.93N (TFA) (Sigma-Aldrich, catalog number: T62200 )
  4. Myo-inositol 2 mM (Sigma-Aldrich, catalog number: I5125 )
  5. Methanolic-HCl 3N (Sigma-Aldrich, catalog number: 33051 Supelco )
  6. Methanol (Thermo Fisher Scientific, catalog number: 10010280 )
  7. Silylation reagent (HMDS:TMCS:Pyridine, 3:1:9 SylonTM HTP) (Sigma-Aldrich, catalog number: 33038 Supelco )
  8. Cyclohexane (Acros Organics, catalog number: 279590010 )
  9. Monosaccharides standards
    Arabinose (Sigma-Aldrich, catalog number: A3256 )
    Fucose (Sigma-Aldrich, catalog number: F8150 )
    Galactose (Sigma-Aldrich, catalog number: G0750 )
    Galacturonic acid (Sigma-Aldrich, catalog number: 857289 )
    Glucose (Merck Millipore, catalog number: 8337.0250 )
    Glucuronic acid (Sigma-Aldrich, catalog number: G8645 )
    Mannose (Sigma-Aldrich, catalog number: M4625 )
    Rhamnose (Sigma-Aldrich, catalog number: R3875 )
    Xylose (Sigma-Aldrich, catalog number: X2126 )
  10. Arabidopsis pollen germination medium (see Recipes)
  11. Trifluoroacetic acid (2 N) (see Recipes)

Equipment

  1. Inverted microscope Olympus CK2
  2. Centrifuge Allegra® X-15R Beckmann Coulter
  3. Hot-water bath (70 °C and 90 °C)
  4. 40-ml glass potter homogenizer
  5. Freeze-dryer
  6. Incubator (22 °C, 80 °C and 110 °C)
  7. Sample concentrator under air flow (Techne, catalog number: FSC400D )
  8. GC-EI-MS instrument is composed of a Zebron Z5-MSi (30 m, 0.25 mm id, 0.25 µm film thickness, Phenomenex) capillary column for chromatographic separations, a Hewlett-Packard 6890 series gas chromatograph coupled to an Autospec mass spectrometer of EBE geometry (Micromass, Manchester, UK) equipped with a Opus 3.1 data system
  9. 1.5 ml Eppendorf tube
  10. Vortex
  11. Pair of tweezers
  12. 50-ml Falcon
  13. Microliter syringe (10 µl, Hamilton)

Procedure

  1. In vitro Arabidopsis pollen tubes growth and fixation
    The protocol described below is similar to the one available at Wang and Jiang (2014) with several differences.
    1. Grow in vitro Arabidopsis pollen according to the method described by Boavida and McCormick (2007).
      1. Harvest 40 freshly open Arabidopsis thaliana flowers (morning is the best).
      2. Submerge in 1 ml of pollen germination medium in a 1.5 ml Eppendorf tube.
      3. Shake the tube vigorously manually and using a vortex for 5 min to release the pollen grains from the anthers.
      4. Check the pollen density with an inverted microscope.
      5. Remove all the flower debris with a pair of tweezers.
      6. Spin-down the pollen suspension at 3,200 x g for 7 min (a yellowish pellet should be visible).
      7. Remove the supernatant.
      8. Add 250 µl of pollen germination medium and resuspend the pellet.
      9. Place the tube horizontally in the incubator at 22 °C in the dark for 6 h.
      10. After 6 h, check for pollen germination and pollen tube growth with the inverted microscope (Figure 1).
    2. Fix the pollen culture by adding 750 µl absolute EtOH (final concentration 75% EtOH) and keep at 4 °C (no longer than one month).
      Note: Around 90 pollen cultures (3,600 flowers) are needed for the detection of minor sugars such as the Kdo (2-keto-3-deoxy-D-manno-octulosonic acid), one of the constituents of rhamnogalacturonan-II (RG-II). For other major cell wall components, pollen tubes from 400 flowers are generally sufficient.


      Figure 1. 6 h-old Arabidopsis pollen tubes. Note that the pollen grain density is important to obtain a good germination rate.

  2. Pectin extraction and analysis
    1. Hot water pectin extraction:
      1. Pool all the pollen cultures in a 50-ml Falcon® tube and centrifuge at 4,700 x g for 5 min. Repeat the centrifugation step if the total volume of the pollen cultures exceeds 45 ml.
      2. Discard the supernatant and wash with 15 mL EtOH 70% to remove salt and sucrose from the germination medium.
      3. Incubate in hot-water bath at 70 °C for 15 min.
      4. Grind the material manually with a 40-ml glass potter homogenizer until no debris are visible.
      5. Centrifuge at 4,700 x g for 5 min and discard the supernatant.
      6. Allow the pellet to dry until no supernatant is visible (it might take no more than 2 h using a stream of dry air).
      7. Resuspend the pellet in 5 ml distilled water.
      8. Incubate in a hot-water bath at 90 °C for 1 h.
      9. Centrifuge at 4,700 x g for 5 min.
      10. Freeze-dry the supernatant. No liquid must be visible (it might take one day).
    2. Sample preparation for gas chromatography analysis:
      Note: Glassware tubes are preferentially used for the sample preparation. A standard sample containing the most common cell wall monosaccharides (Ara, Fuc, Gal, GalUA, Glc, GlcUA, Man, Rha, Xyl) is prepared at the same time without the TFA hydrolysis step.
      1. Hydrolyze the extract with 250 µl TFA 2 N for 2 h at 110 °C.
      2. Add 50 µl of myo-inositol 2 mM in the sample. Myo-inositol is used as an internal standard.
      3. Perform a methanolysis for 16 h at 80 °C using 250 µl of dried 1 M methanolic-HCl (dilute the 3 M stock solution with methanol).
      4. Wash the sample twice using 500 µl methanol.
      5. Convert the methyl glycosides into their trimethylsilyl derivatives at 110 °C for 20 min with 200 µl of the silylation reagent (HMDS: TMCS: Pyridine, 3:1:9, Supelco).
      6. Wash twice the sample using 500 µl cyclohexane.
        Note: Air dry the sample using a stream of dry air with a sample concentrator between each step (a nitrogen flow can be used as well). Add 1 ml cyclohexane and vortex the tube.
    3. Monosaccharides analysis:
      1. Inject 0.5 µl of the sample in the GC-EI-MS instrument, using a 10 µl syringe, in splitless mode. Helium is the carrier gas and the flow-rate is 0.8 ml/min.
      2. Start the temperature programming at 120 °C for 2 min, ramp to 160 °C at 10 °C/min, then to 220 °C at 1.5 °C/min and finally ramp to 280 °C at 15 °C/min (maintain at 270 °C for 1 min). The temperatures of the injector, the interface and the lines are 250 °C. Record the EI mass spectra using an electron energy of 70 eV, an acceleration voltage of 8 kV and a resolving power of 1,000. The trap current of 200 µA and the magnet scan rate is 1 s/decade over a m/z range 600-38. The temperature of ion source is 250 °C.
      3. The data are processed with the software STAR Workstation Varian 5.52. First of all, it is important to check that peak and peak areas (A) are assigned correctly for each spectrum by comparing with the standard sample before proceeding. The response factor (Rf) for each monosaccharide has been determined previously by calibration of the instrument for each standard sugar. Peak area is integrated on the total chromatogram for each sugar and for the internal standard (inositol). The amount of each sugar is calculated by the formula:

      Figure 2 gives an overview of the complete protocol.


      Figure 2. Schematic representation of the protocol for sugar composition of a hot water extract from Arabidopsis thaliana pollen tubes
      1. Recipes Arabidopsis thaliana pollen germination medium
        5 mM CaCl2
        0.01% H3BO3
        1 mM MgSO4
        5 mM KCl
        10% (w/v) sucrose
        pH 7.5
      2. Trifluoroacetic acid (2 N)
        0.15 volume of Trifluoroacetic acid 12.93 N
        0.85 volume of fresh distilled water
        Note: Use glassware.

Acknowledgments

This protocol was initially described in Dumont et al. (2014). This work was supported by the University of Rouen, the region Haute-Normandie, and the “Trans Channel Wallnet” project that has been selected by the INTERREG IVA program France (Channel) – England European cross-border cooperation programme, which is co-financed by the ERDF. The authors also thank the Labex SynOrg (ANR-11-LABX-0029, CRUNCh network, and the European Regional Development Fund (ERDF 31708).

References

  1. Boavida, L. C. and McCormick, S. (2007). TECHNICAL ADVANCE: Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana. Plant J 52(3): 570-582.
  2. Dumont, M., Lehner, A., Bouton, S., Kiefer-Meyer, M. C., Voxeur, A., Pelloux, J., Lerouge, P. and Mollet, J. C. (2014). The cell wall pectic polymer rhamnogalacturonan-II is required for proper pollen tube elongation: implications of a putative sialyltransferase-like protein. Ann Bot 114(6): 1177-1188.
  3. Wang, H. and Jiang, L. (2014). Immunofluorescence labeling of pollen tubes. Bio-protocol 4(10): e1131.

简介

用热水提取是用于从醇不溶性残余物提取物中回收果胶的最古老和最简单的方法,尽管该方法尚未广泛用于花粉管的细胞壁分析,所述花粉管是用于研究细胞壁的模型。 该方案描述了该方法应用于从6h-旧拟南芥花粉管中提取果胶,然后通过气相色谱质谱法进行糖组成分析。

关键字:花粉管, 细胞壁, 单糖组成, 气相色谱-质谱联用

材料和试剂

  1. 根据Boavida和McCormick(2007)的拟南芥 day-0花
  2. 用于分析的无水乙醇(EtOH)(Merck Millipore,目录号:107017)
  3. 三氟乙酸12.93N(TFA)(Sigma-Aldrich,目录号:T62200)
  4. 肌醇2mM(Sigma-Aldrich,目录号:I5125)。
  5. 甲醇-HCl 3N(Sigma-Aldrich,目录号:33051 Supelco)
  6. 甲醇(Thermo Fisher Scientific,目录号:10010280)
  7. 甲硅烷化试剂(HMDS:TMCS:吡啶,3:1:9 Sylon TM HTP)(Sigma-Aldrich,目录号:33038 Supelco)
  8. 环己烷(Acros Organics,目录号:279590010)
  9. 单糖标准
    阿拉伯糖(Sigma-Aldrich,目录号:A3256) 岩藻糖(Sigma-Aldrich,目录号:F8150) 半乳糖(Sigma-Aldrich,目录号:G0750) 半乳糖醛酸(Sigma-Aldrich,目录号:857289) 葡萄糖(Merck Millipore,目录号:8337.0250)
    葡萄糖醛酸(Sigma-Aldrich,目录号:G8645) 甘露糖(Sigma-Aldrich,目录号:M4625) 鼠李糖(Sigma-Aldrich,目录号:R3875) 木糖(Sigma-Aldrich,目录号:X2126)
  10. 拟南芥花粉萌发培养基(参见食谱)
  11. 三氟乙酸(2N)(参见配方)

设备

  1. 倒置显微镜Olympus CK2
  2. 离心机Allegra® X-15R Beckmann Coulter
  3. 热水浴(70℃和90℃)
  4. 40-ml玻璃波特均化器
  5. 冷冻干燥机
  6. 培养箱(22℃,80℃和110℃)
  7. 气流下的样品浓缩器(Techne,目录号:FSC400D)
  8. GC-EI-MS仪器由用于色谱分离的Zebron Z5-MSi(30μm,0.25mm内径,0.25μm膜厚度,Phenomenex)毛细管柱,Hewlett-Packard 6890系列气相色谱仪与Autospec质谱仪 EBE几何(Micromass,Manchester,UK),装备有Opus 3.1数据系统
  9. 1.5 ml Eppendorf管
  10. 涡流
  11. 双镊子
  12. 50毫升Falcon
  13. 微量注射器(10μl,Hamilton)

程序

  1. 体外 拟南芥花粉管生长和固定
    下面描述的协议类似于Wang和Jiang(2014)提供的协议,有几个区别。
    1. 根据Boavida和McCormick(2007)描述的方法,在体外 拟南芥花粉。
      1. 收获40新鲜开放的拟南芥 拟南芥花(早上是最好的)。
      2. 浸没在1ml花粉萌发培养基在1.5ml Eppendorf管中。
      3. 剧烈手动摇动管,并使用涡旋5分钟从花药释放花粉粒。
      4. 用倒置显微镜检查花粉密度。
      5. 用一对镊子去除所有的花碎屑。
      6. 将花粉悬浮液在3,200×g下旋转7分钟(微黄色颗粒应该是可见的)。
      7. 除去上清液。
      8. 加入250微升花粉萌发培养基,并重悬沉淀
      9. 将管水平放置在孵化器中在22°C在黑暗中6小时。
      10. 6小时后,用倒置显微镜检查花粉萌发和花粉管生长(图1)。
    2. 通过加入750微升无水乙醇固定花粉培养(最终 浓度75%EtOH)并保持在4℃(不超过一个月)。
      注意:需要约90个花粉培养物(3,600朵花) 检测次要糖如Kdo (2-酮基-3-脱氧-D-甘露 - 辛糖酸),其中一种成分 鼠李半乳糖醛酸聚糖-II(RG-II)。 对于其他主要细胞壁组件, 花粉管从400朵花一般就足够了。


      图1. 6 h-old拟南芥花粉管。请注意,花粉粒密度对于获得良好的发芽率很重要。

  2. 果胶提取和分析
    1. 热水果胶提取:
      1. 池中所有的花粉文化 50-ml Falcon管中并在4,700×g离心5分钟。 重复 离心步骤,如果花粉培养物的总体积超过 45 ml。
      2. 弃去上清液并用15mL EtOH 70%洗涤以从萌发培养基中除去盐和蔗糖。
      3. 在70℃的热水浴中孵育15分钟
      4. 使用40ml玻璃波特均化器手动研磨材料,直到没有可见的碎屑
      5. 以4,700×g离心5分钟,弃去上清液
      6. 让沉淀干燥,直到没有上清液可见(使用干燥空气流可能需要不超过2小时)。
      7. 将沉淀重悬于5ml蒸馏水中。
      8. 在90℃的热水浴中孵育1小时。
      9. 以4,700×g离心5分钟。
      10. 冷冻干燥上清液。 没有液体必须可见(可能需要一天)。
    2. 气相色谱分析的样品制备:
      注意:玻璃器皿管优先用于样品 制备。 含有最常见细胞壁的标准样品 单糖(Ara,Fuc,Gal,GalUA,Glc,GlcUA,Man,Rha,Xyl)是 在没有TFA水解步骤的同时制备。
      1. 在110℃下用250μlTFA 2 N水解提取物2小时。
      2. 在样品中加入50μl的myo - 肌醇2mM。 Myo - 肌醇被用作内标。
      3. 使用250μl干燥的1M在80℃进行甲醇分解16小时 甲醇-HCl(用甲醇稀释3M原液)。
      4. 使用500μl甲醇洗涤样品两次。
      5. 将甲基糖苷转化成它们的三甲基甲硅烷基衍生物 在110℃下用200μl甲硅烷化试剂(HMDS:TMCS: 吡啶,3:1:9,Supelco)
      6. 使用500μl环己烷洗涤样品两次。
        注意:使用带有样品的干燥空气流对样品进行空气干燥 浓缩器在每个步骤之间(也可以使用氮气流)。 加入1ml环己烷,涡旋管。
    3. 单糖分析:
      1. 在GC-EI-MS仪器中注入0.5μl样品,使用10 μl注射器,在不分流模式。氦是载气和 流速为0.8ml/min。
      2. 在开始温度编程 120℃2分钟,以10℃/min升温至160℃,然后在1.5℃升温至220℃ ℃,最后以15℃/min升温至280℃(在270℃保持1小时)  min)。喷射器,接口和管线的温度  250℃。使用70eV的电子能量记录EI质谱, 加速电压为8kV,分辨率为1000。陷阱  电流为200μA,磁体扫描速率在m/z范围600-38上为1s/decade。离子源的温度为250℃
      3. 数据 使用软件STAR Workstation Varian 5.52进行处理。首先 所有,重要的是检查峰和峰面积(A)是否分配 通过与之前的标准样品比较,对每个光谱正确  进行。每种单糖的响应因子(R sub) 通过对每个标准的仪器校准来确定   糖。 峰面积积分在每种糖的总色谱图上   和内标(肌醇)。 每种糖的量是 由公式计算:

      图2给出了完整协议的概述

      图2.糖协议的示意图 来自拟南芥花粉的热水提取物的组成 管
      1. 拟南芥花粉萌发培养基
        5mM CaCl 2
        0.01%H 3 BO 3 sub。 1mM MgSO 4 5 mM KCl
        10%(w/v)蔗糖 pH 7.5
      2. 三氟乙酸(2N)
        0.15体积的三氟乙酸12.93N
        0.85体积的新鲜蒸馏水
        注意:使用玻璃器皿。

致谢

该协议最初在Dumont等人(2014)中描述。这项工作得到了鲁昂大学,上海法国大学的支持,以及由INTERREG IVA项目选定的"Trans Channel Wallnet"项目。法国(海峡) - 英国欧洲跨境合作项目,由ERDF资助。作者还感谢Labex SynOrg(ANR-11-LABX-0029,CRUNCH网络和欧洲区域发展基金(ERDF 31708)。

参考文献

  1. Boavida,L.C。和McCormick,S。(2007)。 技术进步:温度作为增加和可再现性的决定因素< (3):570-582。 in vitro pollen germination in Arabidopsis thaliana
  2. Dumont,M.,Lehner,A.,Bouton,S.,Kiefer-Meyer,M.C.,Voxeur,A.,Pelloux,J.,Lerouge,P.and Mollet,J.C。 细胞壁果胶聚合物rhamnogalacturonan-II是正常花粉管延长所必需的:推定的唾液酸转移酶like protein。 Ann Bot 114(6):1177-1188。
  3. Wang,H.和Jiang,L。(2014)。 花粉管的免疫荧光标记生物协议 4(10): e1131。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Dumont, M., Lehner, A., Loutelier-Bourhis, C., Mollet, J. and Lerouge, P. (2015). Analysis of Sugar Component of a Hot Water Extract from Arabidopsis thaliana Pollen Tubes Using GC-EI-MS. Bio-protocol 5(12): e1503. DOI: 10.21769/BioProtoc.1503.
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