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Detection of Mitogen-activated Protein Kinase (MAPK) Activation upon Exogenous Chemical Application in Arabidopsis Protoplasts
外源化学物质处理后拟南芥原生质体中促分裂原活化蛋白激酶(MAPK)的检测   

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Abstract

The mitogen activated protein kinase cascade is a highly conserved signal pathway in plants. The exogenous chemicals, like hormones, can trigger a series of signalling cascades, including MAPK pathway, to modulate the plant physiology. Upon activation, some MAPKs are phosphorylated. It is important to develop methods that can detect changes in the phosphorylation status of MAPKs in plants when they come in contact with external chemicals. This method describes the exogenous treatment of Arabidopsis protoplasts with Kinetin and subsequent detection of the activated MAPKs. This method is useful for studying the effect of exogenously applied chemical compounds on the MAPK signaling cascade in Arabidopsis.

Keywords: MAP kinase(MAP激酶), Phosphorylation(磷酸化), In vitro kinase assay(体外激酶测定), Signal transduction(信号转导), Immunoprecipitation(免疫共沉淀)

Materials and Reagents

  1. Plants: Arabidopsis thaliana (Col-0) obtained from NASC The European Arabidopsis Stock Centre
  2. Sodium chloride (NaCl) (Carl Roth, catalog number: 3957 )
  3. MgCl2 (Thermo Fisher Scientific, catalog number: M35 )
  4. CaCl2 (Thermo Fisher Scientific, catalog number: C69 )
  5. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  6. D-Mannitol (Sigma-Aldrich, catalog number: M4125 )
  7. MES hydrate (Sigma-Aldrich, catalog number: 8250 )
  8. Albumin bovine modified Cohn Fraction V, pH 7.0 (BSA) (SERVA Electrophoresis GmbH, catalog number: 11943 )
  9. Macerozyme ONOZUKA R10 (Yakult Pharmaceutical Industry Co)
  10. Cellulase ONOZUKA R10 (Yakult Pharmaceutical Industry Co)
  11. Kinetin (Sigma-Aldrich, catalog number: K3378 )
  12. Phospho-p44/42 MAPK (ERK1/2) Antibody (pTEpY Antibody) (Cell Signaling Technology, catalog number: 9101 )
  13. PierceTM ECL Western Blotting Substrate (Thermo Fisher Scientific, catalog number: 32109 )
  14. Protein free blocking solution (PFBS) (Thermo Fisher Scientific)
  15. 0.45 µm acetate filter (Carl Roth, catalog number: AH53.1 )
  16. Petri Dishes (Greiner Bio-One GmbH, catalog number: 628103 )
  17. Nitrocellulose Hybond ECL membrane (GE Healthcare, Amersham biosciences®, model: RPN82D )
  18. Stock solutions (see Recipes)
  19. Enzyme solution (see Recipes)
  20. W5 solution (see Recipes)
  21. WI solution (see Recipes)
  22. 0.5 mM mannitol (see Recipes)
  23. Kinetin (see Recipes)
  24. 1 L transfer buffer (see Recipes)
  25. 1 LTBST (Tris-Buffered Saline and Tween 20) (see Recipes)
  26. 5x SDS-Loading buffer (see Recipes)
  27. 10% SDS-PAGE gel (see Recipes)

Equipment

  1. Phytochambers or light room or greenhouse (23 °C, 16 h dark / 8 h light)
  2. Sharp razor blades (Carl Roth, catalog number: CK07.1 )
  3. Polycarbonate dessicator (Carl Roth, catalog number: PK30.1 )
  4. Nylon Net Filter, Hydrophilic (100 µm, 25 mm) (EMD Millipore, catalog number: NY1H02500 )
  5. Round bottom 12 ml cell culture polystyrene tubes (Greiner Bio-One GmbH, catalog number: 163160 )
  6. Swing bucket refrigerated Centrifuge (Eppendorf, model: 6810R )
  7. Haemocytometer
  8. SDS-PAGE gel running apparatus (Bio-Rad Laboratories)
  9. Semi-dry blotting apparatus (Bio-Rad Laboratories)

Procedure

  1. Arabidopsis protoplast isolation and chemical treatment
    1. Choose five week old and well-expanded leaves for protoplast isolation. Use a fresh sharp razor blade to cut <0.5 mm leaf strips and transfer them into the freshly prepared filtered enzyme solution (see Recipes for enzyme solution preparation) (around 20 leaves in 10 ml). Dip them completely into the solution by using a pair of flat-tip forceps. 20 leaves should yield 8-10 ml protoplast suspension for the assay (2 x 105 protoplast/ml). The general outline of protoplast isolation technique is shown in Figure 2.
    2. Vacuum-infiltrate leaf strips for 30 min in the dark using a desiccator (covered with a black cloth to prevent any possible effect of light on protoplast).
    3. Continue the digestion, without shaking, in the dark for at least 2.5 h at 20-22 °C.
    4. Gently shake the enzyme solution for 30 min on a lab shaker at 70-100 rpm at room temperature to release the protoplasts (solution should turn green and at least half of the leaf strips should become transparent). Add 10 ml W5 solution per 10 ml enzyme solution.
    5. Carefully filter suspension through a nylon mesh (100 µm) into 12 ml cell culture tubes on ice.
    6. Centrifuge the protoplast suspension for 1 min at 200 x g (4 °C) and remove as much supernatant as possible using a narrow bottom 5 ml tip.
    7. Add 2 ml of W5 solution to the tubes and resuspend the protoplast by gently inverting the tubes.
    8. Invert tube and immediately take 8 µl to determine protoplast concentration with haemocytometer. Dilute the protoplast with W5 solution to the working concentration of 2 x 105 protoplast/ml.
    9. Leave protoplasts on ice for 40 min in the dark. Protoplasts settle on the bottom of the tube by gravity.
    10. Remove supernatant from protoplast pellet and wash again with 2 ml W5 solution. Leave on ice for another 40 min (in the dark).
    11. Treat 300 µl protoplasts with an equal volume of kinetin stocks to get the required final concentration of 1 to 25 µM used in this experiment. This step can be performed in 1.5 or 2 ml microcentrifuge tubes as well.
    12. Mix well and incubate overnight at 22 °C in the dark.
    13. Pellet protoplasts by short pulse centrifuging and remove as much supernatant as possible. Immediately freeze samples in liquid nitrogen.
    14. Add 12 µl SDS-loading buffer to frozen protoplast pellets, thaw while vortexing vigorously.
    15. Boil for 5 min at 95 °C and run on freshly made 10% SDS-PAGE.

  2. Immunoblotting with anti p44/42- ERK (extracellular signal-regulated kinase) antibody
    1. Transfer the proteins from the gel to nitrocellulose membrane using semi-dry blotting apparatus at 25 V, 130 mA for 1 h at room temperature.
    2. Block membrane with protein free blocking solution (PFBS) for 1 h at room temperature.
    3. Incubate p42/44 ERK antibody (1:1,000 dilution) in PFBS overnight at 4 °C.
    4. Wash the membrane 4 times in 0.1x TBST buffer for 5 min each at room temperature.
    5. Add Pierce Goat anti-rabbit HRP-conjugate antibody (1:5,000 dilution) in PFBS and incubate for 1 h at room temperature.
    6. Wash the membrane 4 times with 0.1x TBST for 5 min each.
    7. Develop the membrane using the normal ECL kit as per manufacturer’s instructions.

Representative data

Representative data is shown in Figure 1.


Figure 1. Western blot detection of MPK3 and MPK6 phosphorylation in Arabidopsis protoplasts after kinetin treatment. Arabidopsis mesophyll protoplasts were isolated and treated overnight with the indicated concentrations of kinetin. Protoplasts were harvested and isolated proteins were analyzed by Western blotting with the anti-p42/44 ERK antibody. The activation of the MAP kinases was observed when kinetin is applied at the lower concentrations. The bottom panel shows the amido black staining of RuBisCo (~55 KD) performed after the Western of the same blot as loading control. Source: Sheikh et al. (2014)


Figure 2. General graphical overview of the protoplast isolation. The figure highlights the carefully cut leaves in the enzyme solution and the use of cut tips for handling the protoplast to minimize the damage.

Notes

  1. Preheat enzyme solution at 55 °C for better dissolution of cellulose and cell wall, then cool on ice and add CaCl2 before digestion.
  2. Always cut tip ends when pipetting protoplasts (see Figure 2).

Recipes

  1. Stock solutions (stored at RT)
    5 M NaCl
    1 M CaCl2
    0.1 M KCl
    0.8 M mannitol
    0.2 M MES pH 5.7
  2. Enzyme solution (freshly prepared)
    20 mM MES (pH 5.7)
    0.4 M mannitol
    20 mM KCl
    0.4% (w/v) Macerozyme R10
    1.5% (w/v) Cellulase R10
    10 mM CaCl2
    0.1% (w/v) BSA
  3. W5 solution (can be stored at -20 °C)
    2 mM MES (pH 5.7)
    154 mM NaCl
    125 mM CaCl2
    5 mM KCl
  4. WI solution (freshly prepared)
    4 mM MES (pH 5.7)
  5. 0.5 mM mannitol
    20 mM KCl
  6. Kinetin
    1 to 25 µM Kinetin solution made in 0.1 N NaOH
  7. 1 L transfer buffer
    Tris base 5.8 g
    Glycine 2.9 g
    SDS 0.37 g
    Methanol 200 ml
  8. 1 LTBST
    Dissolve the following in 1,000 ml of distilled H2O and adjust pH to 7.4
    8.8 g of NaCl
    0.2 g of KCl
    3 g of Tris base
    Add 500 µl of Tween-20
  9. 5x SDS-Loading buffer
    10% w/v SDS
    20% v/v glycerol
    200 mM Tris-HCl (pH 6.8)
    0.05% Bromophenol blue
    10 mM DTT (add at last)
  10. 10% SDS-PAGE gel (for BioRad Mini Protean system)
    For 2 gels: (15 ml of resolving buffer and 8 ml of stacking buffer)
    Resloving 15 ml
    10%
    Stacking 8 ml
    5%
    1.5 M Tris (pH 8.8)
    3.4 ml
    1.5 M Tris (pH 6.8)
    2 ml
    30% acrylamide
    5 ml
    30% acrylamide
    1.3 ml
    Water
    6.3 ml
    Water
    4.7 ml
    20% SDS
    150 µl
    20% SDS
    80 µl
    10% APS
    150 µl
    10% APS
    80 µl
    TEMED
    8 µl
    TEMED
    4 µl

Acknowledgments

Authors thank Prof. Dr. Sabine Rosahl at Leibniz IPB, Halle Germany for providing Kinetin solution and Dr. Lennart Eschen Lippold for kind help with Protoplasts. Authors thank Dhammaprakash Wankhede and Nicole Bauer for technical help. Financial support from Department of Biotechnology, Government of India is acknowledged.

References

  1. Sheikh, A. H., Raghuram, B., Eschen-Lippold, L., Scheel, D., Lee, J. and Sinha, A. K. (2014). Agroinfiltration by cytokinin-producing Agrobacterium sp. strain GV3101 primes defense responses in Nicotiana tabacum. Mol Plant Microbe Interact 27(11): 1175-1185.
  2. Yoo, S. D., Cho, Y. H. and Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2(7): 1565-1572.

简介

丝裂原活化蛋白激酶级联是植物中高度保守的信号通路。 外源化学物质,如激素,可以触发一系列信号级联,包括MAPK途径,以调节植物生理。 在激活时,一些MAPK被磷酸化。 开发能够检测植物中MAPK在与外部化学品接触时磷酸化状态的变化的方法是重要的。 该方法描述了用激动素对随后检测激活的MAPK的拟南芥原生质体的外源处理。 该方法可用于研究外源施加的化合物对拟南芥中MAPK信号级联的影响。

关键字:MAP激酶, 磷酸化, 体外激酶测定, 信号转导, 免疫共沉淀

材料和试剂

  1. 植物:拟南芥(Col-0)从NASC获得欧洲拟南芥库存中心
  2. 氯化钠(NaCl)(Carl Roth,目录号:3957)
  3. MgCl 2(Thermo Fisher Scientific,目录号:M35)
  4. CaCl 2(Thermo Fisher Scientific,目录号:C69)
  5. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  6. D-甘露醇(Sigma-Aldrich,目录号:M4125)
  7. MES水合物(Sigma-Aldrich,目录号:8250)
  8. 白蛋白牛改良的Cohn组分V,pH 7.0(BSA)(SERVA Electrophoresis GmbH,目录号:11943)
  9. Macerozyme ONOZUKA R10(Yakult Pharmaceutical Industry Co)
  10. 纤维素酶ONOZUKA R10(Yakult Pharmaceutical Industry Co.)
  11. 激动素(Sigma-Aldrich,目录号:K3378)
  12. Phospho-p44/42MAPK(ERK1/2)抗体(pTEpY抗体)(Cell Signaling Technology,目录号:9101)
  13. Pierce TM ECL Western Blotting Substrate(Thermo Fisher Scientific,目录号:32109)
  14. 无蛋白封闭溶液(PFBS)(Thermo Fisher Scientific)
  15. 0.45μm乙酸盐过滤器(Carl Roth,目录号:AH53.1)
  16. 培养皿(Greiner Bio-One GmbH,目录号:628103)
  17. 硝化纤维素Hybond ECL膜(GE Healthcare,Amersham biosciences ,型号:RPN82D)
  18. 库存解决方案(参见配方)
  19. 酶溶液(见配方)
  20. W5解决方案(参见配方)
  21. WI解决方案(参见配方)
  22. 0.5mM甘露醇(参见配方)
  23. Kinetin(见食谱)
  24. 1 L转移缓冲区(参见配方)
  25. 1 LTBST(Tris-缓冲盐水和Tween 20)(参见配方)
  26. 5x SDS加载缓冲液(参见配方)
  27. 10%SDS-PAGE凝胶(见配方)

设备

  1. 植物室或光室或温室(23℃,16小时黑暗/8小时光照)
  2. 锋利的剃刀刀片(Carl Roth,目录号:CK07.1)
  3. 聚碳酸酯干燥剂(Carl Roth,目录号:PK30.1)
  4. 尼龙网过滤器,亲水性(100μm,25mm)(EMD Millipore,目录号:NY1H02500)
  5. 圆底12ml细胞培养聚苯乙烯管(Greiner Bio-One GmbH,目录号:163160)
  6. 回转桶冷冻离心机(Eppendorf,型号:6810R)
  7. 血细胞计数器
  8. SDS-PAGE凝胶电泳装置(Bio-Rad Laboratories)
  9. 半干印迹装置(Bio-Rad Laboratories)

程序

  1. 拟南芥原生质体分离和化学处理
    1. 选择五周老和良好扩张叶为原生质体分离。 使用新鲜的锋利的剃刀刀片切割<0.5毫米叶条和转移   它们进入新鲜制备的过滤的酶溶液中(参见Recipes 用于酶溶液制备)(在10ml中约20片叶)。 蘸他们 通过使用一对平头钳子完全进入溶液。 20 叶应该产生8-10ml用于测定的原生质体悬浮液(2×10 5原生质体/ml)。 原生质体分离技术的一般概述 如图2所示。
    2. 真空渗透叶条30分钟 在黑暗中使用干燥器(用黑布覆盖防止 光对原生质体的任何可能的影响)
    3. 继续消化,不摇动,在黑暗中至少2.5小时,在20-22℃
    4. 在实验室摇床上轻轻摇动酶溶液30分钟 70-100rpm,以释放原生质体(溶液 应变绿,至少一半的叶条应变成 透明)。 每10ml酶溶液加入10ml W5溶液。
    5. 通过尼龙网(100μm)将悬浮液小心地过滤到在冰上的12ml细胞培养管中
    6. 在200×g(4℃)和4℃离心原生质体悬浮液1分钟   使用窄的底部5毫升吸头尽可能多地除去上清液
    7. 加入2毫升的W5溶液到管中,通过轻轻倒转管重悬原生质体
    8. 倒置管,立即取8μl,以确定原生质体 用血球计浓度。 用W5稀释原生质体 溶液至2×10 5原生质体/ml的工作浓度
    9. 离开原生质体在冰上40分钟在黑暗中。 原生质体通过重力沉降在管的底部。
    10. 从原生质体沉淀去除上清液,并再次用2毫升 W5溶液。 在冰上再放置40分钟(在黑暗中)。
    11. 治疗300微升原生质体与等体积的激动素股票得到 在本实验中使用的所需最终浓度为1至25μM。 该步骤也可以在1.5或2ml微量离心管中进行。
    12. 充分混合,在暗处22℃下孵育过夜
    13. 粒子原生质体通过短脉冲离心并除去尽可能多的 上清液。 立即将样品冷冻在液氮中。
    14. 加入12μlSDS加载缓冲液到冷冻的原生质体沉淀,解冻,同时剧烈涡旋
    15. 在95℃下煮沸5分钟,并在新鲜制备的10%SDS-PAGE上运行

  2. 用抗p44/42-ERK(细胞外信号调节激酶)抗体进行免疫印迹
    1. 使用转移蛋白质从凝胶到硝酸纤维素膜 半干印迹装置,在25V,130mA,室温下1小时
    2. 在室温下用无蛋白封闭溶液(PFBS)封闭膜1小时
    3. 在4℃下,在PFBS中孵育p42/44 ERK抗体(1:1000稀释)过夜
    4. 在室温下,用0.1x TBST缓冲液洗涤膜4次,每次5分钟
    5. 在PFBS中加入Pierce山羊抗兔HRP-缀合物抗体(1:5000稀释),并在室温下孵育1小时。
    6. 用0.1×TBST洗涤膜4次,每次5分钟
    7. 根据制造商的说明使用正常ECL试剂盒开发膜。

代表数据

代表数据如图1所示。


图1.在激动素处理后在拟南芥原生质体中的MPK3和MPK6磷酸化的Western印迹检测。分离拟南芥叶肉原生质体,并用所示的 激动素的浓度。 收获原生质体,并通过用抗p42/44ERK抗体的Western印迹分析分离的蛋白质。 当激动素应用于时,观察到MAP激酶的活化 较低浓度。 底部图显示在与加样对照相同的印迹的Western后进行的RuBisCo(〜55KD)的酰胺黑染色。 资料来源:Sheikh (2014)


图2.原生质体分离的一般图形概述该图突出显示了酶溶液中仔细切割的叶子和使用切割尖端处理原生质体以最小化损伤。

笔记

  1. 在55℃预热酶溶液以更好地溶解纤维素和细胞壁,然后在冰上冷却并在消化前加入CaCl 2。
  2. 在吸移原生质体时,始终切割尖端(见图2)

食谱

  1. 库存解决方案(在RT存储)
    5 M NaCl
    1 M CaCl 2
    0.1 M KCl
    0.8 M甘露醇 0.2 M MES pH 5.7
  2. 酶溶液(新鲜制备)
    20mM MES(pH 5.7)
    0.4M甘露醇 20 mM KCl
    0.4%(w/v)Macerozyme R10
    1.5%(w/v)纤维素酶R10
    10mM CaCl 2
    0.1%(w/v)BSA
  3. W5溶液(可储存于-20°C) 2mM MES(pH 5.7)
    154 mM NaCl 125mM CaCl 2。 5 mM KCl
  4. WI解决方案(新鲜制备)
    4mM MES(pH 5.7)
  5. 0.5mM甘露醇 20 mM KCl
  6. Kinetin
    1至25μM在0.1N NaOH中制备的蛋白酶溶液
  7. 1 L转移缓冲器
    Tris碱5.8g
    甘氨酸2.9克
    SDS 0.37g
    甲醇200ml
  8. 1 LTBST
    将以下物质溶解在1,000ml蒸馏H 2 O中,并将pH调节至7.4 8.8g NaCl
    0.2克KCl
    3克Tris碱
    加入500μlTween-20
  9. 5x SDS装载缓冲区
    10%w/v SDS
    20%v/v甘油 200mM Tris-HCl(pH6.8)
    0.05%溴酚蓝
    10 mM DTT(最后添加)
  10. 10%SDS-PAGE凝胶(对于BioRad Mini Protean系统) 对于2个凝胶:(15ml分辨缓冲液和8ml堆积缓冲液)
    重新洗15ml
    10%
    堆叠8毫升
    5%
    1.5 M Tris(pH 8.8)
    3.4 ml
    1.5 M Tris(pH 6.8)
    2 ml
    30%丙烯酰胺 5 ml
    30%丙烯酰胺 1.3 ml

    6.3 ml

    4.7 ml
    20%SDS
    150μl
    20%SDS
    80μl
    10%APS
    150μl
    10%APS
    80μl
    TEMED
    8微升
    TEMED
    4微升

致谢

作者感谢Sabine Rosahl教授在Leibniz IPB,德国Halle提供Kinetin解决方案和Dr. Lennart Eschen Lippold帮助Protoplasts。作者感谢Dhammaprakash Wankhede和Nicole Bauer的技术帮助。承认印度政府生物技术部的财政支持。

参考文献

  1. Sheikh,A.H.,Raghuram,B.,Eschen-Lippold,L.,Scheel,D.,Lee,J.and Sinha,A.K。(2014)。 通过细胞分裂素产生农杆菌介导的农杆菌渗透。菌株GV3101在烟草中引发防御反应。 Mol Plant Microbe Interact 27(11):1175-1185。
  2. Yoo,S.D.,Cho,Y.H。和Sheen,J。(2007)。 /em>叶肉原生质体:用于瞬时基因表达分析的多功能细胞系统。 Nat Protoc 2(7):1565-1572。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sheikh, A. H. and Sinha, A. K. (2015). Detection of Mitogen-activated Protein Kinase (MAPK) Activation upon Exogenous Chemical Application in Arabidopsis Protoplasts. Bio-protocol 5(18): e1588. DOI: 10.21769/BioProtoc.1588.
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