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Cyclohexane Diamine Tetraacetic Acid (CDTA) Extraction of Plant Cell Wall Pectin
环己烷二胺四乙酸(CDTA)法提取植物细胞壁中的果胶   

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Abstract

The goal of this procedure is to extract pectin from plant cell walls. Pectins are galacturonic acid containing polymeric sugars that are important components of plant cell walls. Various procedures aimed at studying plant cell wall components require the extraction of pectin. Pectin is synthesized in the Golgi apparatus in a highly esterified fashion and is de-esterified in the cell wall (Mohnen, 2008). Pectin is generally water soluble. De-esterified pectin can form so-called “egg-box structures” in the presence of Ca2+ ions (Mohnen, 2008; Harholt et al., 2010). Pectin in these “egg-box structures” is cross-linked and less soluble. Cyclohexane diamine tetraacetic acid (CDTA) chelates Ca2+ ions and hence allows extraction of Ca2+ cross-linked pectin from cell walls.

Materials and Reagents

  1. Plant material or cell wall preparation of choice (see Note 1)
  2. Cyclohexane diamine tetraacetic acid (CDTA) (Fluka, catalog number: 34588 )
  3. Tris-base (Sigma-Aldrich, catalog number: T6066 )
  4. Sodium hydroxide (Thermo Fisher Scientific, catalog number: S318 )
  5. Liquid nitrogen
  6. CDTA extraction buffer (see Recipes)

Equipment

  1. Liquid nitrogen container
  2. Paint shaker (e.g. Harbil, model: 5G-HD) and ball bearings (e.g. 3 mm diameter steel beads) (mortar and pestle or bead beater)
  3. Media bottle
  4. Single-channel pipettor
  5. Microfuge tubes appropriate for use at 95 °C and for freezing in liquid N2 (Thermo Fisher Scientific, catalog number: 02-707-355 )
    Note: We use 2 ml screw cap tubes.
  6. Pipette tips
  7. Water bath or incubator
  8. Vortex mixer
  9. Microcentrifuge capable of spinning at 10,000 x g
  10. Optional: Screw cap tubes or lid locks

Procedure

  1. Pre-heat water bath or incubator to 95 °C and pre-heat CDTA extraction buffer.
  2. Collect plant tissue in tubes - we use leaf material from approx. 4 week old Arabidopsis thaliana plants. Three fully expanded Arabidopsis leaves weigh about 250 mg.
  3. Freeze tubes containing the tissue in liquid nitrogen (tissue can be stored at -80 °C).
  4. Pulverize frozen tissue using ball bearings and a paint shaker, mortar and pestle or a bead beater. The method used to pulverize tissue is not critical. Keep tissue frozen. If paint shaker is used three cycles of three minutes each are generally sufficient to pulverize tissue.
  5. Add 1 ml of CDTA extraction buffer to 250 mg of frozen pulverized Arabidopsis tissue.
  6. Place tubes at 95 °C. Use caution, the liquid will become very hot and lids may open; therefore use screw cap tubes or lid locks to secure lids.
  7. Incubate for 15 min, vortex every 5 min.
  8. Centrifuge at 10,000 x g for 10 min at room temperature.
  9. The supernatant contains the pectin.
  10. Supernatant can be frozen and freeze dried for downstream applications.

Notes

  1. We directly use pulverized Arabidopsis leaf material (Bethke et al., 2014). Alternatively crude cell wall preparations like alcohol insoluble residue (Gille et al., 2009) preparations etc. can be used (pre-heat the water bath and extraction buffer and proceed to step 5).
  2. Different groups have reported different extraction times and temperatures e.g. 15 min at 95 °C (Siedlecka et al., 2008) or 4 h at room temperature (Moller et al., 2008). In our experience about twice the amount of pectin could be extracted when extraction was performed for 15 min at 95 °C as compared to the longer extraction at room temperature.
  3. Different groups have utilized similar protocols for the extraction of pectin from various monocotyledonous and dicotyledonous plants e.g. cucumber, tomato, celery (Jarvis et al., 1982), poplar (Siedlecka et al., 2008), soybean (Huisman et al., 2001) or wheat (Wiethölter et al., 2003).
  4. The extraction buffer can be stored at room temperature for several weeks.
  5. Pectin extracted using this procedure can be used for various downstream application including dot-blot analysis with pectin specific antibodies, sugar analysis, ion-exchange chromatography or determination of degree of esterification of pectin (Bethke et al., 2014; MacDougall et al., 1997).

Recipes

  1. CDTA extraction buffer
    50 mM CDTA
    50 mM Tris-base
    Adjust pH to 7.2 using sodium hydroxide

Acknowledgments

This work was funded by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy through Grant DE-FG02-05ER15670 to J.G.
Many labs have used similar protocols in the past. We have adapted this protocol from Siedlecka et al. (2008).

References

  1. Bethke, G., Grundman, R. E., Sreekanta, S., Truman, W., Katagiri, F. and Glazebrook, J. (2014). Arabidopsis PECTIN METHYLESTERASEs contribute to immunity against Pseudomonas syringae. Plant Physiol 164(2): 1093-1107.
  2. Gille, S., Hansel, U., Ziemann, M. and Pauly, M. (2009). Identification of plant cell wall mutants by means of a forward chemical genetic approach using hydrolases. Proc Natl Acad Sci U S A 106(34): 14699-14704.
  3. Harholt, J., Suttangkakul, A. and Vibe Scheller, H. (2010). Biosynthesis of pectin. Plant Physiol 153(2): 384-395.
  4. Huisman, M. M. H., Fransen, C. T. M., Kamerling, J. P., Vliegenthart, J. F. G., Schols, H. A. and Voragen, A. G. J. (2001). The CDTA-soluble pectic substances from soybean meal are composed of rhamnogalacturonan and xylogalacturonan but not homogalacturonan. Biopolymers 58(3): 279-294.
  5. Jarvis, M. C. (1982). The proportion of calcium-bound pectin in plant cell walls. Planta 154(4): 344-346.
  6. MacDougall, A. J., Rigby, N. M., Ring, S. G. (1997). Phase separation of plant cell wall polysaccharides and its implications for cell wall assembly. Plant Physiol 114(1): 353-362.
  7. Mohnen, D. (2008). Pectin structure and biosynthesis. Curr Opin Plant Biol 11(3): 266-277.
  8. Moller, I., Marcus, S. E., Haeger, A., Verhertbruggen, Y., Verhoef, R., Schols, H., Ulvskov, P., Mikkelsen, J. D., Knox, J. P. and Willats, W. (2008). High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles. Glycoconj J 25(1): 37-48.
  9. Siedlecka, A., Wiklund, S., Peronne, M. A., Micheli, F., Lesniewska, J., Sethson, I., Edlund, U., Richard, L., Sundberg, B. and Mellerowicz, E. J. (2008). Pectin methyl esterase inhibits intrusive and symplastic cell growth in developing wood cells of Populus. Plant Physiol 146(2): 554-565.
  10. Wiethölter, N., Graeβner, B., Mierau, M., Willats, W. G. T., Knox, J. P., Moerschbacher, B. M. (2003). Isolation and characterisation of the homogalacturonan from type II cell walls of the commelinoid monocot wheat using HF-solvolysis. Carbohydrate Res 338(5):423-431.

简介

该程序的目的是从植物细胞壁提取果胶。 果胶是含有聚合糖的半乳糖醛酸,其是植物细胞壁的重要组分。 旨在研究植物细胞壁组分的各种方法需要提取果胶。 果胶以高度酯化的方式在高尔基体中合成,并在细胞壁中脱酯(Mohnen,2008)。 果胶通常是水溶性的。 脱酯的果胶在Ca 2+ 2+离子存在下可形成所谓的"蛋盒结构"(Mohnen,2008; Harholt等人,2010)。 这些"蛋盒结构"中的果胶是交联的并且溶解性较差。 环己烷二胺四乙酸(CDTA)螯合Ca 2+ 2+离子,因此允许从细胞壁提取Ca 2+ 2+交联的果胶。

材料和试剂

  1. 选择植物材料或细胞壁制剂(见注1)
  2. 环己烷二胺四乙酸(CDTA)(Fluka,目录号:34588)
  3. Tris-碱(Sigma-Aldrich,目录号:T6066)
  4. 氢氧化钠(Thermo Fisher Scientific,目录号:S318)
  5. 液氮
  6. CDTA提取缓冲液(参见配方)

设备

  1. 液氮容器
  2. 油漆振动器(例如Harbil,型号:5G-HD)和滚珠轴承(例如直径3mm的钢珠)(灰浆和杵或珠磨机)
  3. 媒体瓶
  4. 单通道移液器
  5. 适于在95℃下使用和用于在液体N 2中冷冻的Microfuge管(Thermo Fisher Scientific,目录号:02-707-355)
    注意:我们使用2毫升螺帽管。
  6. 移液器提示
  7. 水浴或培养箱
  8. 涡流搅拌器
  9. 能够以10,000 x g 旋转的微量离心机
  10. 可选:螺旋盖管或盖锁

程序

  1. 预热水浴或培养箱至95°C和预热CDTA提取缓冲液
  2. 收集植物组织在管 - 我们使用叶材料从约。 4周龄拟南芥植物。 三个完全扩展的拟南芥叶重约250mg
  3. 在液氮中冷冻含有组织的管(组织可以储存在-80℃)
  4. 使用球轴承和油漆搅拌器,研钵和杵或珠磨机粉碎冷冻的组织。 用于粉碎组织的方法不是关键的。 保持组织冷冻。 如果使用油漆搅拌器,三次循环三分钟,通常足以粉碎组织
  5. 向250mg冷冻粉碎的拟南芥组织中加入1ml CDTA提取缓冲液。
  6. 将管置于95℃。 使用注意,液体会变得很热,盖子可能打开; 因此使用螺旋盖管或盖锁来固定盖子
  7. 孵育15分钟,每5分钟涡旋一次。
  8. 在室温下以10,000×g离心10分钟
  9. 上清液含有果胶
  10. 上清液可以冷冻和冷冻干燥用于下游应用

笔记

  1. 我们直接使用粉碎的拟南芥叶片材料(Bethke ,,2014)。或者粗制细胞壁制剂,如醇不溶性残留物(Gille等人,2009)制备物等。可以使用(预热水浴和提取缓冲液,然后进行步骤5)
  2. 不同的组已报道了不同的提取时间和温度,例如在95℃(Siedlecka等人,2008)下15分钟或在室温下4小时(Moller等人al。,2008)。根据我们的经验,当在95℃下进行提取15分钟时,与在室温下更长的提取相比,可以提取两倍的果胶量。
  3. 不同的组利用类似的方案从各种单子叶和双子叶植物中提取果胶, 黄瓜,番茄,芹菜(Jarvis等人,1982),杨树(Siedlecka等人,2008),大豆(Huisman等人。,2001)或小麦(Wiethölter等人,2003)。
  4. 提取缓冲液可以在室温下储存数周
  5. 使用该程序提取的果胶可以用于各种下游应用,包括用果胶特异性抗体的点印迹分析,糖分析,离子交换层析或测定果胶的酯化程度(Bethke等人, 2014; MacDougall等人,1997)。

食谱

  1. CDTA提取缓冲区
    50mM CDTA
    50mM Tris-碱
    使用氢氧化钠将pH调节至7.2

致谢

这项工作由美国能源部基础能源科学办公室的化学科学,地球科学和生物科学司通过授予DE-FG02-05ER15670的J.G.
许多实验室在过去使用类似的协议。我们已经从Siedlecka等人(2008)修改了该协议。

参考文献

  1. Bethke,G.,Grundman,R.E.,Sreekanta,S.,Truman,W.,Katagiri,F.and Glazebrook,J。(2014)。 拟南芥 PECTIN METHYLESTERASE有助于针对丁香假单胞菌的免疫植物生理学 164(2):1093-1107。
  2. Gille,S.,Hansel,U.,Ziemann,M。和Pauly,M。(2009)。 通过使用水解酶的正向化学遗传方法鉴定植物细胞壁突变体。 Proc Natl Acad Sci USA 106(34):14699-14704。
  3. Harholt,J.,Suttangkakul,A。和Vibe Scheller,H。(2010)。 果胶的生物合成 植物生理学 153(2) :384-395。
  4. Huisman,M.M.H.,Fransen,C.T.M.,Kamerling,J.P.,Vliegenthart,J.F.G.,Schols,H.A.and Voragen,A.G.J。(2001)。 钙结合果胶在植物细胞壁中的比例 /em> 154(4):344-346。
  5. MacDougall,A.J.,Rigby,N.M.,Ring,S.G。(1997)。 植物细胞壁多糖的相分离及其对细胞壁组装的影响 Plant Physiol 114(1):353-362。
  6. Mohnen,D。(2008)。 果胶结构和生物合成 Curr Opin Plant Biol 11 (3):266-277。
  7. Moller,I.,Marcus,S.E.,Haeger,A.,Verhertbruggen,Y.,Verhoef,R.,Schols,H.,Ulvskov,P.,Mikkelsen,J.D.,Knox,J.P.and Willats, 通过其碳水化合物微阵列结合谱的分层聚类,高通量筛选针对植物细胞壁聚糖的单克隆抗体。 Glycoconj J 25(1):37-48。
  8. Siedlecka,A.,Wiklund,S.,Peronne,MA,Micheli,F.,Lesniewska,J.,Sethson,I.,Edlund,U.,Richard,L.,Sundberg,B.and Mellerowicz,EJ(2008) 。 果胶甲酯酶抑制毛白杨木材细胞中的侵入性和对称性细胞生长 >。 Plant Physiol 146(2):554-565。
  9. Wiethölter,N.,Graeβner,B.,Mierau,M.,Willats,W.G.T.,Knox,J.P.,Moerschbacher,B.M。(2003)。 使用HF溶剂分解从类胡萝卜素单子叶植物小麦的II型细胞壁分离和表征同聚半乳糖醛酸/a>。 Carbohydrate Res 338(5):423-431。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Bethke, G. and Glazebrook, J. (2014). Cyclohexane Diamine Tetraacetic Acid (CDTA) Extraction of Plant Cell Wall Pectin. Bio-protocol 4(24): e1357. DOI: 10.21769/BioProtoc.1357.
  2. Bethke, G., Grundman, R. E., Sreekanta, S., Truman, W., Katagiri, F. and Glazebrook, J. (2014). Arabidopsis PECTIN METHYLESTERASEs contribute to immunity against Pseudomonas syringae. Plant Physiol 164(2): 1093-1107.
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