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Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds

拟南芥种子总粘液提取物中单糖的分析

Cătălin Voiniciuc Cătălin Voiniciuc
MG Markus Günl

发布: 2016年05月05日第6卷第9期 DOI: 10.21769/BioProtoc.1801 浏览次数: 10634

评审: Marisa RosaHarrie van ErpFernanda Salvato

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参见作者原研究论文

The authors used this protocol in:

Cover of Plant Physiology, featuring study using the protocol.

Sep 2015

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Abstract

The Arabidopsis thaliana seed coat epidermis produces copious amounts of mucilage polysaccharides (Haughn and Western, 2012). Characterization of mucilage mutants has identified novel genes required for cell wall biosynthesis and modification (North et al., 2014). The biochemical analysis of seed mucilage is essential to evaluate how different mutations affect cell wall structure (Voiniciuc et al., 2015c). Here we describe a robust method to screen the monosaccharide composition of Arabidopsis seed mucilage using ion chromatography (IC). Mucilage from up to 48 samples can be extracted and prepared for IC analysis within 24 h (only 4 h hands-on). Furthermore, this protocol enables fast separation (31 min per sample), automatic detection and quantification of both neutral and acidic sugars.

Keywords: Matrix polysaccharides (矩阵多糖) search Seed coat (种子大衣) search Plant cell wall (植物细胞壁) search HPAEC-PAD (hpaec垫) search Ion chromatography (离子色谱法) search

Materials and Reagents

  1. Sterile Petri dishes
  2. Filter paper (MACHEREY-NAGEL GmbH & Co., catalog number: 434009 or similar types)
  3. 3M Micropore paper tape (VWR International, catalog number: 115-8172 )
  4. Square (7 x 7 cm) or round (Ø 5 cm; 35 multi-well insets) plastic pots, and trays
  5. Peat-sand-pumice substrate (SoMi 513 Dachstauden) (HAWITA GRUPPE GmbH)
  6. ARACON cone and tube (Betatech bvba)
  7. Large (sandwich-style) brown paper bags
  8. Small white paper bags (Baumann Saatzuchtbedarf, catalog number: 3.065.002 , or similar style)
  9. 15 ml Falcon tubes (VWR International, catalog number: 734-0452 )
  10. 2 ml Eppendorf safe-lock tubes (VWR International, catalog number: 211-2165 )
  11. 2 ml screw-cap tubes (VWR International, catalog number: 211-0093 )
  12. Chromatography vials with inserts (VWR International, catalog number: 548-0120 )
  13. Snap cap for chromatography vials (VWR International, catalog number: 548-1151 )
  14. Manual pipettes tips
  15. Arabidopsis thaliana seeds
  16. Murashige and Skoog (MS) basal salts (Sigma-Aldrich, catalog number: M5519-10L )
  17. Agar (Carl Roth GmbH + Co, catalog number: 4807.2 )
  18. D-(-)-Ribose (Rib) (Sigma-Aldrich, catalog number: R7500-5 G )
  19. L-(+)-Arabinose (Ara) (Sigma-Aldrich, catalog number: A3256-25 G )
  20. L-(-)-Fucose (Fuc) (Sigma-Aldrich, catalog number: F2252-5 G )
  21. D-(+)-Galactose (Gal) (Sigma-Aldrich, catalog number: G0750-25 G )
  22. D-(+)-Galacturonic acid monohydrate (GalA) (Sigma-Aldrich, catalog number: 48280-5G-F )
  23. D-(+)-Glucose (Glc) (Sigma-Aldrich, catalog number: G8270-100 G )
  24. D-Glucuronic acid (GlcA) (Sigma-Aldrich, catalog number: G5269-10 G )
  25. D-(+)-Mannose (Man) (Sigma-Aldrich, catalog number: M8574-25 G )
  26. L-Rhamnose monohydrate (Rha) (Sigma-Aldrich, catalog number: R3875-5 G )
  27. D-(+)-Xylose (Xyl) (Sigma-Aldrich, catalog number: X3877-25 G )
  28. Ultrapure water (18.2 MΩ cm at 25 °C)
  29. Sodium hydroxide (NaOH) (VWR International, catalog number: BAKR3727.2500 )
  30. Trifluoroacetic acid (TFA) (Carl Roth GmbH + Co., catalog number: 6957.1 )
  31. ½ MS plates (see Recipes)
  32. Sugar standard stocks (see Recipes)
  33. 9-Sugar mix (see Recipes)
  34. 2 M TFA (see Recipes)
  35. 10 mM NaOH (see Recipes)
  36. 733 mM NaOH (see Recipes)

Equipment

  1. Laminar flow clean bench
  2. Growth chamber (Johnson Controls)
  3. Autoclave
  4. Water purification system (Milli-Q or similar style)
  5. Manual pipettes (Eppendorf AG, Research plus and Repeater Plus style)
  6. Analytical balance (Mettler-Toledo, model: XSE205DU )
  7. Ball mill (Retsch GmbH, catalog number: MM400 )
  8. Two 24 TissueLyser adapters for ball mill (QIAGEN, catalog number: 69982 )
  9. Safety glasses
  10. Lab coat
  11. Chemical resistant gloves (Honeywell, Dermatril, catalog number: 740 , or similar style)
  12. Fume hood
  13. Sample concentrator (Bibby Scientific Limited, Techne, catalog number: FSC400D ), equipped with
    1. 127 mm needles (Bibby Scientific Limited, Techne, catalog number: F7210 )
    2. A Dri-block heater (Bibby Scientific Limited, Techne, catalog number: DB200/3 )
    3. Three aluminium blocks (Bibby Scientific Limited, Techne, catalog number: F3505 )
    4. Connected to a central air supply, in a fume hood
  14. Ice machine
  15. Ion chromatography (IC): Dionex DX-600 system equipped with
    1. AS50 autosampler
    2. GP50 gradient pump
    3. ED50 electrochemical detector
    4. CarboPac PA20 guard column (Thermo Fisher Scientific, Dionex Softron, catalog number: 060144 )
    5. CarboPac PA20 analytical column (Thermo Fisher Scientific, Dionex Softron, catalog number: 60142 )
  16. Vortex mixer (Scientific Industries Inc., model: Vortex-Genie 2 , or similar style)
  17. Benchtop centrifuge (compatible with 2 ml tubes)
  18. Racks with lids for 2 ml tubes (VWR International, catalog number: 211-0215 )
  19. Boxes with lids for vials (neoLab, catalog number: 2-2580 )
  20. Fine-tipped forceps (VWR International; catalog number: 232-0107 , or similar types)
  21. Serological pipettes (Thermo Fisher Scientific, Nunc-type or similar style)
  22. 2 L volumetric flask
  23. Helium gas tank

Software

  1. Chromeleon 6.8 chromatography data system software (Thermo Fisher Scientific, Dionex)
  2. Microsoft Excel with the Real Statistics Resource Pack (http://www.real-statistics.com/)

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Voiniciuc, C. and Günl, M. (2016). Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds. Bio-protocol 6(9): e1801. DOI: 10.21769/BioProtoc.1801.
  2. Voiniciuc, C., Schmidt, M. H., Berger, A., Yang, B., Ebert, B., Scheller, H. V., North, H. M., Usadel, B. and Gunl, M. (2015b). MUCILAGE-RELATED10 produces galactoglucomannan that maintains pectin and cellulose architecture in Arabidopsis seed mucilage. Plant Physiol 169(1): 403-420.

    3. ris ris Download Citation in RIS Format

分类

植物科学 > 植物生物化学 > 糖类

生物化学 > 糖类 > 多糖

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