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Quantification of the Mucilage Detachment from Arabidopsis Seeds
拟南芥种子粘液释放的定量测定

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Abstract

The Arabidopsis thaliana seed coat produces large amounts of cell wall polysaccharides, which swell out of the epidermal cells upon hydration of the mature dry seeds. While most mucilage polymers immediately diffuse in the surrounding solution, the remaining fraction tightly adheres to the seed, forming a dense gel-like capsule (Macquet et al., 2007). Recent evidence suggests that the adherence of mucilage is mediated by complex interactions between several cell wall components (Griffiths et al., 2014; Voiniciuc et al., 2015a). Therefore, it is important to evaluate how different cell wall mutants impact this mucilage property. This protocol facilitates the analysis of monosaccharides in sequentially extracted mucilage fractions, and quantifies the detachment of each component from seeds.

Keywords: Mucilage adherence(粘液的粘附), Sequential extractions(顺序提取), Ion chromatography(离子色谱法), Monosaccharides(单糖), Plant cell wall(植物细胞壁)

Materials and Reagents

  1. 15 ml Falcon tubes (VWR International, catalog number: 734-0452 )
  2. 2 ml Eppendorf safe-lock tubes (VWR International, catalog number: 211-2165 )
  3. 2 ml screw-cap tubes (VWR International, catalog number: 211-0093 )
  4. Chromatography vials with inserts (VWR International, catalog number: 548-0120 )
  5. Snap cap for chromatography vials (VWR International, catalog number: 548-1151 )
  6. Manual pipettes tips
  7. Arabidopsis thaliana seeds
  8. 2-Deoxy-D-glucose (2-deoxy-Glc) (Sigma-Aldrich, catalog number: D6134-1 G )
  9. D-(-)-Ribose (Rib) (Sigma-Aldrich, catalog number: R7500-5 G )
  10. L-(+)-Arabinose (Ara) (Sigma-Aldrich, catalog number: A3256-25 G )
  11. L-(-)-Fucose (Fuc) (Sigma-Aldrich, catalog number: F2252-5 G )
  12. D-(+)-Galactose (Gal) (Sigma-Aldrich, catalog number: G0750-25 G )
  13. D-(+)-Galacturonic acid monohydrate (GalA) (Sigma-Aldrich, catalog number: 48280-5G-F )
  14. D-(+)-Glucose (Glc) (Sigma-Aldrich, catalog number: G8270-100 G )
  15. D-Glucuronic acid (GlcA) (Sigma-Aldrich, catalog number: G5269-10 G )
  16. D-(+)-Mannose (Man) (Sigma-Aldrich, catalog number: M8574-25 G )
  17. L-Rhamnose monohydrate (Rha) (Sigma-Aldrich, catalog number: R3875-5 G )
  18. D-(+)-Xylose (Xyl) (Sigma-Aldrich, catalog number: X3877-25 G )
  19. Ultrapure water (18.2 MΩ•cm at 25 °C)
  20. Sodium hydroxide (NaOH) (VWR International, catalog number: BAKR3727.2500 )
  21. Trifluoroacetic acid (TFA) (Carl Roth GmbH + Co., catalog number: 6957.1 )
  22. Sugar standard stocks (see Recipes)
  23. 9-Sugar mix (see Recipes)
  24. 2 M TFA (see Recipes)
  25. 10 mM NaOH (see Recipes)
  26. 733 mM NaOH (see Recipes)

Equipment

  1. Autoclave
  2. Water purification system (Milli-Q or similar style)
  3. Manual pipettes (Eppendorf AG, Research plus and Repeater Plus style)
  4. Analytical balance (Mettler-Toledo, model: XSE205DU )
  5. Ball mill (Retsch GmbH, catalog number: MM400 )
  6. Two 24 TissueLyser adapters for ball mill (QIAGEN, catalog number: 69982 )
  7. Lab coat
  8. Safety glasses
  9. Chemical resistant gloves (Honeywell, Dermatril, catalog number: 740 , or similar style)
  10. Fume hood
  11. 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
  12. Ice machine
  13. 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 )
  14. Vortex mixer (Scientific Industries Inc., model: Vortex-Genie 2 , or similar style)
  15. Benchtop centrifuge (compatible with 2 ml tubes)
  16. Racks with lids for 2 ml tubes (VWR International, catalog number: 211-0215 )
  17. Serological pipettes (Thermo Fisher Scientific, Nunc-type or similar style)
  18. 2 L volumetric flask
  19. 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
    Since mucilage phenotypes are influenced by how seeds are produced, harvested and stored (see recent review, Voiniciuc et al., 2015c), it is essential to analyze seeds grown under the same conditions. I recommend the procedures described by Voiniciuc and Günl (2016), which yield consistent seed quality and mucilage chemotypes across multiple generations (Voiniciuc et al., 2015a; Voiniciuc et al., 2015b; Voiniciuc et al., 2015c)

  2. Extraction of non-adherent mucilage
    1. For each experiment (up to 48 samples), prepare a spreadsheet with the relevant fields (e.g. Sample #; Seed Weight; Seed Bag; Genotype). To simplify labeling, assign a two-digit number to each sample (e.g. 01 to 48).
      Note: Two sequential extractions will be performed for each sample.
    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 the standards listed in Table 1, by adding the shown volumes to 2 ml Screw-Cap Tubes.

      Table 1. Dilution series of 9-Sugar mix (10 mg/ml) 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 #1) solutuion 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 #1 to all samples and standards.
    6. Manually invert the rack with the sample tubes to suspend all seeds in solution. Place the samples horizontally in a box (large enough to have a single layer of tubes). Shake for 30 min at 200 rpm using an orbital shaker, at room temperature (~24 °C).
      Note: This treatment detaches only the non-adherent mucilage from seeds (Figure 1B).


      Figure 1. Mucilage staining with ruthenium red (RR) after sequential extractions. Wild-type (Col-0) seeds stained directly in RR, without shaking (A), or after the non-adherent (B) and adherent (C) sequential extractions. Bars = 0.4 mm

    7. Let the 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 with the letter N (for non-adherent mucilage extraction) followed by the sample number.
      Note: Do not transfer any seeds, and do not cap the tubes at this point.
    8. Evaporate the non-adherent mucilage samples and standards under air flow at 45 °C using the sample concentrator.
    9. Wash the seeds in the 2 ml Safe Lock tubes with 1 ml water, and discard the solution.
      Note: Ensure that no seeds are removed.
    10. Repeat step B9 once. Remove as much of the remaining water as possible, without losing seeds.
      Note: The seeds can then be used immediately for the Adherent Mucilage Extraction if a second sample concentrator is available. Alternatively, store the rinsed seeds at -20 °C, and complete the analysis of the non-adherent mucilage samples (drying, hydrolysis, and final elution) before starting the second extraction.

  3. Extraction of adherent mucilage
    1. Prepare a new set of 10 standards in 2 ml Screw-Cap Tubes for the adherent mucilage quantification, by adding the volumes listed in Table 1.
    2. Prepare enough 30 µg/ml 2-deoxy-Glc solution (Internal Standard #2) to add 1 ml to all samples and sugar standards.
    3. Using a repeater pipette, add 1 ml of Internal Standard #2 to the new standards, and to the rinsed seeds in Safe Lock tubes (from non-adherent mucilage extraction; completely thawed, if previously frozen).
    4. Remove the adherent mucilage by essentially performing a total mucilage extraction (Voiniciuc and Günl, 2016). Shake the seed-containing tubes for 15 min at 30 Hz in a ball mill using two 24 TissueLyser Adapters, at room temperature (~24 °C).
    5. While the seeds are shaking, pre-label the sides of 2 ml Screw-Cap tubes with the letter A (for adherent mucilage extraction) followed by each sample number.
    6. Rotate block 180° and shake for another 15 min at 30 Hz to finish the mucilage extraction.
      Note: This detaches all the adherent mucilage from seeds (Figure 1C).
    7. Let the seeds settle for at least 30 sec. For each sample, transfer 800 µl of the supernatant to the corresponding 2 ml Screw-Cap tube (pre-labeled in step C5).
      Note: Do not transfer any seeds, and do not screw the caps on the tubes at this point.
    8. Dry the adherent mucilage samples and the new standards under air flow at 45 °C using the sample concentrator. Process the samples and standards identically from this point onwards.

  4. Hydrolysis of matrix polysaccharides
    Note: The same steps are used for the preparation of both mucilage extracts. Only hydrolyze the two extracts in parallel if there is sufficient space in the Techne Dri-blocks for all samples and standards.
    1. Using a repeater pipette, add 300 µl of 2 M TFA to all tubes.
    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.

  5. 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.

  6. Separation and quantification of monosaccharides
    1. Perform 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, essentially as described by Voiniciuc and Günl (2016).
    2. Ensure that sufficient volumes of the three eluents (ultrapure water, 10 mM NaOH, and 733 mM NaOH) are available to run all the samples and standards for both mucilage extractions.
    3. Equilibrate the columns for 10 min with 2 mM NaOH (80% water, 20% 10 mM NaOH).
    4. Inject 10 µl of each sample.
    5. Separate neutral sugars over 18 min with 2 mM NaOH (80% water, 20% 10 mM NaOH).
    6. Pump 513 mM NaOH (70% 733 mM NaOH, 30% water) for 7.5 min to separate uronic acids.
    7. Finally, rinse the column with 733 mM NaOH for 4 min.
      Note: Repeat the methods in steps F3-7 for each sample and standard.
    8. Automatically annotate the monosaccharide peaks by configuring the Peak Table and Detection Parameters in the Chromeleon Chromatography Data System software (Dionex) as described by Voiniciuc and Günl (2016).
      Note: Minor adjustments are required to annotate the peak of 2-deoxy-Glc (retention time of 2.5 to 3.0 min), and to set it as the internal standard for the adherent mucilage samples and the corresponding standards.
    9. Inspect chromatograms in Chromeleon to ensure that all peaks are correctly labeled.
      Note: If necessary, manually correct any peaks that are mislabeled.
    10. All monosaccharide amounts are normalized to the internal standard in each extraction, and quantified as µg based on calibration plots of the standard dilution series (Table 1).  
      Note: Only trace levels of Rib are detected in the adherent mucilage samples, indicating that two water washes effectively remove carryover of the non-adherent mucilage solution. Therefore, 30 µg Rib could be safely used as the internal standard for the adherent mucilage extracts, eliminating the need to adjust the detection parameters for 2-deoxy-Glc.
    11. Export the amount of monosaccharides in each sample (expressed in µg) to Microsoft Excel for the final calculations.

  7. Final calculations and statistical analyses
    The amounts of monosaccharides detected in the sequential extracts can be expressed as absolute levels (Figure 2A), or as the relative detachment of each sugar from the seed (Figure 2B).


    Figure 2. Quantification of mucilage detachment based on sequential extractions. A. Absolute amounts of mucilage sugars in the non-adherent and adherent extracts. B. The detachment of individual mucilage sugars or their combined sum, expressed as a percent of the total amount detected in two sequential extracts. Graphs show means of + SD of five biological replicates for previously analyzed wild type (Col-0) seeds (Voiniciuc et al., 2015b).

    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: Perform this calculation separately for the non-adherent and adherent extractions.
    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 G1 by the molecular mass of the respective sugar (Table 2), and multiply the result by 1,000.
      Note: Perform this calculation separately for each extraction (Figure 2A).

      Table 2. 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. The relative detachment of each mucilage component (expressed as a percentage) is equal to its non-adherent amount divided by the sum of that particular monomer in the non-adherent and adherent fractions. The overall detachment of seed mucilage is the ratio of non-adherent sugars to the total amount of sugars sequentially extracted (based on values calculated in step G1).
    4. Statistical analyses to compare the mucilage detachment of wild-type and mutants can be performed in Excel. The absolute amount of particular monosaccharide in a mucilage fraction, or its relative detachment from the seed can be compared between two genotypes with multiple biological replicates using the Excel 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 detachment is affected by two independent mutations (via the analysis of wild-type, single and double mutant samples).
      Note: In several cell wall mutants, affecting genes with distinct functions, around 90% of Rha and GalA molecules (the most abundant sugars in mucilage) are non-adherent (Voiniciuc et al., 2015a). Although the loss of different genes caused similar increases in mucilage detachment, the precise mechanism that anchors mucilage to the seed surface remains to be elucidated.

Recipes

  1. 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 2-deoxy-Glc and Rib stocks into 2 ml Safe Lock tubes since they are frequently used.
    Store all stocks at -20 °C.
  2. 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.
  3. 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.
  4. 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
  5. 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 sequential extraction method presented was derived from (Voiniciuc et al., 2015b), and also briefly described by (Voiniciuc et al., 2015a). This work extends the protocol by Voiniciuc and Günl (2016), and was supported by the Natural Sciences and Engineering Research Council of Canada (PGS-D3 grant to C. V.).

References

  1. Griffiths, J. S., Tsai, A. Y., Xue, H., Voiniciuc, C., Sola, K., Seifert, G. J., Mansfield, S. D. and Haughn, G. W. (2014). SALT-OVERLY SENSITIVE5 mediates Arabidopsis seed coat mucilage adherence and organization through pectins. Plant Physiol 165(3): 991-1004.
  2. Macquet, A., Ralet, M. C., Kronenberger, J., Marion-Poll, A. and North, H. M. (2007). In situ, chemical and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage. Plant Cell Physiol 48(7): 984-999.
  3. Voiniciuc, C. and Günl, M. (2016). Analysis of monosaccharides in total mucilage extractable from Arabidopsis seeds. Bio-protocol 6(9): e1801.
  4. 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.
  5. 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.
  6. 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

拟南芥种子产生大量的细胞壁多糖,其在成熟干种子水合时从表皮细胞中膨胀。 虽然大多数粘液聚合物立即在周围溶液中扩散,但剩余部分紧密粘附于种子,形成致密的凝胶状胶囊(Macquet等人,2007)。 最近的证据表明粘液的粘附由几个细胞壁组分之间的复杂相互作用介导(Griffiths等人,2014; Voiniciuc等人,2015a)。 因此,重要的是评估不同的细胞壁突变体如何影响这种粘液性质。 该协议有助于分析顺序提取的粘液部分中的单糖,并且量化每种成分从种子中的脱离。

关键字:粘液的粘附, 顺序提取, 离子色谱法, 单糖, 植物细胞壁

材料和试剂

  1. 15ml Falcon管(VWR International,目录号:734-0452)
  2. 2ml Eppendorf安全锁管(VWR International,目录号:211-2165)
  3. 2ml螺旋盖管(VWR International,目录号:211-0093)
  4. 具有插入物的色谱小瓶(VWR International,目录号:548-0120)
  5. 用于色谱小瓶(VWR International,目录号:548-1151)的快速盖子
  6. 手动移液器提示
  7. 拟南芥种子
  8. 2-脱氧-D-葡萄糖(2-脱氧-Glc)(Sigma-Aldrich,目录号:D6134-1G)
  9. D - ( - ) - 核糖(Rib)(Sigma-Aldrich,目录号:R7500-5G)
  10. L - (+) - 阿拉伯糖(Ara)(Sigma-Aldrich,目录号:A3256-25G)
  11. L - ( - ) - 岩藻糖(Fuc)(Sigma-Aldrich,目录号:F2252-5G)
  12. D - (+) - 半乳糖(Gal)(Sigma-Aldrich,目录号:G0750-25G)
  13. D - (+) - 半乳糖醛酸一水合物(GalA)(Sigma-Aldrich,目录号:48280-5G-F)
  14. D - (+) - 葡萄糖(Glc)(Sigma-Aldrich,目录号:G8270-100G)
  15. D-葡萄糖醛酸(GlcA)(Sigma-Aldrich,目录号:G5269-10G)
  16. D - (+) - 甘露糖(Man)(Sigma-Aldrich,目录号:M8574-25G)
  17. L-鼠李糖一水合物(Rha)(Sigma-Aldrich,目录号:R3875-5G)
  18. D - (+) - 木糖(Xyl)(Sigma-Aldrich,目录号:X3877-25G)
  19. 超纯水(25℃时为18.2MΩ·cm)
  20. 氢氧化钠(NaOH)(VWR International,目录号:BAKR3727.2500)
  21. 三氟乙酸(TFA)(Carl Roth GmbH + Co.,目录号:6957.1)
  22. 糖标准储备(见配方)
  23. 9糖混合(见配方)
  24. 2 M TFA(参见配方)
  25. 10 mM NaOH(参见配方)
  26. 733mM NaOH(参见配方)

设备

  1. 高压灭菌器
  2. 水净化系统(Milli-Q或类似风格)
  3. 手动移液器(Eppendorf AG,Research plus和Repeater Plus样式)
  4. 分析天平(Mettler-Toledo,型号:XSE205DU)
  5. 球磨机(Retsch GmbH,目录号:MM400)
  6. 两个用于球磨机的24个TissueLyser适配器(QIAGEN,目录号:69982)
  7. 实验室外套
  8. 安全眼镜
  9. 耐化学性手套(Honeywell,Dermatril,目录号:740或类似风格)
  10. 通风橱
  11. 样品浓缩器(Bibby Scientific Limited,Techne,目录号:FSC400D),配备
    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. 连接到中央气源,在通风橱
  12. 制冰机
  13. 离子色谱(IC):配有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)
  14. 涡流混合器(Scientific Industries Inc.,型号:Vortex-Genie 2或类似型)
  15. 台式离心机(与2 ml管相容)
  16. 带有用于2ml管的盖的架(VWR International,目录号:211-0215)
  17. 血清移液管(Thermo Fisher Scientific,Nunc型或类似样式)
  18. 2升容量瓶
  19. 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
    由于粘液表型受到种子如何产生,收获和储存的影响(参见最近的综述,Voiniciuc等人,2015c),因此必须分析在相同条件下生长的种子。我推荐Voiniciuc和Günl(2016)描述的程序,其在多代中产生一致的种子质量和粘液化学型(Voiniciuc等人,2015a; Voiniciuc等人, ,2015b; Voiniciuc 等人,2015c)

  2. 非粘连胶浆的提取
    1. 对于每个实验(最多48个样本),请准备包含相关字段的电子表格(例如样品#;种子重量;种子袋;基因型)。为了简化标记,请为每个样本指定两位数字(例如 01至48)。
      注意:每个样品将执行两次连续提取。
    2. 使用样品编号预先标记2 ml Safe Lock试管的侧面。使用分析天平为每个管添加4-6 mg种子,并在电子表格中记录精确重量。  
    3. 通过将所示体积加到2ml螺旋盖管中来制备表1中列出的标准
      表1.用于单糖定量的9-糖混合物(10 mg/ml)的稀释系列
      标签
      S000
      S001
      S002
      S005
      S010
      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(内部标准#1)解决方案,在一个实验中添加1毫升到所有样品和糖标准。对于具有48个样品和10个标准品的典型实验,加入180μl的10mg/ml肋,并用超纯水填充至60ml。
    5. 使用中继移液器,向所有样品和标准品中加入1ml内标#1
    6. 用样品管手动倒置样品架,将所有种子悬浮在溶液中。将样品水平放置在一个盒子(足够大,有单层管)。在室温(〜24℃)下使用轨道振荡器在200rpm下摇动30分钟 注意:这种处理只从种子上剥离非粘性胶浆(图1B)。 。

      图1.在RR中直接染色的野生型(Col-0)种子,没有振荡(A),或在非粘附物(B)之后, )和粘附(C)连续提取。条= 0.4mm

    7. 让种子沉淀至少30秒。对于每个样品,将800μl上清液转移到2ml预先用字母N标记的螺旋帽管(用于非粘附胶浆提取)随后是样品编号。
      注意:请勿转移任何种子,并且不要在这一点上盖住管。
    8. 使用样品浓缩器在45℃的气流下蒸发非粘附的粘液样品和标准品。
    9. 用1ml水洗涤2ml Safe Lock试管中的种子,弃去溶液 注意:请确保不删除种子。
    10. 重复步骤B9一次。 尽可能多地去除剩余的水,而不损失种子。
      注意:如果有第二个样品浓缩器,那么可以立即将种子用于粘附粘性提取。 或者,将冲洗的种子储存在-20℃,并在开始第二次提取之前完成非粘附胶浆样品的分析(干燥,水解和最终洗脱)。

  3. 提取粘附胶浆
    1. 通过添加表1中列出的体积,在2 ml螺旋盖管中制备一套新的10个标准品用于粘附胶浆定量。
    2. 准备足够的30μg/ml 2-脱氧-Glc溶液(内标#2),以添加1毫升到所有样品和糖标准。
    3. 使用中继器移液管,添加1毫升内标#2到新的标准,并在安全锁管中(从非粘附胶浆提取;完全解冻,如果以前冷冻)冲洗的种子。
    4. 通过基本上进行全部粘液提取去除粘附的粘液(Voiniciuc和Günl,2016)。在室温(〜24℃)下,在球磨机中使用两个24个TissueLyser适配器在30Hz下将含种子管摇动15分钟。
    5. 当种子振动时,用信号A(用于粘附胶浆提取),随后为每个样品编号预先标记2ml螺旋盖管的侧面。
    6. 旋转块180°,并在30 Hz下再振动15分钟,以完成粘液提取。
      注意:这会从种子上剥离所有的粘性胶浆(图1C)。
    7. 让种子沉淀至少30秒。对于每个样品,将800μl上清液转移到相应的2ml螺旋帽管(在步骤C5中预先标记)。
      注意:不要转移任何种子,此时不要将瓶盖拧在管子上。
    8. 使用样品浓缩器在45℃的空气流下干燥粘附的粘液样品和新标准品。从这一点向前处理样品和标准相同。

  4. 基质多糖水解
    注意:相同的步骤用于制备两种粘液提取物。如果Techne Dri-blocks中有足够的空间用于所有样品和标准,则只能平行水解两种提取物。
    1. 使用中继器移液管,加入300μl的2M TFA到所有管。
    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。

  5. 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:如果使用中振动强度和小心,管不需要加盖此步骤,因为解决方案不会溢出。
    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. 以升序将管置于Dionex DX-600系统的自动进样器中,但将样品和标准品的注射顺序随机(例如使用Microsoft Excel)。

  6. 单糖的分离和定量
    1. 基本上如Voiniciuc和Günl(2016)所述,使用CarboPac PA20 Guard和分析柱在40℃和0.4ml/min的恒定流速下进行基于脉冲安培检测(HPAEC-PAD)的高效阴离子交换层析, 。
    2. 确保足够体积的三种洗脱液(超纯水,10mM NaOH和733mM NaOH)可用于运行所有的样品和标准用于两种粘液提取。
    3. 用2mM NaOH(80%水,20%10mM NaOH)平衡柱10分钟
    4. 注入10μl每个样品。
    5. 用2mM NaOH(80%水,20%10mM NaOH)在18分钟内分离中性糖
    6. 泵513mM NaOH(70%733mM NaOH,30%水)7.5分钟以分离糖醛酸
    7. 最后,用733mM NaOH冲洗该柱4分钟。
      注意:对于每个样品和标准,重复步骤F3-7中的方法。
    8. 如Voiniciuc和Günl(2016)所述,通过配置Chromeleon色谱数据系统软件(Dionex)中的峰表和检测参数自动注释单糖峰。
      注意:需要进行少量调整以注释2-脱氧-Glc的峰(保留时间为2.5至3.0分钟),并将其设定为粘附的粘液样品和相应标准品的内标。
    9. 检查Chromeleon中的色谱图,以确保所有峰都正确标记。
      注意:如有必要,请手动更正任何错误标记的峰。
    10. 在每次提取中,将所有单糖量标准化为内标,并基于标准稀释系列的校准曲线以μg表示(表1)。  
      注意:在粘附的粘液样品中仅检测到痕量的Rib,表明两次水洗有效地去除了非粘附的粘液溶液的携带。因此,30μgRib可以安全地用作粘附胶浆提取物的内标,无需调整2-脱氧-Glc的检测参数。
    11. 将每个样品中的单糖数量(以μg表示)导出到Microsoft Excel中进行最终计算

  7. 最终计算和统计分析
    在连续提取物中检测到的单糖的量可以表示为绝对水平(图2A),或者表示为每种糖与种子的相对脱离(图2B)。


    图2.基于连续提取的粘液脱离的定量 A.非粘附和粘附提取物中的粘液糖的绝对量。 B.单独的粘液糖或它们的组合总和的脱离,表示为在两个连续提取物中检测到的总量的百分比。图表显示先前分析的野生型(Col-0)种子的五个生物学重复的平均值±SD(Voiniciuc等人,2015b)。
    1. 糖量(以μg表示)可除以用于每次粘液提取的种子量(以mg表示),以计算绝对组成(μg糖/mg种子)。
      注意:对于非粘附和粘附提取单独执行此计算。
    2. 绝对单糖水平也可以表示为相对于所用种子的量标准化的分子数。 为了计算每mg种子的nmol糖,将步骤G1中获得的值除以相应糖的分子量(表2),并将结果乘以1000。
      注意:对每个提取单独执行此计算(图2A)。

      表2.最终计算的单糖的摩尔质量

      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. 每种粘液成分的相对脱离(以百分比表示)等于其非粘附量除以非粘附部分和粘附部分中的特定单体的总和。种子粘液的整体脱离是非附着糖与顺序提取的糖总量(基于步骤G1中计算的值)的比率。
    4. 比较野生型和突变体的粘液脱离的统计分析可以在Excel中进行。可以使用Excel内置的T.TEST函数比较具有多个生物学复制的两种基因型之间的粘液级分中特定单糖的绝对量或其与种子的相对脱离。当许多突变体(及其各种糖组分)与野生型比较时,可以使用条件格式化(例如 突出显示具有 P - 值<0.05的细胞)揭示哪些组件被显着改变。双因素方差分析(ANOVA)也可以在Excel中使用真实统计资源包( http://www.real-statistics.com/),以评估两个独立突变(通过野生型,单个和双重突变体样品的分析)如何影响粘液分离。
      注意:在几种细胞壁突变体中,影响具有不同功能的基因,约90%的Rha和GalA分子(粘液中最丰富的糖)是非粘附的(Voiniciuc等人,2015a)。尽管不同基因的损失导致了粘液脱离的类似增加,但是将粘液锚定到种子表面的精确机制仍有待阐明。

食谱

  1. 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。
    将2-脱氧-Glc和Rib储液分装到2ml Safe Lock试管中,因为它们经常使用。
    将所有原液储存在-20°C。
  2. 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℃
  3. 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。
  4. 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
  5. 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

所提出的顺序提取方法来自(Voiniciuc等人,2015b),并且也简要地描述(Voiniciuc等人,2015a)。 这项工作扩展了Voiniciuc和Günl(2016)的协议,并得到加拿大自然科学和工程研究委员会(PGS-D3授予C.V.)的支持。

参考文献

  1. Griffiths,J.S.,Tsai,A.Y.,Xue,H.,Voiniciuc,C.,Sola,K.,Seifert,G.J.,Mansfield,S.D.and Haughn,G.W。 SALT-OVERLY SENSITIVE5介导拟南芥种皮粘液粘附和通过果胶的组织。 Plant Physiol 165(3):991-1004。
  2. Macquet,A.,Ralet,M.C.,Kronenberger,J.,Marion-Poll,A.and North,H.M。(2007)。 原位,化学和大分子研究的组成 Arabidopsis thaliana seed coat mucilage 。植物细胞生理学48(7):984-999。
  3. Voiniciuc,C。和Günl,M.(2016)。 可从拟南芥种子提取的总粘液中的单糖分析 生物协议 6(9):e1801。
  4. 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。
  5. 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
  6. 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。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Voiniciuc, C. (2016). Quantification of the Mucilage Detachment from Arabidopsis Seeds. Bio-protocol 6(9): e1802. DOI: 10.21769/BioProtoc.1802.
  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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