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Cytokinin Analysis: Sample Preparation and Quantification
细胞分裂素分析:样本制备和定量测定   

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Abstract

Cytokinins are a group of phytohormones discovered about half a decade ago by Miller et al. (1955) and Skoog et al. (1965). Since then they were found to participate in many plant physiological processes, including the regulation of the source/sink transitions, plant growth and organ development, responses to environmental conditions such as light, nutrient and water availability and biotic interactions with mutualists, pathogens and herbivores (Werner and Schmülling, 2009; Giron et al., 2013). To aid the quantification of cytokinins for analyzing their changes after environmental stress conditions, we developed this cytokinin extraction and analysis method. This protocol is based on the cytokinin extraction with an acidic methanol-water solution and purification with a mixed-mode solid phase extraction procedure described by Dobrev and Kamı́nek (2002) and the modifications of Kojima et al. (2009). The protocol was successfully used to verify cytokinin overproduction in transgenic Nicotiana attenuata plants expressing the cytokinin biosynthesis gene Tumor morphology root (Tmr) from Agrobacterium tumefaciens under the control of the chemical inducible expression system pOp6/LhGR in the glasshouse and under field conditions (Schäfer et al., 2013) to study the role of cytokinins in plant-herbivore interactions.

Materials and Reagents

  1. Plant tissue
  2. MeOH
  3. 1 N HCOOH
  4. 0.35 N NH4OH
  5. 0.35 N NH4OH in 60% MeOH
  6. 0.1% (v/v) acetic acid
  7. 0.05% (v/v) HCOOH (for mass spectrometry) in Milli-Q H2O
  8. Acetonitrile (gradient grade)
  9. [2H5] tZ (Olchemim, catalog number: 030 0301 )
  10. [2H5] tZR (Olchemim, catalog number: 030 0311 )
  11. [2H5] tZROG (Olchemim, catalog number: 030 5131 )
  12. [2H5] tZ7G (Olchemim, catalog number: 030 5111 )
  13. Extraction buffer (see Recipes)
  14. Extraction buffer + deuterated standards (see Recipes)

Equipment

  1. 96-well BioTubes (1.1 ml individual tubes) (Arctic White LLC, catalog number: AWTS-X22100 )
  2. Steel balls (ASKUBAL, catalog number: 3 MM-G100-1.4034 )
  3. Caps for 96-well BioTubes (strips of 8 plug caps) (Arctic White LLC, catalog number: AWSM-T100-30 )
  4. Pipet
  5. ArctiSeal 96 Round Well Sealing Mats for 96-well BioTubes (Arctic White LLC, catalog number: AWSM-2002RB )
  6. Nunc 96-well Deep Well Plates (Thermo Fisher Scientific, catalog number: 278752 )
  7. Nunc 96-Well Cap Mats (Thermo Fisher Scientific, catalog number: 276002 )
  8. 96-well PCR plates (Kaneka Corporation, Eurogentec, catalog number: RT-PL96-MQ )
  9. Sealing film (OMNILAB-LABORZENTRUM, Schubert & Weiss, catalog number: 5420203 )
  10. Machery Nagel Multi 96 HR-X (96 x 25 mg) (MACHEREY-NAGEL, catalog number: 738530.025M )
  11. Machery Nagel Multi 96 HR-XC (96 x 25 mg) (MACHEREY-NAGEL, catalog number: 738540.025M )
  12. Chromabond Multi 96 vacuum manifold (MACHEREY-NAGEL, catalog number: 738630.M )
  13. Evaporator system (Glas-Col, catalog number: 099A EV9624S )
  14. Geno/Grinder 2000 (SPEX SamplePrep)
  15. Eppendorf Centrifuge 5804 R equipped with a Swing-bucket rotor A-2-DWP (Eppendorf)
  16. Ultrasonic bath Bransonic Models 1200 (BRANDSONTM)
  17. Agilent 1200 HPLC system (Agilent)
  18. Zorbax Eclipse XDB-C18 column (50 x 4.6 mm, 1.8 µm) (Agilent)
  19. API 5000 tandem mass spectrometer (Applied Biosystems®) equipped with a Turbospray ion source

Procedure

  1. Preparation of plant material
    1. Collect plant tissue and immediately freeze it in liquid nitrogen. Stored at -80 °C.
    2. Homogenize plant tissue under liquid nitrogen.
    3. Aliquot 100 mg plant tissue per sample in liquid nitrogen-precooled 96-well BioTubes (with two steel balls per tube, closed with caps).
      Note: Make small holes in the caps to prevent tubes from exploding.
    4. Keep at least for 30 min at -20 °C (for longer storage at -80 °C) before starting with the extraction to evaporate liquefied gases from the tubes.

  2. Extraction
    Note: In-between the steps, while buffer addition and sample collection samples are kept on ice.
    1. Add 800 µl precooled (-20 °C) extraction buffer + deuterated standards to each tube, and cover with a precooled sealing mat.
    2. Shake in Geno/Grinder for 60 s (1,150 strokes *min-1).
    3. Incubate over night at -20 °C.
    4. Shake in the Geno/Grinder for 60 s (1,150 strokes *min-1).
    5. Centrifuge (20 min, 1,913 x g, 4 °C).
    6. Collect 600 µl supernatant in 96-well BioTubes and keep it at -20 °C.
    7. Add 600 µl extraction buffer to the pellet.
    8. Shake in the Geno/Grinder for 60 s (1,150 strokes *min-1). 
    9. Incubate for 30 min at -20 °C.
    10. Centrifuge (20 min, 1,913 x g, 4 °C).
    11. Collect 600 µl supernatant and combine it with the supernatant from the first extraction steps. Cover samples with a sealing mat.
    12. Centrifuge supernatants (20 min, 1,913 x g, 4 °C) and continue with the sample purification.

  3. Purification
    Note: If not further mentioned the following steps are performed at room temperature.

    HR-X
    1. Condition the HR-X column with 0.6 ml MeOH. Discard flow-through.
      Note: Use the Vacuum manifold to suck the samples and buffers through the column. Same accounts for the following steps.
    2. Condition the column with 0.6 ml extraction buffer. Discard flow-through.
    3. Load the samples to the column (collect flow-through) and subsequent wash with 0.2 ml extraction buffer (collect flow-through). Collection of both flow-through can be done in a Nunc 96-well Deep Well Plate.
    4. Evaporate the samples at 45 °C under a constant nitrogen stream to remove MeOH from the samples, utilizing the evaporator system (see Equipment).
      Note: MeOH evaporates fastest and after evaporation of the MeOH, approximately 350 µl liquid should be left, which can be tested with a pipet. Samples only have to be evaporated until this point.
    5. Add 850 µl 1 N HCOOH per sample.
    6. Cover the plate with a sealing mat and shake in the Geno/Grinder for 30 s (1,000 strokes *min-1).
    7. Centrifuge (20 min, 1,913 x g, 4 °C).

    HR-XC
    1. Condition the HR-XC column with 0.6 ml MeOH. Discard flow-through.
    2. Condition the column with 0.6 ml 1 M HCOOH. Discard flow-through.
    3. Load the samples to the column. Discard flow-through.
    4. Wash column with 1 ml 1 M HCOOH. Discard flow-through.
    5. Wash column with 1 ml MeOH. Discard flow-through.
    6. Wash column with 1 ml 0.35 N NH4OH. Discard flow-through.
    7. Elute cytokinin-nucleobases, -ribosides and -glucosides from the column with 0.35 N NH4OH in 60% MeOH. Collect flow-through in 96-well BioTubes.
    8. Completely evaporate the samples at 45 °C under a constant nitrogen stream utilizing the evaporator system. 
    9. Reconstitute samples in 50 µl 0.1% (v/v) acetic acid.
    10. Cover the plate with a sealing mat and shake in the Geno/Grinder for 60 s (1,000 strokes *min-1).
    11. Sonicate the samples for 10 min in an Ultrasonic bath.
      Note: Remove the bottom plate of the 96-well rack and place the whole rack in the Ultrasonic bath. Do not use 96-well racks without bottom plate for shaking and centrifugation! 
    12. Centrifuge (20 min, 1,913 x g, 4 °C).
    13. Fill samples in 96-well PCR plates and cover with the sealing film.
    14. Centrifuge (20 min, 1,913 x g, 4 °C) and continue with Ultra-performance LC coupled MS/MS.

  4. Ultra-performance LC coupled MS/MS
    Inject 2 µl per sample in an Agilent 1200 HPLC system equipped with a Zorbax Eclipse XDB-C18 column. The mobile phase comprises of 0.05% (v/v) HCOOH in Milli-Q H2O as solvent A and acetonitrile as solvent B with following settings:
    Flow rate: 1.1 ml min–1
    Column temperature: 25 °C
    Solvent gradient for chromatographic separation:

    Analysis is done with an API 5000 tandem mass spectrometer equipped with a Turbospray ion source. Multiple-reaction-monitoring mode is used with following settings for quantification:  
    Ionization mode: positive
    Ion spray voltage: 5,500 eV
    Turbo gas temperature: 700 °C
    Nebulizing gas: 70 psi
    Curtain gas: 25 psi
    Heating gas: 60 psi
    Collision gas: 6 psi
    Precursor-to-product ion transitions used for cytokinin analysis:   

    tZ, trans-zeatin, tZR, trans-zeatin riboside, tZROG, trans-zeatin riboside O-glucoside, tZ7G, trans-zeatin N7-glucoside
    1Declustering potential, 2Collision energy, 3Collision cell exit potential

Representative data

  1. Chromatograms


    Figure 1. Separation of deuterated internal standards in the ultra-performance LC coupled MS/MS analysis. The red arrows indicate the respective peaks.


    Figure 2. Separation of cytokinins from the plant tissue in the ultra-performance LC coupled MS/MS analysis. The red arrows indicate the respective peaks.

  2. Calculation
    Exemplary tZ quantification in 0.1 g leaf tissue, supplemented with 1 ng deuterated tZ as internal standard. The obtained peak areas were 23,700 counts and 38,400 counts for tZ and [2H5] tZ, respectively.
    ConcentrationCompound = Peak areaCompound/PeakareaInternal Standard/mExtracted tissue * mInternal Standard
    ConcentrationtZ = Peak areatZ/Peak area[2H5] tZ/mLeaf tissue * m[2H5] tZ
    ConcentrationtZ = 23,700 counts/38,400 counts/0.1 g * 1 ng = 6.2 ng tZ per g leaf tissue

Recipes

  1. Extraction buffer
    750 ml MeOH
    200 ml ddH2O
    50 ml HCOOH
  2. Extraction buffer + deuterated standards
    Prepare Extraction buffer spiked with 1 ng [2H5] tZ, 0.1 ng [2H5] tZR, 4 ng [2H5] tZROG and 2 ng [2H5] tZ7G per 800 µl extraction buffer.
    Note: Deuterated cytokinins are used as internal standards for quantification.

Acknowledgments

The presented work was adopted from Schäfer et al. (2013) and is based on the procedure described by Dobrev and Kamı́nek (2002) and the modifications of Kojima et al. (2009). We thank Radomira Vanková for valuable help with the method development. MS, MR and IB are funded by the Max-Planck-Society and SM is funded by Advanced Grant no. 293926 of the European Research Council to IB. Additional financial support was given by the Human Frontier Science Program (RGP0002/2012) and the Global Research Lab program (2012055546) from the National Research Foundation of Korea.

References

  1. Dobrev, P. I. and Kaminek, M. (2002). Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction. J Chromatogr A 950(1-2): 21-29.
  2. Giron, D., Frago, E., Glevarec, G., Pieterse, C. M. and Dicke, M. (2013). Cytokinins as key regulators in plant–microbe–insect interactions: connecting plant growth and defence. Funct Ecol 27(3): 599-609.
  3. Kojima, M., Kamada-Nobusada, T., Komatsu, H., Takei, K., Kuroha, T., Mizutani, M., Ashikari, M., Ueguchi-Tanaka, M., Matsuoka, M., Suzuki, K. and Sakakibara, H. (2009). Highly sensitive and high-throughput analysis of plant hormones using MS-probe modification and liquid chromatography-tandem mass spectrometry: an application for hormone profiling in Oryza sativa. Plant Cell Physiol 50(7): 1201-1214.
  4. Miller, C. O., Skoog, F., Von Saltza, M. H. and Strong, F. (1955). Kinetin, a cell division factor from deoxyribonucleic acid1. J American Chem Soci 77(5): 1392-1392.
  5. Schäfer, M., Brütting, C., Gase, K., Reichelt, M., Baldwin, I. and Meldau, S. (2013). ‘Real time’genetic manipulation: a new tool for ecological field studies. Plant J 6(3): 506-518.
  6. Skoog, F., Strong, F. M. and Miller, C. O. (1965). Cytokinins. Science 148(3669): 532-533.
  7. Werner, T. and Schmulling, T. (2009). Cytokinin action in plant development. Curr Opin Plant Biol 12(5): 527-538.

简介

细胞分裂素是由Miller等人(1955)和Skoog等人(1965)在约十年前发现的一组植物激素。从那时起,他们被发现参与许多植物生理过程,包括源/汇过渡,植物生长和器官发育的调节,对环境条件的反应,例如光,营养物和水的可用性以及与共生物,病原体和草食动物的生物相互作用(Werner和Schmülling,2009; Giron等人,2013)。为了帮助定量分析细胞分裂素在环境胁迫条件后的变化,我们开发了这种细胞分裂素提取和分析方法。该方案基于用酸性甲醇 - 水溶液的细胞分裂素提取和由Dobrev和Kamınek(2002)和Kojima等人的修改描述的混合模式固相提取程序进行纯化。 (2009)。该方案被成功地用于验证表达细胞分裂素生物合成基因的转基因烟草(Nicotiana attenuata)植物中的细胞分裂素过度产生肿瘤形态学根( Tmr 在温室和田间条件下在化学诱导型表达系统pOp6/LhGR的控制下培养根癌土壤杆菌(Schäfer等人,2013),以研究细胞分裂素在植物 - 草食动物中的作用互动。

材料和试剂

  1. 植物组织
  2. MeOH
  3. 1 N HCOOH
  4. 0.35 N NH 4 OH
  5. 0.35N NH 4 OH在60%MeOH中的溶液
  6. 0.1%(v/v)乙酸
  7. 在Milli-Q H 2 O中的0.05%(v/v)HCOOH(对于质谱)
  8. 乙腈(梯度级)
  9. [ 2 5 ] t Z(Olchemim,目录号:030 0301)
  10. [ 2 H ] ZR(Olchemim,目录号:030 0311)
  11. [ 2 H ] t ZROG(Olchemim,目录号:030 5131)
  12. [ 2 H ] Z7G(Olchemim,目录号:030 5111)
  13. 提取缓冲液(参见配方)
  14. 提取缓冲液+氘标准品(参见配方)

设备

  1. 96孔BioTubes(1.1ml单管)(Arctic White LLC,目录号:AWTS-X22100)
  2. 钢球(ASKUBAL,目录号:3 MM-G100-1.4034)
  3. 用于96孔BioTubes的帽(8个塞帽的条)(Arctic White LLC,目录号:AWSM-T100-30)
  4. Pipet
  5. 用于96孔BioTubes的ArctiSeal 96圆井密封垫(Arctic White LLC,目录号:AWSM-2002RB)
  6. Nunc 96孔深孔板(Thermo Fisher Scientific,目录号:278752)
  7. Nunc 96孔帽垫(Thermo Fisher Scientific,目录号:276002)
  8. 96孔PCR板(Kaneka Corporation,Eurogentec,目录号:RT-PL96-MQ)
  9. 密封膜(OMNILAB-LABORZENTRUM,Schubert& Weiss,目录号:5420203)
  10. Machery Nagel Multi 96 HR-X(96×25mg)(MACHEREY-NAGEL,目录号:738530.025M)
  11. Machery Nagel Multi 96 HR-XC(96×25mg)(MACHEREY-NAGEL,目录号:738540.025M)
  12. Chromabond Multi 96真空歧管(MACHEREY-NAGEL,目录号:738630.M)
  13. 蒸发器系统(Glas-Col,目录号:099A EV9624S)
  14. Geno/Grinder 2000(SPEX SamplePrep)
  15. 装备有旋转叶片转子A-2-DWP(Eppendorf)的Eppendorf离心机5804R
  16. 超声波浴Bransonic Models 1200(BRANDSON TM
  17. Agilent 1200 HPLC系统(Agilent)
  18. Zorbax Eclipse XDB-C18柱(50×4.6mm,1.8μm)(Agilent)
  19. API 5000串联质谱仪(Applied Biosystems ),装备有Turbospray离子源

程序

  1. 植物材料的制备
    1. 收集植物组织,立即在液氮中冷冻。 储存于-80℃。
    2. 在液氮下均化植物组织。
    3. 在液氮预冷96孔BioTubes(每管两个钢球,用盖封闭)中,每份样品等分100mg植物组织。
      注意:在盖子上制作小孔,以防止管道爆炸。
    4. 在开始萃取前从管中蒸发液化气体,在-20℃下保存至少30分钟(以便在-80℃下更长时间保存)。

  2. 提取
    注意:步骤之间,缓冲液添加和样品收集样品保存在冰上。
    1. 向每个管中加入800μl预冷(-20℃)提取缓冲液+氘标准品,并盖上预冷的密封垫。
    2. 在Geno/Grinder中摇动60秒(1,150冲程* min <-1> )。
    3. 在-20°C孵育过夜。
    4. 在Geno/Grinder中摇动60s(1,150冲程* min <-1> )。
    5. 离心(20分钟,1,913×g,4℃)。
    6. 收集600μl上清液在96孔BioTubes,并保持在-20°C。
    7. 向沉淀中加入600μl提取缓冲液。
    8. 在Geno/Grinder中摇动60秒(1,150次*分钟 -1 )。
    9. 在-20°C孵育30分钟。
    10. 离心(20分钟,1,913×g,4℃)。
    11. 收集600μl上清液,并与来自第一次提取步骤的上清液组合。 用密封垫覆盖样品。
    12. 离心上清液(20分钟,1,913×g,4℃),继续样品纯化。

  3. 净化
    注意:如果没有进一步提及,以下步骤在室温下进行。

    HR-X
    1. 用0.6ml MeOH调节HR-X柱。 丢弃流通。
      注意:使用真空歧管吸取样品和缓冲液通过色谱柱。 相同帐户用于以下步骤。
    2. 用0.6ml提取缓冲液调节柱子。 丢弃流通。
    3. 将样品加载至色谱柱(收集流出物),然后用0.2ml萃取缓冲液(收集流出物)洗涤。 两种流通的收集可以在Nunc 96孔深孔中进行 盘子。
    4. 使用蒸发器系统(参见设备)在45℃下在恒定氮气流下蒸发样品以从样品中除去MeOH。
      注意:MeOH蒸发最快,在MeOH蒸发后,应留下约350μl液体,其可用移液管测试。 样品只有在这一点之前才需要蒸发。
    5. 每个样品加入850μl1N HCOOH。
    6. 用密封垫覆盖板并在Geno/Grinder中摇动30秒(1,000冲程*分钟<-1>)。
    7. 离心(20分钟,1,913×g,4℃)
    HR-XC
    1. 用0.6ml MeOH调节HR-XC柱。 丢弃流通。
    2. 用0.6ml 1M HCOOH调节柱。 丢弃流通。
    3. 将样品装入色谱柱。 丢弃流通。
    4. 用1ml 1M HCOOH洗涤柱。 丢弃流通。
    5. 用1ml MeOH洗涤柱。 丢弃流通。
    6. 用1ml 0.35N NH 4 OH洗涤柱。 丢弃流通。
    7. 用0.35N NH 4 OH在60%MeOH中洗脱来自柱的细胞分裂素 - 核碱基, - 核糖苷和 - 糖苷。 在96孔BioTubes中收集流通液。
    8. 使用蒸发器系统在恒定氮气流下在45℃下完全蒸发样品。
    9. 重组样品在50μl0.1%(v/v)乙酸。
    10. 用密封垫覆盖板并在Geno/Grinder中振动60秒(1,000冲程*分钟<-1>)。
    11. 在超声波浴中超声处理样品10分钟。
      注意:取下96孔架的底板,将整个架放在超声波浴中。 不要使用没有底板的96孔架进行摇动和离心!
    12. 离心(20分钟,1,913×g,4℃)。
    13. 在96孔PCR板中填充样品,并用密封膜覆盖。
    14. 离心(20分钟,1,913×g <4℃,4℃),继续进行超高性能LC耦合MS/MS。
  4. 超高性能LC耦合MS/MS
    在配有Zorbax Eclipse XDB-C18色谱柱的Agilent 1200 HPLC系统中注入2μl样品。流动相包含溶剂A中的0.05%(v/v)HCOOH和Milli-Q H 2 O中的HCOOH,以及作为溶剂B的乙腈,具有以下设置:
    流速:1.1ml min
    柱温:25℃

    使用装备有Turbospray离子源的API 5000串联质谱仪进行分析。多反应监测模式与以下定量设置配合使用:
    电离模式:正电
    离子喷雾电压:5,500eV
    涡轮气体温度:700℃
    雾化气体:70 psi
    幕帘气体:25psi
    加热气体:60 psi
    碰撞气体:6 psi
    用于细胞分裂素分析的前体至产物离子跃迁:  

    Z,反式 - 玉米醇溶蛋白,ZR,反式玉米素核糖核苷,ZROG,反式玉米素核苷O-葡萄糖苷, t7G,反式玉米素N7-葡萄糖苷 1 去星电位, 2 碰撞能量, 3 碰撞池退出电位

代表数据

  1. 色谱图


    图1.超性能LC耦合MS/MS分析中氘代内标的分离。红色箭头表示相应的峰。


    图2.在超性能LC偶联的MS/MS分析中从植物组织中分离细胞分裂素。红色箭头表示相应的峰。

  2. 计算
    在0.1g叶组织中的示例性t定量,补充有1ng氘化t/z作为内标。所得到的峰面积为23,700计数和38,400计数,其中t和t分别为z和[分别 浓度化合物 =峰面积化合物/峰面积内部标准/m 提取的组织/sub>
    浓度峰面积 Z峰面积 /峰面积 叶片组织 t t t t 每g叶组织的浓度 zZ = 23,700计数/38,400计数/0.1g * 1ng = 6.2ng/t Z

食谱

  1. 提取缓冲区
    750ml MeOH
    200ml ddH 2 O
    50ml HCOOH
  2. 提取缓冲液+氘代标准品
    制备提取缓冲液,其中加入1ng [2 H] H 5 S] t,0.1ng [sup] 2 H 5 ] t ZR,4ng [ 2 5 ] t [ 2 H ] t Z7G /800μl提取缓冲液。 注意:氘化细胞分裂素用作内标准定量。

致谢

所提出的工作从Schafer等人(2013)获得,并且基于Dobrev和Kamınek(2002)描述的程序以及Kojima等人的修改( 2009)。我们感谢RadomiraVanková对方法开发的宝贵帮助。 MS,MR和IB由Max-Planck-Society资助,SM由Advanced Grant No。资助。 293926欧洲研究理事会到IB。来自韩国国家研究基金会的人类前沿科学计划(RGP0002/2012)和全球研究实验室计划(2012055546)提供了额外的财政支持。

参考文献

  1. Dobrev,P.I。和Kaminek,M。(2002)。 从生长素和脱落酸快速有效地分离细胞分裂素,并使用混合模式固相 A 950(1-2):21-29。
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引用:Schäfer, M., Reichelt, M., Baldwin, I. . and Meldau, S. (2014). Cytokinin Analysis: Sample Preparation and Quantification. Bio-protocol 4(13): e1167. DOI: 10.21769/BioProtoc.1167.
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