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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

Martin Schäfer Martin Schäfer
MR Michael Reichelt
IB Ian T. Baldwin
SM Stefan Meldau

发布: 2014年07月05日第4卷第13期 DOI: 10.21769/BioProtoc.1167 浏览次数: 16325

评审: Tie LiuAnonymous reviewer(s)

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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

English
中文翻译

文章信息

版权信息

© 2014 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Schäfer, M., Reichelt, M., Baldwin, I. T. and Meldau, S. (2014). Cytokinin Analysis: Sample Preparation and Quantification. Bio-protocol 4(13): e1167. DOI: 10.21769/BioProtoc.1167.

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分类

植物科学 > 植物生物化学 > 植物激素 >

植物科学 > 植物生理学 > 组织分析

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