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Endogenous ABA Extraction and Measurement from Arabidopsis Leaves
拟南芥叶片中内源ABA的提取和测定   

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Abstract

The endogenous messenger, the phytohormone abscisic acid (ABA) plays a major role in plant’s adaption to drought, salinity, cold and other abiotic stresses. In addition to abiotic stress signaling, ABA is involved also in developmental regulation and in responses to diverse biotic stresses. Dehydration stress results in a strong increase in endogenous ABA levels, which can be perceived by RCAR/PYR1/PYL receptors, initiating the ABA signaling pathway to coordinate the genome-wide gene expression, and plants adaptive physiological responses. ABA biosynthesis triggered by environmental cues as well as developmental signals occurs predominantly in vascular parenchyma cells. The measurement of ABA content in different organ/tissues is required to understandings how ABA is produced and delivered within the plants upon various stress conditions and to elucidate its regulatory role in both physiological and transcriptional responses.
Quantitation of ABA can be achieved by two approaches: 1) the use of gas chromatography–tandem mass spectrometry (GC-MS) and 2) the use of immunoassays. Both methods are sensitive to trace amount of ABA down to the low pico-gram (10-12 g/ml FW) range. The GC-MS method needs special facilities, however the antibody based method is relatively simple and can be carried out in laboratory. Here we describe an easy method for ABA extraction from Arabidopsis seedlings, and further determination of ABA levels by competitive ELISA kit.

Materials and Reagents

  1. 3-week old Arabidopsis thaliana (A. thaliana) plants.
  2. Methanol (Thermo Fisher Scientific, Acros Organics, catalog number: AC124790010 )
  3. Sterile deionized H2O
  4. Sodium diethyldithiocarbamate trihydrate (Sigma-Aldrich, catalog number: D3506 )
  5. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  6. Magnesium chloride (Sigma-Aldrich, catalog number: M8266 )
  7. Sodium chloride (Sigma-Aldrich, catalog number: S3104 )
  8. Phytodtek ELISA kit (Agdia, catalog number: PDK 09347 )
  9. Extraction buffer (see Recipes)
  10. Methanolic Tris buffer (see Recipes)

Equipment

  1. Microcentrifuge (Eppendorf, model: 5415R )
  2. Tweezers or forceps
  3. Mortar and pestle
  4. Siliconized borosilicate tube (VWR International, catalog number: EPCTS-13100 )
  5. Liquid nitrogen
  6. pH meter (Corning, catalog number: 443i )
  7. Refrigerator 4 °C
  8. Refrigerated CentriVap Benchtop Vacuum Concentrator (Labconco, catalog: 7310020 )
  9. Vertical light path photometer for microtiter plate (Dynex Semiconductor, MRX revelation microplate reader)

Procedure

  1. Arabidopsis seedlings were grown in potting soil under regular growth conditions (at 22 °C with a 12-h light photoperiod and light intensity of 180 μmol/m2/s) until rosettes were fully expanded (~4 cm diameter).
  2. Collect ~15 rosette leaves (~200 mg) from ~3 week old Arabidopsis seedlings with sharp tweezers, and measure the fresh weight (FW).
  3. Freeze leaf tissues with liquid nitrogen immediately.
  4. Carefully grind them in a mortar and pestle to get the fine powder.
  5. Add 500 µl extraction buffer to mix them thoroughly by gently pipetting.
  6. Transfer extracts to a covered, siliconized borosilicate tube.
  7. Incubate the extracts overnight in darkness at 4 °C.
  8. Centrifuge at 8, 000 x g for 10 min at 4 °C.
  9. Transfer supernatant to pre-cold new 1.5 ml Eppendorf tube.
  10. Vacuum centrifuge at 4 °C to evaporate the supernatant.
  11. Dissolve the residue with 500 µl methanolic Tris buffer (if necessary, gently pipette up and down a few times).
  12. Measure the ABA content with Phytodtek ELISA kit according the manufacturer’s instructions.

Notes

  1. In our procedure, the extractions tube should always be kept on ice during steps 5-11, and a lightproof cover is recommended to prevent extracts from degradation.
  2. In addition to Arabidopsis leaf, other tissues like root, flower, seed, etc., can be used for extraction. Depending on the type of tissues, sufficient material should be collected until ~200 mg of fresh weight.

Recipes

  1. Extraction buffer
    90% (v/v) methanol
    200 mg/L sodium diethyldithiocarbamate trihydrate
  2. Methanolic tris buffer
    10% methanol
    50 mM Tris (pH 8.0)
    1 mM MgCl2
    150 mM NaCl

Acknowledgments

This work was supported by NSF award MCB-1121898 to Z.A. and M.F. The authors thank Dr. Laetitia Virlouvet (University of Nebraska-Lincoln) for technical assistance and valuable discussions.

References

  1. Ding, Y., Avramova, Z. and Fromm, M. (2011). The Arabidopsis trithorax-like factor ATX1 functions in dehydration stress responses via ABA-dependent and ABA-independent pathways. Plant J 66 (5): 735-744.

简介

内源性信使,植物激素脱落酸(ABA)在植物适应干旱,盐分,寒冷和其他非生物胁迫中发挥重要作用。除了非生物胁迫信号转导,ABA也参与发育调节和对不同生物胁迫的反应。脱水胁迫导致内源性ABA水平的强烈增加,其可以被RCAR/PYR1/PYL受体感知,启动ABA信号传导途径以协调全基因组基因表达,并植物适应性生理反应。由环境信号以及发育信号触发的ABA生物合成主要发生在血管实质细胞中。需要测量不同器官/组织中的ABA含量以理解ABA在各种胁迫条件下如何在植物内产生和递送,并阐明其在生理学和转录反应中的调节作用。
可以实现ABA的定量通过两种方法:1)使用气相色谱 - 串联质谱(GC-MS)和2)使用免疫测定。这两种方法对痕量的ABA至低的皮克(10-12g/ml FW)范围敏感。 GC-MS方法需要特殊的设备,然而基于抗体的方法相对简单并且可以在实验室中进行。在这里我们介绍一种简单的ABA提取拟南芥幼苗的方法,并通过竞争性ELISA试剂盒进一步确定ABA水平。

材料和试剂

  1. 3周龄拟南芥植物( thaliana)。
  2. 甲醇(Thermo Fisher Scientific,Acros Organics,目录号:AC124790010)
  3. 无菌去离子H 2 O 2 /
  4. 二乙基二硫代氨基甲酸钠三水合物(Sigma-Aldrich,目录号:D3506)
  5. (Sigma-Aldrich,目录号:T1503)
  6. 氯化镁(Sigma-Aldrich,目录号:M8266)
  7. 氯化钠(Sigma-Aldrich,目录号:S3104)
  8. Phytodtek ELISA试剂盒(Agdia,目录号:PDK09347)
  9. 提取缓冲液(参见配方)
  10. 甲醇Tris缓冲液(见配方)

设备

  1. 微量离心机(Eppendorf,型号:5415R)
  2. 镊子或镊子
  3. 砂浆和杵
  4. 硅化硼硅管(VWR International,目录号:EPCTS-13100)
  5. 液氮
  6. pH计(Corning,目录号:443i)
  7. 冰箱4°C
  8. 冷冻CentriVap台式真空浓缩机(Labconco,目录:7310020)
  9. 微量滴定板(Dynex半导体,MRX显示酶标仪)的垂直光路光度计

程序

  1. 在正常生长条件下(在22℃,12小时光光周期和180μmol/m 2/s的光强度)在盆栽土壤中生长拟南芥幼苗,直到 玫瑰花完全膨胀(〜4cm直径)
  2. 使用尖锐的镊子从〜3周龄的拟南芥幼苗中收集〜15个莲座叶(〜200mg),并测量鲜重(FW)。
  3. 立即用液氮冷冻叶组织。
  4. 用研钵和杵仔细研磨,得到细粉
  5. 加入500μl提取缓冲液,通过轻轻吹打彻底混合
  6. 将提取液转移到有盖的硅化硼硅管上
  7. 将提取物在4℃下在黑暗中孵育过夜
  8. 在4℃下以8,000×g离心10分钟。
  9. 将上清液转移至预冷的新1.5ml Eppendorf管中
  10. 在4℃下真空离心以蒸发上清液
  11. 用500μl甲醇Tris缓冲液溶解残留物(如果需要,轻轻地上下吸移数次)。
  12. 使用Phytodtek ELISA试剂盒根据制造商的说明测量ABA含量

笔记

  1. 在我们的程序中,提取管在第5-11步骤期间应始终保持在冰上,建议使用防光罩以防止提取物降解。
  2. 除了拟南芥叶之外,其他组织如根,花,种子等可以用于提取。 根据组织的类型,应收集足够的材料,直到〜200毫克的鲜重

食谱

  1. 提取缓冲区
    90%(v/v)甲醇 200mg/L二乙基二硫代氨基甲酸钠三水合物
  2. 甲醇tris缓冲液
    10%甲醇
    50mM Tris(pH8.0) 1mM MgCl 2
    150mM NaCl

致谢

这项工作是由NSF奖MCB-1121898支持Z.A. 和M.F. 作者感谢Laetitia Virlouvet博士(内布拉斯加大学林肯分校)提供技术帮助和宝贵的讨论。

参考文献

  1. Ding,Y.,Avramova,Z.和Fromm,M。(2011)。 拟南芥 trithorax-like因子ATX1通过ABA在脱水应激反应中起作用 - 依赖性和ABA非依赖性途径。植物杂志66(5):735-744。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liu, N., Ding, Y., Fromm, M. and Avramova, Z. (2014). Endogenous ABA Extraction and Measurement from Arabidopsis Leaves. Bio-protocol 4(19): e1257. DOI: 10.21769/BioProtoc.1257.
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