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Camalexin Quantification in Arabidopsis thaliana Leaves Infected with Botrytis cinerea
灰葡萄孢菌感染拟南芥叶片后Camalexin的定量测定   

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Abstract

Phytoalexins are heterogeneous low molecular mass secondary metabolites with antimicrobial activity produced at the infection site in response to pathogen invasion and represent an important part of the plant defense repertoire. Camalexin (3-Thiazol-2′-yl-indole) is a known phytoalexin first detected and isolated in Camelina sativa, from which it takes its name, infected with Alternaria brassicae (Browne et al., 1991). Production of camalexin is also induced in Arabidopsis thaliana leaves by a range of biotrophic and necrotrophic plant pathogens (bacteria, oomycetes, fungi and viruses) (Ahuja et al., 2012) as well as by abiotic stresses, such as UV and chemicals (e.g. acifluorfen, paraquat, chlorsulfuron and α-amino butyric acid) (Zhao et al., 1998; Tierens et al., 2002). Camalexin originates from tryptophan and CYP79B2 and CYP71B15 (PAD3) are P450 enzymes that catalyze important steps in its biosynthetic pathway (Glawischnig, 2007).
The detection and quantification of camalexin content is required to understand how it is produced upon various stress conditions. Here we describe an easy method for camalexin extraction from Arabidopsis leaves infected with the necrotrophic fungus Botrytis cinerea, and further determination of camalexin levels by liquid chromatography–mass spectrometry (LC-MS). The method is sensitive enough to trace amount of camalexin down to the low pico-gram (10 pg/mg FW) range.


Figure 1. The structural formula of camalexin

Materials and Reagents

  1. Arabidopsis leaves
  2. Liquid nitrogen
  3. Dichloromethane (DCM) (Sigma-Aldrich, catalog number: 270997 )
  4. Methanol (LC/MS grade) (Carlo Erba Reagents, catalog number: 414831 )
  5. Formic acid (gradient grade) (Sigma-Aldrich, catalog number: F0507 )
  6. Water with 0.1% formic acid (LC/MS grade) (Sigma-Aldrich, catalog number: 34673 )
  7. Camalexin standard (Sigma-Aldrich, catalog number: SML1016 )
  8. Extraction buffer (see Recipes)

Equipment

  1. Mortar and pestle
  2. Spatula
  3. Glass Pasteur pipettes
  4. Tube adapters
  5. Screw cap round bottom borosilicate glass culture tubes (16 x 125 mm) (Pyrex) (Sigma-Aldrich, catalog number: Z653594-40EA )
  6. Disposable borosilicate glass culture tubes (Pyrex)
  7. Spin-X centrifuge tube filters with nylon membranes (Sigma-Aldrich, catalog number: CLS8170-200EA )
  8. Glass vials with cap (capacity: 100 µl)
  9. Vortex mixer
  10. Rocking shaker
  11. Nitrogen evaporator (e.g. sample concentrator with dry block DB-3D) (Techne) (Figure 1)
  12. Balance
  13. Variable speed refrigerated centrifuge with rotor having number of rotor cavities x nominal largest load (capacity: 8 x 50 ml) (e.g. Beckman Coulter)
  14. HPLC (e.g. Thermo Fisher Scientific, model: UltiMate 3000 HPLC )
  15. High performance reversed-phase columns C18 for the separation of small molecules (e.g. Acclaim120 C18 Reverse-phase column-3 μm, 200 Å, 2.1 x 150 mm) (Thermo Fisher Scientific, catalog number: 059130 )
  16. ESI-Mass Spectrometer (e.g. Orbitrap XL Discovery, Thermo Fisher Scientific)

Software

  1. Analysis software (e.g. Thermo Xcalibur software 2.10, Thermo Fisher Scientific)

Procedure

  1. Preparation of plant material
    Notes:
    1. At least 50-100 mg of 4-week-old Arabidopsis plant leaves are required.
    2. Arabidopsis leaves should be weighed before freezing in liquid nitrogen.
    3. At least 4 biological replicates for each sample should be analyzed.
    1. Prepare and pre-cool the extraction buffer.
    2. Pre-cool centrifuge rotor at 4 °C.
    3. Freeze weighed Arabidopsis leaves in liquid nitrogen.
    4. Grind the leaf tissue into powder by using mortar and pestle.
    5. Transfer each sample into a round bottom screw-capped borosilicate glass tube (Pyrex) by using the spatula.
    6. Keep the sample in ice.

  2. Camalexin extraction
    1. Add extraction buffer in each tube, keeping the ratio 1:10 [tissue (mg): extraction buffer (µl)].
    2. Vortex the samples for 20 sec.
    3. Shake tubes on the rocking shaker (30 min, 100 rpm, 4 °C).
    4. Add DCM in each sample keeping the ratio 1:2 [extraction buffer (µl): DCM (µl)].
    5. Shake tubes on the rocking shaker (30 min, 100 rpm, 4 °C).
    6. Centrifuge (10 min, 5,000 x g, 4 °C).
      Note: After centrifugation two phases will form. Camalexin is contained in the lower green phase while the upper phase contains mostly DCM. Plant debris will be present between the two layers.
    7. Collect the lower phase by using a glass Pasteur pipette and transfer it into disposable borosilicate glass culture tubes (Pyrex).
      Note: Special attention should be paid not to recover any material floating in the interface between the two phases.
    8. Concentrate the collected lower phase under nitrogen gas flow (10 bar) and heating the tubes at 42 °C.
    9. Allow sample to completely dry and then suspend it in methanol with a ratio 1:1 [tissue (mg): methanol (µl)].
    10. Filter samples by using spin-X centrifuge tube (5 min, 10,000 x g, 4 °C).
    11. Transfer the filtered samples into glass-capped vials.

  3. Camalexin detection and quantification
    HPLC coupled to MS
    1. Inject 20 µl per sample in an Ultimate3000 HPLC system equipped with a Acclaim120 C18 Reverse-phase column. The mobile phase consists of water with 0.1% formic acid (LC/MS grade), as solvent A, and methanol with 0.1% formic acid (LC/MS grade), as solvent B. Settings:
      Flow rate: 0.2 ml/min
      Column temperature: 35 °C
      Solvent gradient for chromatographic separation:
      In these conditions camalexin will elute with a retention time of 15 min (see Figure 3).
    2. Electrospray the effluent from the HPLC directly into the mass spectrometer (Orbitrap XL Discovery) operating in full-scan MS with resolution R = 30,000. Collect data in centroid mode at mass range of m/z 120-310. Settings:
      Ionization mode: positive
      Ion spray voltage: 4.5 kV
      Capillary temperature: 250 °C
      Sheath Gas Flow rate 35 (arbitrary units)
      Aux Ga flow rate 25 (arbitrary units)
    3. Determine the abundance of camalexin by using the calibration curve method. Use a concentration of camalexin 800-400-200-100-50-25-12.5-6.25-3.12 ng/ml for the preparation of standard curve solutions (see Figure 4).

Representative data



Figure 2. Sample concentration. Evaporation is increased by passing an inert gas (N2) over the surface of the sample to remove the solvent. The gas travels through the gas chamber to the samples via the needles.

A

B

Figure 3. LC-MS analyisis of camalexin from external standard (upper panels) and from the plant tissue (lower panels). A. Total ion chromatograms (TICs). B. ESI mass spectra in positive ionization mode. The mass of 201.048 corresponds to C11H9N2S, the protonated form of camalexin [M+H]+ (red arrows).


Figure 4. Camalexin standard curve. A linear standard curve was obtained in the concentration range between 3.12 - 800 ng/ml.

Recipes

  1. Extraction buffer
    2-propanol: H2O: HCl 37% (2:1:0.002, vol/vol/vol)

Acknowledgments

This work was supported by the European Research Council (advanced grant no. 233083). Part of the procedures were adapted from a previously described protocol for quantitative analysis of major plant hormones from crude plant extracts (Pan et al., 2010).

References

  1. Ahuja, I., Kissen, R. and Bones, A. M. (2012). Phytoalexins in defense against pathogens. Trends Plant Sci 17(2): 73-90.
  2. Browne, L. M., Conn, K. L., Ayert, W. A. and Tewari, J. P. (1991). The camalexins: New phytoalexins produced in the leaves of camelina sativa (cruciferae). Tetrahedron 47(24): 3909-3914.
  3. Glawischnig, E. (2007). Camalexin. Phytochemistry 68(4): 401-406.
  4. Pan, X., Welti, R. and Wang, X. (2010). Quantitative analysis of major plant hormones in crude plant extracts by high-performance liquid chromatography-mass spectrometry. Nat Protoc 5(6): 986-992.
  5. Tierens, K. F. J., Thomma, B. P., Bari, R. P., Garmier, M., Eggermont, K., Brouwer, M., Penninckx, I. A., Broekaert, W. F. and Cammue, B. (2002). Esa1, an Arabidopsis mutant with enhanced susceptibility to a range of necrotrophic fungal pathogens, shows a distorted induction of defense responses by reactive oxygen generating compounds. Plant J 29(2): 131-140.
  6. Zhao, J., Williams, C. C. and Last, R. L. (1998). Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor. Plant Cell 10(3): 359-370.

简介

植物素是具有抗菌活性的异质低分子量次级代谢物,其在感染部位响应于病原体侵入产生并且代表植物防御所有组成部分的重要部分。 Camalexin(3-噻唑-2'-基 - 吲哚)是已知的植物抗毒素,首先在Camelina sativa中检测和分离,从其命名,感染了链格孢菌 (Browne等人,1991)。 camalexin的产生也通过一系列生物营养和坏死的植物病原体(细菌,卵菌,真菌和病毒)在拟南芥叶中诱导(Ahuja等人,2012)以及非生物胁迫,例如紫外线和化学品(例如氟锁草醚,百草枯,氯磺隆和α-氨基丁酸)(Zhao等人,1998; Tierens等人, et al。,,2002)。 Camalexin源自色氨酸,CYP79B2和CYP71B15(PAD3)是催化其生物合成途径中的重要步骤的P450酶(Glawischnig,2007)。
camalexin含量的检测和定量需要了解它是如何在各种应激条件下产生的。在这里我们介绍camalexin提取从拟南芥叶片感染的坏死性真菌灰霉病,和进一步通过液相色谱 - 质谱(LC-MS)的camalexin水平的测定方法。该方法足够敏感,足以将痕量的camalexin降至低皮克(10pg/mg FW)范围。


图1. camalexin的结构式

材料和试剂

  1. 拟南芥叶子
  2. 液氮
  3. 二氯甲烷(DCM)(Sigma-Aldrich,目录号:270997)
  4. 甲醇(LC/MS级)(Carlo Erba Reagents,目录号:414831)
  5. 甲酸(梯度级)(Sigma-Aldrich,目录号:F0507)
  6. 含有0.1%甲酸的水(LC/MS级)(Sigma-Aldrich,目录号:34673)
  7. Camalexin标准(Sigma-Aldrich,目录号:SML1016)
  8. 提取缓冲液(参见配方)

设备

  1. 砂浆和杵
  2. 小铲
  3. 玻璃巴斯德移液器
  4. 管适配器
  5. 螺旋盖圆底硼硅酸盐玻璃培养管(16×125mm)(Pyrex)(Sigma-Aldrich,目录号:Z653594-40EA)
  6. 一次性硼硅酸盐玻璃培养管(Pyrex)
  7. 具有尼龙膜的Spin-X离心管过滤器(Sigma-Aldrich,目录号:CLS8170-200EA)
  8. 带盖的玻璃小瓶(容量:100μl)
  9. 涡流搅拌器
  10. 摇床
  11. 氮气蒸发器(例如具有干燥块DB-3D的样品浓缩器)(Techne)(图1)
  12. 余额
  13. 变速冷冻离心机,转子数量 转子腔×标称最大负载(容量:8×50ml)(例如,Beckman Coulter)
  14. HPLC(例如Thermo Fisher Scientific,型号:UltiMate 3000HPLC)
  15. 高效反相色谱柱C18用于分离小   分子(例如Acclaim 120C8反相柱-3μm,200,2.1x 150mm)(Thermo Fisher Scientific,目录号:059130)
  16. ESI-质谱仪(例如 Orbitrap XL Discovery,Thermo Fisher Scientific)

软件

  1. 分析软件(例如Thermo Xcalibur软件2.10,Thermo Fisher Scientific)

程序

  1. 植物材料的制备
    注意:
    1. 需要至少50-100mg的4周龄拟南芥植物叶。
    2. 在冷冻于液氮中之前应称重拟南芥叶。
    3. 应分析每个样品至少4个生物学重复。
    1. 准备并预冷提取缓冲液。
    2. 预冷离心机转子在4°C
    3. 冷冻称重的拟南芥叶在液氮中
    4. 使用研钵和杵将叶组织粉碎成粉末
    5. 使用刮刀将每个样品转移到圆底螺旋盖的硼硅酸盐玻璃管(Pyrex)中。
    6. 将样品保存在冰上。

  2. 卡马西林提取
    1. 在每个管中加入提取缓冲液,保持比例1:10 [组织(mg):提取缓冲液(μl)]。
    2. 涡旋样品20秒。
    3. 在振荡器上摇动管(30分钟,100rpm,4℃)
    4. 在保持比例1:2 [提取缓冲液(μl):DCM(μl)]的每个样品中加入DCM。
    5. 在振荡器上摇动管(30分钟,100rpm,4℃)
    6. 离心(10分钟,5,000x g,4℃)。
      注意:离心后将形成两相。 含有Camalexin   在下部绿色相中,而上部相主要含有DCM。 植物碎屑将存在于两层之间。
    7. 收集 下相通过使用玻璃巴斯德吸管并将其转移 一次性硼硅酸盐玻璃培养管(Pyrex) 注意:应特别注意不要恢复在两个阶段之间的界面中浮动的任何材料。
    8. 在氮气流(10巴)下浓缩收集的下层相并在42℃下加热管
    9. 使样品完全干燥,然后将其悬浮在甲醇中,比例为1:1 [组织(mg):甲醇(μl)]。
    10. 通过使用spin-X离心管(5分钟,10,000×g,4℃)过滤样品。
    11. 将过滤的样品转移到玻璃瓶小瓶中。

  3. Camalexin检测和定量
    HPLC与MS偶联
    1. 在配有的Ultimate3000 HPLC系统中,每个样品注入20μl   Acclaim 120 C18反相柱。 流动相由 含有0.1%甲酸的水(LC/MS级),作为溶剂A和甲醇 用0.1%甲酸(LC/MS级)作为溶剂B.设置:
      流速:0.2ml/min
      柱温:35℃
      用于色谱分离的溶剂梯度:
      在这些条件下,camalexin将以15分钟的保留时间洗脱(见图3)
    2. 将来自HPLC的流出物直接电喷射到物料中 光谱仪(Orbitrap XL发现)在全扫描MS操作 分辨率R = 30,000。 在质心范围内以质心模式收集数据 m/z 120-310。 设置:
      电离模式:正电
      离子喷雾电压:4.5kV
      毛细管温度:250℃
      护套气体流量35(任意单位)
      Aux Ga流速25(任意单位)
    3. 使用校准曲线确定camalexin的丰度 方法。 使用浓度的camalexin 800-400-200-100-50-25-12.5-6.25-3.12 ng/ml用于制备 标准曲线解(见图4)。

代表数据



图2.样品浓度。通过通过惰性增加蒸发 在样品表面上沉积气体(N 2)以除去溶剂。 气体 穿过气体室经由针行进到样品。

A

B

图3.来自外标的camalexin的LC-MS分析(上图) 和来自植物组织(下图)。 A.总离子色谱图 (TIC)。 B.正电离模式中的ESI质谱。 质量的 201.048对应于camalexin的质子化形式[M + H] + sup/+的C 11 H 11 NH 2 S, (红色箭头)。


图4.卡马西平标准曲线在3.12-800ng/ml的浓度范围内获得线性标准曲线。

食谱

  1. 提取缓冲区
    2-丙醇:H 2 O:HCl 37%(2:1:0.002,体积/体积/体积)。

致谢

这项工作得到了欧洲研究委员会(高级补助金233083)的支持。 部分程序改变自先前描述的用于定量分析来自粗植物提取物的主要植物激素的方案(Pan等人,2010)。

参考文献

  1. Ahuja,I.,Kissen,R。和Bones,A.M。(2012)。 Phytoalexins防御病原体 趋势植物科学 17 (2):73-90。
  2. Browne,L.M.,Conn,K.L.,Ayert,W.A.and Tewari,J.P。(1991)。 camalexins:在camelina sativa的叶片中产生的新植物抗毒素(十字花科 )。 Tetrahedron 47(24):3909-3914。
  3. Glawischnig,E。(2007)。 Camalexin。 植物化学 68(4):401- 406。
  4. Pan,X.,Welti,R。和Wang,X。(2010)。 通过高效液相色谱 - 质谱法定量分析粗植物提取物中的主要植物激素。/a> Nat Protoc 5(6):986-992
  5. Tierens,K.F.J.,Thomma,B.P.,Bari,R.P.,Garmier,M.,Eggermont,K.,Brouwer,M.,Penninckx,I.A.,Broekaert,W.F.and Cammue,B。(2002)。 Esa1,一个拟南芥突变体增强对一系列坏死性真菌病原体的易感性,显示通过活性氧产生化合物的防御反应的扭曲诱导。植物J 29(2):131-140。
  6. Zhao,J.,Williams,C.C。和Last,R.L。(1998)。 通过氨基酸饥饿,氧化应激和非生物激发剂诱导拟南芥色氨酸途径酶和卡马西平。 植物细胞 10(3):359-370。
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Copyright: © 2015 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. Savatin, D. V., Bisceglia, N. G., Gravino, M., Fabbri, C., Pontiggia, D. and Mattei, B. (2015). Camalexin Quantification in Arabidopsis thaliana Leaves Infected with Botrytis cinerea. Bio-protocol 5(2): e1379. DOI: 10.21769/BioProtoc.1379.
  2. Savatin, D. V., Bisceglia, N. G., Marti, L., Fabbri, C., Cervone, F. and De Lorenzo, G. (2014). The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-Related Protein Kinases Are Required for Elicitor-Induced Oxidative Burst and Immunity. Plant Physiol 165(3): 1188-1202.
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