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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

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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), Seed coat(种子大衣), Plant cell wall(植物细胞壁), HPAEC-PAD(hpaec垫), Ion chromatography(离子色谱法)

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: 0 60144 )
    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

  1. Plant growth, seed harvest and storage
    The manner in which seeds are produced, harvested and stored can impact the content and composition of mucilage (see recent review, Voiniciuc et al., 2015c). To facilitate comparisons between different genotypes, it is necessary to always grow plants under the same conditions. We recommend the following procedures, which yield high quality seeds and consistent mucilage chemotypes (Voiniciuc et al., 2015a; Voiniciuc et al., 2015b; Voiniciuc et al., 2015c).
    1. Using filter paper, sprinkle seeds on ½ MS plates, and seal plates with 3M Micropore Paper Tape. Germinate seeds under continuous light.
    2. After five to seven days (when the cotyledons fully open), transfer seedlings using forceps to individual pots filled with wet peat-sand-pumice substrate. Cover with plastic dome for one week.
    3. Grow plants under constant light (~170 µE m-2 s-1), temperature (20 °C) and relative humidity (60%), watering every two to three days as needed.
    4. Before flowering, cover each plant with an ARACON cone and tube to prevent cross-pollination and seed dispersal. Remove any branches that cannot be contained within the ARACON tubes.
    5. When most of the siliques turn yellow (almost 60 days after step A1), cut plants from their base and harvest mature seeds by shaking each plant into large brown paper bags for 10 sec.
      Note: To preserve the biological variation, use one bag per plant and do not pool seeds.
    6. Empty the contents of each brown bag onto a clean piece of filter paper and carefully remove the vegetative material using forceps. Transfer only seeds to small white paper bags.

  2. Total mucilage extraction
    1. Prepare a spreadsheet for each experiment, with the relevant information (e.g. Sample #; Seed Weight; Seed Bag; Genotype). Up to 48 unknown samples can be processed at once. To simplify labeling and later processing, samples should be assigned a letter (e.g. experiment A) and a two-digit number, making sure that single digit numbers are preceded by a zero (e.g. A01 to A48).
    2. Pre-label the sides of 2 ml Safe Lock tubes with the sample numbers. Use an analytical balance to add 4-6 mg seeds to each tube and record precise weight in the spreadsheet.
    3. Prepare a dilution series of 9-Sugar mix standards by adding the volumes listed in Table 1 to 2 ml Screw-Cap Tubes.

      Table 1. 9-Sugar mix (10 mg/ml) dilution series for monosaccharide quantification
      Label
      S000
      S001
      S002
      S005
      S010
      S025
      S050
      S075
      S100
      S125
      µl
      0
      1
      2
      5
      10
      25
      50
      75
      100
      125
      µg
      0
      1
      2
      5
      10
      25
      50
      75
      100
      125

    4. Prepare enough 30 µg/ml Rib (Internal Standard) to add 1 ml to all samples and sugar standards in one experiment. For a typical experiment with 48 samples and 10 standards, add 180 µl of the 10 mg/ml Rib and fill up to 60 ml with ultrapure water.
    5. Using a repeater pipette, add 1 ml of Internal Standard to all samples and standards.
    6. Perform the total mucilage extraction by shaking the seed-containing tubes for 15 min at 30 Hz in a ball mill using two 24 Tissue Lyser Adapters, at room temperature (~24 °C).
    7. Rotate block 180° and shake for an additional 15 min at 30 Hz to complete the total mucilage extraction. This detaches all mucilage from wild-type seeds (Figure 1).


      Figure 1. Ruthenium red staining of seeds after mucilage extraction. Wild-type (Col-0) seeds stained in a 24-well plate as previously described (Voiniciuc et al., 2015b), after gentle shaking in water (A) or the total mucilage extraction (B). Bars = 2 mm.

    8. Let seeds settle for at least 30 sec. For each sample, transfer 800 µl of the supernatant to a 2 ml Screw-Cap tube, pre-labeled on its side.
      Note: Do not transfer any seeds, and do not cap the tubes at this point.
    9. Dry samples and standards under air flow at 45 °C using the sample concentrator. Process all tubes identically from this point onwards.

  3. Hydrolysis of matrix polysaccharides
    1. Using a repeater pipette, add 300 µl of 2 M TFA to all tubes.
      Note: Perform TFA work in a fume hood, with the appropriate personal protective equipment.
    2. Cap each tube tightly, and vortex for 3 sec.
    3. Transfer tubes to the Techne Dri-block (preheated to 120 °C) and incubate for 60 min.
    4. Cool heating blocks and tubes on ice. Centrifuge tubes for 30 sec at maximum speed.
    5. Uncap tubes, and evaporate TFA under air flow at 45 °C using sample concentrator.
      Note: Keep caps on a clean paper towel, in the correct order.

  4. Final elution of samples
    1. Add 600 µl of autoclaved, ultrapure water to all tubes using a repeater pipette. Vortex mix for 3 sec.
      Note: If using medium shaking intensity and exercising caution, the tubes do not need to be capped for this step because the solution will not spill over.
    2. Transfer 150 µl from each tube to pre-labeled IC vials with inserts. Seal vials with caps.
    3. Place tubes in the autosampler of a Dionex DX-600 system in ascending order, but randomize (e.g. using Microsoft Excel) the injection order of the sample and standards.

  5. Separation and quantification of monosaccharides
    1. Separate and quantify monosaccharides by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using CarboPac PA20 Guard and Analytical columns, at 40 °C and a constant flow rate of 0.4 ml/min.
    2. Ensure that sufficient volumes of the three IC eluents (ultrapure water, 10 mM NaOH, and 733 mM NaOH; Figure 2) are available to run all samples and standards in an experimental batch.


      Figure 2. The proportion of eluents pumped after sample injection

    3. Equilibrate the columns with 2 mM NaOH (80% water, 20% 10 mM NaOH) for 10 min.
    4. Inject 10 µl of each sample.
    5. Separate neutral sugars with 2 mM NaOH over the course of 18 min (Figure 2).
    6. Afterwards, pump 513 mM NaOH for 7.5 min to separate uronic acids (Figure 2).
    7. Finally, rinse the column with 733 mM NaOH for 4 min (Figure 2).
      Note: Repeat the methods in steps E3-7 for each sample and standard (Figure 2).
    8. Automatically annotate the monosaccharide peaks by configuring the Peak Table (Table 2) and the Detection Parameters (Table 3) in the Chromeleon Chromatography Data System software. These parameters work correctly for most standards and samples (Figure 3), and require only minor adjustments if the retention times shifts slightly between experiments.
      Note: Depending on your individual set-up and column age, you might have to adjust the flow rate or the NaOH concentration during the neutral sugar elution. Do not decrease NaOH concentration too much as this has an effect on sensitivity.

      Table 2. Chromeleon 6.8 Peak Table to identify and calibrate monosaccharides
      Peak name
      Ret. time (min)
      Window
      Standard
      Int. type
      Cal. type
      Peak type
      Fuc
      4.50
      0.270 AN
      Internal rib
      Area
      Quad
      B-M-B
      Rha
      8.00
      0.400 AN
      Internal rib
      Area
      Quad
      B-M
      Ara
      8.70
      0.400 AN
      Internal rib
      Area
      Quad
      M-B
      Gal
      11.00
      0.800 AF
      Internal rib
      Area
      Quad
      B-M-B
      Glc
      12.25
      0.800 AN
      Internal rib
      Area
      Quad
      B-M-B
      Xyl
      14.40
      1.000 AF
      Internal rib
      Area
      Quad
      B-M
      Man
      15.00
      1.000 AG
      Internal rib
      Area
      Quad
      M-B
      Rib
      18.70
      1.300 AN
      ISTD internal
      Area
      Lin
      B-M-B
      GalA
      26.26
      0.800 AG
      Internal rib
      Area
      Lin
      B-M-B
      GlcA
      28.40
      0.800 AG
      Internal rib
      Area
      Lin
      B-M-B

      Table 3. Chromeleon 6.8 Detection Parameters to annotate monosaccharide peaks
      Time
      Param. name
      Param. value
      Channel
      0.00
      Inhibit integration
      On
      All channels
      3.60
      Inhibit integration
      Off
      All channels
      4.10
      Minimum area 
      0.5
      All channels
      4.75
      Minimum area
      1
      All channels
      5.00
      Inhibit integration
      On
      All channels
      6.60
      Inhibit integration
      Off
      All channels
      6.60
      Lock baseline
      On
      All channels
      7.25
      Minimum area
      0.5
      All channels
      9.80
      Lock baseline
      Off
      All channels
      9.80
      Inhibit integration
      On
      All channels
      10.20
      Inhibit integration
      Off
      All channels
      10.40
      Lock baseline
      On
      All channels
      12.70
      Inhibit integration
      On
      All channels
      12.80
      Lock baseline
      Off
      All channels
      13.10
      Inhibit integration
      Off
      All channels
      14.00
      Minimum area
      0.25
      All channels
      14.00
      Lock baseline
      On
      All channels
      16.00
      Lock baseline
      Off
      All channels
      18.00
      Minimum area
      10
      All channels
      20.50
      Inhibit integration
      On
      All channels
      25.50
      Inhibit integration
      Off
      All channels
      25.60
      Minimum area
      0.5
      All channels
      27.00
      Inhibit integration
      On
      All channels
      28.20
      Inhibit integration
      Off
      All channels
      28.20
      Minimum area
      0.5
      All channels
      29.00
      Inhibit integration
      On
      All channels

    9. Inspect chromatograms in Chromeleon to ensure that all peaks are correctly labeled. Manually annotate the peaks of Ara and Man if necessary, which overlap with more abundant sugars.
    10. By defining the standards and their concentrations in Chromeleon (Tables 1 and 2), calibration plots are automatically generated for all monosaccharides, normalized to Rib (Internal Standard).
      Note: Inspect the calibration plots and disable significant outliers if necessary.
    11. The amounts of all monosaccharides (expressed in µg) in a sample are automatically calculated based on the calibration plots and can be exported from Chromeleon for further calculations.
      Note: Fuc is detected in trace amounts in total mucilage extracts from Arabidopsis seeds (Figure 3; Voiniciuc et al., 2015c), while GlcA is generally not detected in these samples.


      Figure 3. Example chromatograms exported from the Chromeleon software. The vertical axes represent detected signals (nC). The horizontal axes show time post-injection (min).

  6. Final calculations and statistical analysis
    Further calculations are performed in Microsoft Excel. The monosaccharide composition of total mucilage extracts can be expressed as absolute (Figure 4A), or relative amounts (Figure 4B).


    Figure 4. Composition of total mucilage extracts from the Col-0 wild type. The data show means of three biological replicates. The error bars in (A) represent standard deviations.

    1. Sugar amounts (expressed in µg) can be divided by the amount of seeds (expressed in mg) used for each mucilage extraction, to calculate the absolute composition (µg sugar/mg seed).
      Note: The total amount of mucilage per mg seed is the sum of all sugar values in a sample.
    2. Absolute monosaccharide levels can also be expressed as the number of molecules normalized to the amount of seeds used. To calculate the nmol sugar per mg seed, divide the value obtained in step F1 by the molecular mass of the respective sugar (Table 4), and multiply the result by 1,000.

      Table 4. Molar mass of the monosaccharides for final calculations
      Sugar
      Fuc
      Rha
      Ara
      Gal
      Glc
      Xyl
      Man
      GalA
      GlcA
      Mass
      164.16
      164.16
      150.13
      180.16
      180.16
      150.13
      180.16
      194.14
      194.14

    3. Relative monosaccharide composition in mucilage can be expressed as a molar percentage (mol%), and equals the amount of each monomer divided by the sum of all sugars (both from step F2).
    4. Statistical analyses to compare the mucilage composition of wild-type and mutants can be performed in Excel. Significant changes in a particular monosaccharide (expressed either in absolute or relative amounts) between two genotypes with multiple biological replicates can be identified using the built-in T.TEST function. When many mutants (and their various sugars components) are compared to wild-type, conditional formatting (e.g. highlight cells with P-value < 0.05) can be used to quickly reveal which components are significantly altered. Two-Factor Analysis of Variance (ANOVA) can also be performed in Excel with the Real Statistics Resource Pack (http://www.real-statistics.com/) to evaluate how mucilage composition is affected by two independent mutations (via the analysis of wild-type, single and double mutant samples).

Recipes

  1. ½ MS plates
    For 500 ml solution (yields around 25 plates), mix 1.08 g MS basal salts, 3.5 g agar, and water.
    Autoclave, and pour media (while still warm) into sterile Petri dishes on a clean bench. When kept in a sterile bag, plates can be stored at 4 °C for at least 6 months.
  2. Sugar standard stocks (10 mg/ml)
    For each monosaccharide except GalA and Rha, prepare individual stocks by dissolving 100 mg sugar in 10 ml of autoclaved, ultrapure water in a sterile 15 ml Falcon tube.
    Since they are sold in monohydrate forms, use 109 mg of GalA and 111 mg of Rha rather than the standard 100 mg.
    Aliquot the Rib stock into 2 ml Safe Lock tubes since it is frequently used.
    Store all stocks at -20 °C.
  3. 9-Sugar mix (1 mg/ml)
    For a 10 ml solution, mix 1 ml of water with 1 ml of Fuc, Rha, Ara, Gal, Glc, Xyl, Man, GalA, and GlcA (all 10 mg/ml stocks).
    Aliquot the 9-Sugar mix into 2 ml Safe Lock tubes and stored at -20 °C.
  4. 2 M Trifluoroacetic acid (TFA)
    Prepare 500 ml of a 2 M TFA solution by slowly adding 77 ml of TFA (12.98 M) to 423 ml of ultrapure water
    Note: Perform TFA work in a fume hood, with the appropriate personal protective equipment.
  5. 10 mM NaOH (IC eluent)
    Use a volumetric flask to transfer 2 L of ultrapure water to the appropriate eluent container
    Add 1,040 µl of 50% (w/v) NaOH using a 1 ml serological pipette
    Degas eluent using helium for 5 min
  6. 733 mM NaOH (IC eluent)
    Use a volumetric flask to transfer 2 L of ultrapure water to the appropriate eluent container
    Add a total of 80 ml of 50% (w/v) NaOH using a 50 ml serological pipette
    Degas eluent using helium for 5 min

Acknowledgements

The methods described here were derived from (Voiniciuc et al., 2015c), and were employed by (Voiniciuc et al., 2015a; Voiniciuc et al., 2015b). We thank Björn Usadel for helpful comments on the protocol. This work was supported by the Natural Sciences and Engineering Research Council of Canada (PGS-D3 grant to C. V.). Author Contributions: C. V. and M. G. developed the method. C. V. wrote the protocol.

References

  1. Haughn, G. W. and Western, T. L. (2012). Arabidopsis seed coat mucilage is a specialized cell wall that can be used as a model for genetic analysis of plant cell wall structure and function. Front Plant Sci 3: 64.
  2. North, H. M., Berger, A., Saez-Aguayo, S. and Ralet, M. C. (2014). Understanding polysaccharide production and properties using seed coat mutants: future perspectives for the exploitation of natural variants. Ann Bot 114(6): 1251-1263.
  3. Voiniciuc, C., Gunl, M., Schmidt, M. H. and Usadel, B. (2015a). Highly branched xylan made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 links mucilage to Arabidopsis seeds. Plant Physiol 169(4): 2481-2495.
  4. 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.
  5. Voiniciuc, C., Yang, B., Schmidt, M. H., Gunl, M. and Usadel, B. (2015c). Starting to gel: how Arabidopsis seed coat epidermal cells produce specialized secondary cell walls. Int J Mol Sci 16(2): 3452-3473.

简介

拟南芥种皮表皮产生大量的粘液多糖(Haughn和Western,2012)。 粘液突变体的表征已经鉴定了细胞壁生物合成和修饰所需的新基因(North等人,2014)。 种子粘液的生物化学分析对于评估不同突变如何影响细胞壁结构是必要的(Voiniciuc等人,2015c)。 在这里我们描述了使用离子色谱(IC)筛选拟南芥种子粘液的单糖组成的有力方法。 在24小时内(仅4小时手动)可以提取并准备来自多达48个样品的粘蛋白并进行IC分析。 此外,该协议使快速分离(每个样品31分钟),自动检测和定量的中性和酸性糖。

关键字:矩阵多糖, 种子大衣, 植物细胞壁, hpaec垫, 离子色谱法

材料和试剂

  1. 无菌培养皿
  2. 滤纸(MACHEREY-NAGEL GmbH& Co.,目录号:434009或类似类型)
  3. 3M微孔纸带(VWR International,目录号:115-8172)
  4. 正方形(7 x 7厘米)或圆形(Ø5厘米; 35多孔插图)塑料盆和托盘
  5. 泥炭 - 浮石基质(SoMi 513 Dachstauden)(HAWITA GRUPPE GmbH)
  6. ARACON锥管(Betatech bvba)
  7. 大(夹心式)棕色纸袋
  8. 小白皮书袋(Baumann Saatzuchtbedarf,目录号:3.065.002或类似风格)
  9. 15ml Falcon管(VWR International,目录号:734-0452)
  10. 2ml Eppendorf安全锁管(VWR International,目录号:211-2165)
  11. 2ml螺旋盖管(VWR International,目录号:211-0093)
  12. 具有插入物的色谱小瓶(VWR International,目录号:548-0120)
  13. 用于色谱小瓶(VWR International,目录号:548-1151)的快速盖子
  14. 手动移液器提示
  15. 拟南芥种子
  16. Murashige和Skoog(MS)基础盐(Sigma-Aldrich,目录号:M5519-10L)
  17. 琼脂(Carl Roth GmbH + Co,目录号:4807.2)
  18. D - ( - ) - 核糖(Rib)(Sigma-Aldrich,目录号:R7500-5G)
  19. L - (+) - 阿拉伯糖(Ara)(Sigma-Aldrich,目录号:A3256-25G)
  20. L - ( - ) - 岩藻糖(Fuc)(Sigma-Aldrich,目录号:F2252-5G)
  21. D - (+) - 半乳糖(Gal)(Sigma-Aldrich,目录号:G0750-25G)
  22. D - (+) - 半乳糖醛酸一水合物(GalA)(Sigma-Aldrich,目录号:48280-5G-F)
  23. D - (+) - 葡萄糖(Glc)(Sigma-Aldrich,目录号:G8270-100G)
  24. D-葡萄糖醛酸(GlcA)(Sigma-Aldrich,目录号:G5269-10G)
  25. D - (+) - 甘露糖(Man)(Sigma-Aldrich,目录号:M8574-25G)
  26. L-鼠李糖一水合物(Rha)(Sigma-Aldrich,目录号:R3875-5G)
  27. D - (+) - 木糖(Xyl)(Sigma-Aldrich,目录号:X3877-25G)
  28. 超纯水(25℃时为18.2MΩ·cm)
  29. 氢氧化钠(NaOH)(VWR International,目录号:BAKR3727.2500)
  30. 三氟乙酸(TFA)(Carl Roth GmbH + Co.,目录号:6957.1)
  31. ½MS板(参见配方)
  32. 糖标准储备(见配方)
  33. 9糖混合(见配方)
  34. 2 M TFA(参见配方)
  35. 10 mM NaOH(参见配方)
  36. 733mM NaOH(参见配方)

设备

  1. 层流净化台
  2. 生长室(Johnson Controls)
  3. 高压灭菌器
  4. 水净化系统(Milli-Q或类似风格)
  5. 手动移液器(Eppendorf AG,Research plus和Repeater Plus样式)
  6. 分析天平(Mettler-Toledo,型号:XSE205DU)
  7. 球磨机(Retsch GmbH,目录号:MM400)
  8. 两个用于球磨机的24个TissueLyser适配器(QIAGEN,目录号:69982)
  9. 安全眼镜
  10. 实验室外套
  11. 耐化学性手套(Honeywell,Dermatril,目录号:740或类似风格)
  12. 通风橱
  13. 样品浓缩器(Bibby Scientific Limited,Techne,目录号:FSC400D),装备有
    1. 127mm针(Bibby Scientific Limited,Techne,目录号:F7210)
    2. 驱动块加热器(Bibby Scientific Limited,Techne,目录号:DB200/3)
    3. 三个铝块(Bibby Scientific Limited,Techne,目录号:F3505)
    4. 连接到中央空气供应,在通风橱
  14. 制冰机
  15. 离子色谱(IC):装有
    的Dionex DX-600系统
    1. AS50自动进样器
    2. GP50梯度泵
    3. ED50电化学检测器
    4. CarboPac PA20保护柱(Thermo Fisher Scientific,Dionex Softron,目录号:060144)
    5. CarboPac PA20分析柱(Thermo Fisher Scientific,Dionex Softron,目录号:60142)
  16. 涡流混合器(Scientific Industries Inc.,型号:Vortex-Genie 2或类似型)
  17. 台式离心机(与2 ml管相容)
  18. 带有用于2ml管的盖的架(VWR International,目录号:211-0215)
  19. 带有小瓶盖的盒子(neoLab,目录号:2-2580)
  20. 精尖镊子(VWR International;目录号:232-0107或类似类型)
  21. 血清移液管(Thermo Fisher Scientific,Nunc型或类似样式)
  22. 2升容量瓶
  23. 氦气罐

软件

  1. Chromeleon 6.8色谱数据系统软件(Thermo Fisher Scientific,Dionex)
  2. 具有真实统计资源包的Microsoft Excel( http://www.real-statistics.com/

程序

  1. 植物生长,种子收获和存储
    种子生产,收获和储存的方式可影响粘液的含量和组成(参见最近的综述,Voiniciuc等人,2015c)。为了便于不同基因型之间的比较,有必要在相同条件下总是生长植物。我们推荐以下程序,其产生高质量的种子和一致的粘液化学型(Voiniciuc等人,2015a; Voiniciuc等人,2015b; Voiniciuc等人。,2015c)。
    1. 使用滤纸,将种子撒在½MS板上,并用密封板 3M微孔纸带。在连续光下发芽种子
    2. 五至七天后(当子叶完全打开时),转移 幼苗使用镊子到单个盆充满湿 泥炭 - 砂 - 浮石基质。用塑料圆顶盖一周。
    3. 在恒定光(〜170μEm -2 s -1 s -1)下,温度(20℃)下生长植物,   和相对湿度(60%),每两至三天浇水一次 需要
    4. 开花前,用一个ARACON锥覆盖每个植物 和管,以防止异花授粉和种子扩散。 删除任何 不能包含在ARACON管内的分支。
    5. 什么时候 大多数长角果变黄(在A1步骤后大约60天),切割 植物从其基部并通过摇动每个植物收获成熟的种子 进入大的棕色纸袋中10秒。
      注意:为了保持生物变异,每株植物使用一个袋子,不能混合种子。
    6. 将每个棕色袋子的内容物倒入一块干净的过滤器上 纸,并使用镊子小心地去除植物性材料。 只转移种子到小白皮书袋。

  2. 全部粘液提取
    1. 为每个实验准备一份电子表格以及相关信息   (例如样品#;种子重量;种子袋;基因型)。 最多48个未知 可以立即处理样品。 简化标签和以后 处理,应为样品分配一个字母(例如实验A)和a   两位数字,确保单个数字前面加上   零(例如 A01到A48)。
    2. 预先标记2毫升安全锁的侧面   具有样品编号的管。 使用分析天平添加4-6毫克 种子,并在电子表格中记录精确重量。
    3. 通过将表1所列的体积加入2ml螺旋盖管中制备9-Sugar混合物标准品的稀释系列。

      表1. 9- 糖混合(10 mg/ml)稀释系列用于单糖定量
      标签
      S000
      S 001
      S002
      S005
      S 01 0 S025
      S050
      S075
      S100
      S125
      微博
      0
      1
      2
      5
      10
      25
      50
      75
      100
      125
      μg
      0
      1
      2
      5
      10
      25
      50
      75
      100
      125

    4. 准备足够的30微克/毫升Rib(内标)加入1毫升到所有 样品和糖标准品。 对于典型的实验 用48个样品和10个标准品,加入180μl的10mg/ml Rib和 用超纯水填充至60ml
    5. 使用中继移液器,向所有样品和标准品中加入1ml内标
    6. 通过摇动含种子进行总胶浆提取   在球磨机中使用两个24Tissue Lyser在30Hz下进行15分钟 适配器,在室温(〜24°C)。
    7. 旋转块180°和 在30Hz下再摇动15分钟以完成全部粘液 萃取。 这从所有的粘液从野生型种子(图1)。


      图1.在粘液提取后种子的钌红染色。如先前在24孔板中将野生型(Col-0)种子染色 (Voiniciuc等人,2015b),在水(A)中轻轻摇动后, 或全部粘液提取(B)。 条= 2mm。

    8. 让 种子沉降至少30秒。 对于每个样品,转移800微升 将上清液倒入2ml螺旋帽管中,在其侧面预先标记。
      注意:请勿转移任何种子,并且不要在这一点上盖住管。
    9. 干样品和标准品在45°C空气流下使用样品集中器。 从这一点向前处理所有管相同。

  3. 基质多糖水解
    1. 使用中继器移液管,加入300μl的2M TFA到所有管。
      注意:在通风橱中使用适当的个人防护装备进行TFA。
    2. 紧紧盖上每个管,并涡旋3秒。
    3. 将管转移到Techne Dri块(预热至120℃)并孵育60分钟
    4. 凉快的加热块和管在冰。 以最高速度离心管30秒。
    5. 盖帽管,并在45℃的空气流下使用样品浓缩器蒸发TFA。
      注意:请以正确的顺序将盖子放在干净的纸巾上。

  4. 样品的最终洗脱
    1. 加入600微升的高压灭菌,超纯水的所有管使用中继移液器。 涡旋混合3秒。
      注意:如果使用中等摇晃强度和小心, 管不需要为此步骤加盖,因为溶液会 不会溢出。
    2. 转移150μl从每个管到带有插入物的预标记的IC小瓶。 用盖子密封小瓶。
    3. 将管置于Dionex DX-600系统的自动进样器中 升序,但随机化(例如使用Microsoft Excel) 注射顺序的样品和标准品。

  5. 单糖的分离和定量
    1. 通过高效阴离子交换分离和定量单糖   使用脉冲电流检测(HPAEC-PAD)进行层析 CarboPac PA20保护和分析柱,在40°C和恒定流量   速率为0.4ml/min
    2. 确保三个足够的体积 IC洗脱液(超纯水,10mM NaOH和733mM NaOH;图2) 可用于运行实验批次中的所有样品和标准

      图2.样品注入后抽取的洗脱液的比例

    3. 用2mM NaOH(80%水,20%10mM NaOH)平衡柱10分钟
    4. 注入10μl每个样品。
    5. 在18分钟的过程中用2mM NaOH分离中性糖(图2)。
    6. 然后,泵513mM NaOH 7.5分钟以分离糖醛酸(图2)
    7. 最后,用733mM NaOH冲洗柱4分钟(图2)。
      注意:对每个样品和标准品重复步骤E3-7中的方法(图2)。
    8. 通过配置自动注释单糖峰 峰值表(表2)和检测参数(表3) Chromeleon色谱数据系统软件。这些参数工作 适用于大多数标准品和样品(图3),仅需要 如果保留时间在之间稍微移动,则进行微小调整 实验。
      注意:根据您的个人设置和列 年龄,您可能需要调整流速或NaOH浓度 在中性糖洗脱期间。不要降低NaOH浓度  因为这对灵敏度有影响。

      表2. Chromeleon 6.8峰表,用于识别和校准单糖
      峰名
      Ret。 时间(分钟)
      窗口
      标准
      Int。 类型
      校准。 类型
      峰值类型
      Fuc
      4.50
      0.270 AN
      内肋
      区域
      Quad
      B-M-B
      Rha
      8.00
      0.400 AN
      内肋
      区域
      Quad
      B-M
      Ara
      8.70
      0.400 AN
      内肋
      区域
      Quad
      M-B
      Gal
      11.00
      0.800 AF
      内肋
      区域
      Quad
      B-M-B
      Glc
      12.25
      0.800 AN
      内肋
      区域
      Quad
      B-M-B
      Xyl
      14.40
      1.000 AF
      内肋
      区域
      Quad
      B-M
      男士
      15.00
      1.000公司
      内肋
      区域
      Quad
      M-B

      18.70
      1.300 AN
      ISTD内部
      区域

      B-M-B
      GalA
      26.26
      0.800公司
      内肋
      区域

      B-M-B
      GlcA
      28.40
      0.800公司
      内肋
      区域

      B-M-B

      表3. Chromeleon 6.8 De 部分注释单糖峰的参数
      时间
      参数。 na m e
      参数。 值
      频道
      0.00
      禁止集成

      所有频道
      3.60
      禁止集成
      关闭
      所有频道
      4.10
      最低面积
      0.5
      所有频道
      4.75
      最小区域
      1
      所有频道
      5.00
      禁止集成

      所有频道
      6.60
      禁止集成
      关闭
      所有频道
      6.60
      锁定基准

      所有频道
      7.25
      最小区域
      0.5
      所有频道
      9.80
      锁定基准
      关闭
      所有频道
      9.80
      禁止集成

      所有频道
      10.20
      禁止集成
      关闭
      所有频道
      10.40
      锁定基准

      所有频道
      12.70
      禁止集成

      所有频道
      12.80
      锁定基准
      关闭
      所有频道
      13.10
      禁止集成
      关闭
      所有频道
      14.00
      最小区域
      0.25
      所有频道
      14.00
      锁定基准

      所有频道
      16.00
      锁定基准
      关闭
      所有频道
      18.00
      最小区域
      10
      所有频道
      20.50
      禁止集成

      所有频道
      25.50
      禁止集成
      关闭
      所有频道
      25.60
      最小区域
      0.5
      所有频道
      27.00
      禁止集成

      所有频道
      28.20
      禁止集成
      关闭
      所有频道
      28.20
      最小区域
      0.5
      所有频道
      29.00
      禁止集成

      所有频道

    9. 检查Chromeleon中的色谱图以确保所有峰都是正确标记。 手动注释Ara和Man的峰 必需,与更丰富的糖重叠。
    10. 通过定义   在Chromeleon中的标准和浓度(表1和2), 自动生成所有单糖的校准曲线, 归一化为Rib(内标)。
      注意:如有必要,请检查校准图并禁用重要的异常值。
    11. 样品中所有单糖的量(以μg表示) 基于校准曲线自动计算并且可以 从Chromeleon导出进一步计算。
      注意:Fuc是 在拟南芥的总粘液提取物中以痕量检测 种子(图3; Voiniciuc等,2015c),而GlcA通常不是 在这些样本中检测到。


      图3.导出的示例色谱图 从Chromeleon软件。 垂直轴表示检测到 信号(nC)。横轴表示注射后时间(min)。

  6. 最终计算和统计分析
    进一步的计算在Microsoft Excel中执行。总粘液提取物的单糖组成可以表示为绝对(图4A)或相对量(图4B)。


    图4.来自Col-0野生型的总粘液提取物的组成。数据显示三个生物重复的平均值。 (A)中的误差条表示标准偏差。

    1. 糖的量(以μg表示)可以除以种子的量 (以mg表示)用于每次粘液提取,计算绝对组成(μg糖/mg种子)。
      注意:每mg种子的粘液总量是样品中所有糖值的总和。
    2. 绝对单糖水平也可以表示为数字 的分子归一化到所用种子的量。 计算 nmol糖/mg种子,将步骤F1中获得的值除以 相应糖的分子量(表4),并乘以 结果1000。

      表4.最终计算的单糖的摩尔质量
      S uga r
      Fuc
      Rha
      Ara
      Gal
      Glc
      Xyl
      男士
      GalA
      GlcA
      质量
      164.16
      164.16
      150.13
      180.16
      180.16
      150.13
      180.16
      194.14
      194.14

    3. 粘液中的相对单糖组成可以表示为a   摩尔百分比(mol%),并且等于所分割的每种单体的量 除以所有糖的总和(均来自步骤F2)
    4. 统计 分析以比较野生型和突变体的粘液组成 可以在Excel中执行。 特定的重大变化 单糖(以绝对或相对量表示) 之间的两个基因型与多个生物复制可以 使用内置的T.TEST功能识别。 当许多突变体(和 其各种糖组分)与野生型,有条件的 格式化(例如 使用 P - 值<0.05)突出显示单元格可用于快速揭示哪些组件被显着改变。 双因素 方差分析(ANOVA)也可以在Excel中进行 实际统计资源包( http://www.real-statistics.com/) 评估如何两种独立的粘液组成 突变(通过野生型,单和双突变体的分析 样品)。

食谱

  1. ½MS板
    对于500ml溶液(产生约25个板),混合1.08g MS基础盐,3.5g琼脂和水。
    高压灭菌,并在干净的工作台上将培养基(仍然温热)倒入无菌培养皿中。 当保存在无菌袋中时,板可以在4℃下储存至少6个月。
  2. 糖标准品(10 mg/ml)
    对于除GalA和Rha之外的每种单糖,通过将100mg糖溶解在10ml无菌15ml Falcon管中的经高压灭菌的超纯水中来制备单独的原液。
    由于它们以一水合物形式出售,使用109mg GalA和111mg Rha而不是标准100mg。
    等分Rib股票2毫升安全锁管,因为它经常使用。
    将所有原液储存在-20°C。
  3. 9-糖混合物(1mg/ml)
    对于10ml溶液,将1ml水与1ml Fuc,Rha,Ara,Gal,Glc,Xyl,Man,GalA和GlcA(全部10mg/ml储液)混合。
    将9-Sugar混合物分装到2ml Safe Lock试管中,并储存在-20℃
  4. 2 M三氟乙酸(TFA) 通过向423ml超纯水中缓慢加入77ml TFA(12.98M)制备500ml 2M TFA溶液。
    注意:在通风橱中使用适当的个人防护装备进行TFA。
  5. 10mM NaOH(IC洗脱液)
    使用容量瓶将2 L超纯水转移到适当的洗脱液容器
    使用1ml血清移液管加入1040μl50%(w/v)NaOH 使用氦气脱气洗脱液5分钟
  6. 733mM NaOH(IC洗脱液)
    使用容量瓶将2 L超纯水转移到适当的洗脱液容器
    使用50ml血清移液管加入总共80ml的50%(w/v)NaOH 使用氦气脱气洗脱液5分钟

致谢

本文描述的方法源自(Voiniciuc等人,2015c),并且由(Voiniciuc等人,2015a; Voiniciuc等人,/em>,2015b)。我们感谢BjörnUsadel对协议有帮助的意见。这项工作由加拿大自然科学和工程研究理事会(PGS-D3授予C.V.)支持。作者贡献:C.V.和M.G开发了该方法。 C. V.写了协议。

参考文献

  1. Haughn,G. W.和Western,T.L。(2012)。 拟南芥种衣液是一种特殊的细胞壁,可用作植物细胞壁结构和功能的遗传分析模型。前植物科学 3:64.
  2. North,H.M.,Berger,A.,Saez-Aguayo,S.and Ralet,M.C。(2014)。 使用种皮突变体了解多糖生产和特性:开发天然变体的未来前景。 a> Ann Bot 114(6):1251-1263。
  3. Voiniciuc,C.,Gunl,M.,Schmidt,M. H.和Usadel,B。(2015a)。 由IRREGULAR XYLEM14和MUCILAGE-RELATED21制造的高支化木聚糖将粘液连接至拟南芥 >种子。 植物生理学 169(4):2481-2495。
  4. Voiniciuc,C.,Schmidt,M. H.,Berger,A.,Yang,B.,Ebert,B.,Scheller,H. V.,North,H. M.,Usadel,B.and Gunl, MUCILAGE-RELATED10可产生在拟南芥种子中保持果胶和纤维素结构的半乳葡甘露聚糖粘液。植物生理学169(1):403-420。
  5. Voiniciuc,C.,Yang,B.,Schmidt,M. H.,Gunl,M。和Usadel,B.(2015c)。 开始凝胶:如何拟南芥种皮表皮细胞产生特化的继发细胞 。Int J Mol Sci 16(2):3452-3473。

  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
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.
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