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Root Aliphatic Suberin Analysis Using Non-extraction or Solvent-extraction Methods
不萃取或溶剂萃取法分析根脂肪族木栓质   

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参见作者原研究论文

本实验方案简略版
Plant Cell Reports
Apr 2015

Abstract

Here we describe both non-extraction and solvent-extraction methods for root aliphatic suberin analysis. The non-extraction method is fast as roots are directly depolymerized using acidic transmethylation. However, suberin aliphatic components are isolated together with all the other acyl chains making up the lipids (e.g., membranes) present in roots. For the solvent-extraction method, roots are first delipidated before transmethylation. This method is longer but allows separation of soluble and polymerized root lipids. This protocol is optimized for tissue culture- or soil-grown Arabidopsis thaliana plants, but can be used with roots of other plants.

Keywords: Suberin (木栓质), Lipid polymer (脂质聚合物), Cell wall (细胞壁), Root (根), Lipid extraction (脂质提取), Gas chromatography (气相色谱法), Arabidopsis thaliana (拟南芥)

Background

Suberin is an extracellular plant lipid polymer deposited in the cell walls of various tissues such as endodermis, exodermis and periderm of roots. Suberin acts as a barrier controlling water and solute fluxes and restricting pathogen infections (Ranathunge et al., 2011; Andersen et al., 2015; Vishwanath et al., 2015; Barberon et al., 2016). Suberin is a complex heteropolymer made up of aliphatics, phenolics, and glycerol, which is associated with solvent-extractable waxes (Bernards, 2002). In the model plant Arabidopsis thaliana, the suberin polymer is primarily made of ω-hydroxy acids and α,ω-dicarboxylic acids, but it also contains unsubstituted fatty acids and primary fatty alcohols (Domergue et al., 2010; Vishwanath et al., 2013), whereas the associated waxes are in the form of alkyl hydroxycinnamates (AHCs; Kosma et al., 2012; Delude et al., 2016). The non-extraction method described here allows for high-throughput and rapid analysis of suberin composition, which is particularly advantageous when screening large numbers of plant lines (e.g., mutants, overexpressing transgenic lines, or natural variants). A more traditional and accurate solvent extraction method applicable when soluble and polymerized lipids (i.e., suberin polyester) need to be analyzed separately is included for comparison.

Materials and Reagents

  1. Petri dish (90 mm diameter) (Fisher Scientific)
  2. Peat moss, vermiculite and perlite (3:1:1, v/v/v; Medan S.A.)
  3. Paper towels
  4. 8 ml glass tubes (Dutscher, catalog number: 065307B ) with polytetrafluoroethylene (PTFE)-lined caps (Dutscher, catalog number: 001031 )
  5. Pots for Arabidopsis (Polystyrene, 9 x 9 x 9.5 cm) (SOPARCO, catalog number: 4686 )
  6. 2 ml GC vials with caps (Agilent Technologies, catalog number: 5182-0557 ) and 400 μl flat bottom glass inserts (Agilent Technologies, catalog number: 5181-3377 )
  7. Arabidopsis seeds
  8. 95% ethanol
  9. Sodium hypochlorite (Bleach)
  10. Distilled water
  11. Isopropanol (Fisher Scientific, catalog number: 10315720 )
  12. Chloroform (Sigma-Aldrich, catalog number: 32211 )
  13. Methanol (Sigma-Aldrich, catalog number: 34885 )
  14. Nitrogen (H2O < 3 ppm; CnHm < 0.5 ppm; O2 < 2 ppm)
  15. Sulfuric acid (H2SO4) (CARLO ERBA Reagents, catalog number: E410391 )
  16. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 )
  17. Methyl tert-butyl ether (MTBE) (Sigma-Aldrich, catalog number: 20256 )
  18. Tri(hydroxymethyl)aminomethane (Tris base) (Sigma-Aldrich, catalog number: T6066 )
  19. N,O-Bis(trimethylsilyl)-trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) (Sigma-Aldrich, catalog number: T6381 )
  20. Heptane (Sigma-Aldrich, catalog number: 32287 )
  21. Toluene (Sigma-Aldrich, catalog number: 32249 )
  22. Murashige and Skoog medium (Duchefa Biochemie, catalog number: M0222.0050 )
  23. Vitamins
  24. Plant agar (Duchefa Biochemie, catalog number: P1001.1000 )
  25. 2-(N-Morpholino)-ethane sulfonic acid (MES) (Euromedex, catalog number: EU0033-B )
  26. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: P5958 )
  27. Internal standards:
    Heptadecanoic acid (C17:0) (Sigma-Aldrich, catalog number: H3500 )
    Pentadecanol (C15:0-OH) (Sigma-Aldrich, catalog number: 412228 )
    ω-pentadecalactone (yielding ω-OH-C15:0) (Sigma-Aldrich, catalog number: W284009 )
  28. Murashige and Skoog (MS) medium (see Recipes)
  29. Stock solutions (5 mg/ml) of internal standards (see Recipes)

Equipment

  1. Growth chamber (Fitotron, Weiss Technik)
  2. Pair of scissors (Holtex) and tweezers (Hammacher)
  3. Dry heating block (Fisher Scientific, catalog number: FB15103 )
  4. Polytron (IKA, model: T 25 digital )
  5. Tube rotator (Cole-Parmer, Stuart, model: SB3 )
  6. Fume hood (Delagrave)
  7. Dessicator (Nalgene)
  8. Centrifuge (Hettich Lab Technology, model: ROTORFIX 32 A , 6000RPM max)
  9. Glass Pasteur pipets (e.g., VWR)
  10. Weighing scales (Sartorius, model: SECUR A124-1S )
  11. Temperature-controlled evaporator connected to nitrogen tank (Meyer N-Evap Organomation)
  12. Agilent 6850 (Agilent Technologies, model: Agilent 6850 ) gas chromatograph equipped with an HP-5MS column (length 30 m, id 0.25 mm, film thickness 0.25 μm) and an Agilent 5975 mass spectrometric detector (70 eV, mass-to-charge ratio 50-750) (or equivalent GC-MS)
  13. pH meter (Hanna Instruments, model: Hi4222 )
  14. Autoclave

Procedure

  1. Plant growth and root harvest
    1. Sow sterilized Arabidopsis seeds (1 min in 95% ethanol, 15 min in Bleach [6-14% sodium hypochlorite], and 6 washes with sterilized water) on Petri dish with Murashige and Skoog (MS) medium supplemented with 0.7% agar and 2.5 mM MES, pH 5.7 (see Recipes).
    2. Stratify seeds in the dark for 3 to 4 days at 4 °C, and transfer plates to a controlled-environment growth chamber under long-day conditions (16 h of light and 8 h of darkness) at a temperature of 22 °C. Grow seedlings for up to 4 weeks.
    3. For soil-grown plants, transfer 2 week-old seedlings grown on MS medium plates to pots containing pre-wetted peat moss-vermiculite-perlite (3:1:1, v/v/v) growing medium (about 700 cm3 per pot). Five plants per pots. Place the pots at 22 °C in a controlled-environment growth chamber with ambient humidity under long-day conditions.
    4. For collecting roots from tissue culture-grown seedlings, use a pair of scissors to eliminate the aerial portions of the seedlings, remove the entire agar medium disc from the Petri dish and place it upside down in the dish cover. With tweezers, collect the roots from the agar. It is not necessary to wash the roots after collecting them from the agar medium.
    5. For collecting roots from soil-grown plants, carefully eliminate the soil growing medium by soaking in water, use a pair of scissors to separate roots from other parts of the plant, and rinse roots thoroughly with distilled water to remove as much as possible the remaining growth medium.

  2. Non-extraction method
    1. Pool roots to form bundles of roots. Make 4 to 5 replicates.
    2. Dry roots as much as possible by pressing the material several times between paper towels, (i.e., until paper towels stay dry).
    3. Measure the fresh weight (FW). For Arabidopsis, 5 to 25 mg of FW roots is necessary per replicate.

  3. Solvent-extraction method
    1. Pool roots to form bundles of roots.
    2. Dry roots with a paper towel and measure the fresh weight (FW). For Arabidopsis, 70 to 100 mg of FW roots is necessary per replicate.
    3. Place freshly collected roots in glass extraction tube containing 4 ml hot (85 °C) isopropanol and incubate for 30 min at 85 °C in dry heating block. After cooling to room temperature, reduce to a fine powder with a Polytron when large root or lots of material is analyzed to allow efficient delipidation.
    4. After sedimentation of the residue, transfer isopropanol phase to a new glass tube and delipidate further the roots by extracting soluble lipids successively with:
      1. 4 ml chloroform:methanol (2:1, v/v)
      2. 4 ml chloroform:methanol (1:1, v/v)
      3. 4 ml chloroform:methanol (1:2, v/v)
      4. 4 ml 100% methanol
    5. Perform all delipidation steps at room temperature for 24 h on a tube rotator with the wheel rotating at 40 rpm. Following each delipidation step, collect the solvent fraction in the same 9 ml glass tube, with the newly collected solvent being reduced each time under a gentle stream of nitrogen gas such that no solvent splashes out of the tube (but not completely dry so as to minimize oxidation of lipids). At the end, fully dry the resulting pooled extract, which corresponds to the soluble lipid fraction, resuspend it in 1 ml of chloroform/methanol (1:1; [v:v]), and store it at 4 °C until further analysis as described in the fatty acyl-chain analysis section.
    6. Following final delipidation step with methanol, dry the resulting solvent-extracted root material in a fume hood at room temperature for 2-3 days. This delipidated dry residue will contain the suberin polymer. Place the tubes containing the delipidated roots (without caps) in a dessicator for another 2 days before measuring the dried residues (DR) weight (usually representing 10 to 15% of FW).

  4. Fatty acyl-chain analysis by GC-MS
    1. Add 1 ml of 5% (v/v) sulfuric acid in methanol containing 5 µg each of heptadecanoic acid (C17:0), pentadecanol (C15:0-OH) and C15-hydroxypentadecanoic acid (ω-OH-C15:0) as internal standards to the freshly collected roots (non-extraction method), or the solvent extracted dried residues, or to one fifth (200 µl) of the soluble lipid fraction (solvent extraction method).
    2. Close tubes tightly and incubate for 3 h at 85 °C in a dry heating block without agitation. This transmethylation step depolymerizes the suberin polymer as well as releases the acyl-chains making them all soluble lipids.
    3. After cooling at room temperature, add to each tube 1 ml of NaCl (2.5%, w/v) and 2.2 ml of methyl tert-butyl ether (MTBE) and mix by shaking vigorously.
    4. Centrifuge at 800 x g for 5 min at room temperature to allow phase separation, and transfer the upper MTBE phase to a clean glass tube using a glass Pasteur pipet.
    5. Add 1 ml of 100 mM Tris base pH 8.0 containing 0.09% (w/v) NaCl to the MTBE phase, mix by shaking vigorously, and centrifuge at 800 x g for 5 min at room temperature to allow phase separation.
    6. Collect the upper MTBE phase in a clean glass tube using a glass Pasteur pipet, avoiding any aqueous (lower) phase, and evaporate MTBE under a gentle stream of nitrogen. It is better to leave behind some of the upper MTBE phase than to contaminate with any aqueous (lower) phase.
    7. Add 100 μl of 99% BSTFA (N,O-Bis(trimethylsilyl)-trifluoroacetamide) with 1% TMCS, close tube tightly, and incubate at 110 °C for 15 min in a dry heating block without agitation. This step trimethylsilylates the free hydroxyl groups making compounds containing these groups more amenable to separation by gas chromatography.
    8. After cooling at room temperature, evaporate the solvent under a gentle stream of nitrogen and dissolve the products in 250 μl heptane:toluene (1:1, v/v).
    9. Transfer each sample into a GC vial containing a glass insert.
    10. For GC-MS analysis, a 1 µl aliquot of the sample dissolved in heptane:toluene (1:1) is injected in splitless mode. The temperature of the injector is held at 250 °C. The column oven temperature is held at 50 °C for 1 min and then increased from 50 °C to 200 °C at a rate of 25 °C per minute, followed by a 1 min hold, and then is ramped up again at a rate of 10 °C per minute to a final temperature of 320 °C, which is held for 8 min. The total run time is 28 min. High purity helium is used as the carrier gas at a flow rate of 1.5 ml per min.

Data analysis

  1. Typical chromatograms of total (non-extraction method), soluble and polymerized (solvent extraction method) acyl chains of roots from 4-week old wild-type seedlings grown in tissue culture are shown in Figure 1.


    Figure 1. Representative chromatograms of total, soluble and polymerized acyl chains of roots from 4-weeks old wild-type seedlings grown in tissue culture. The identity of all the major monomers (representing more than 1% of the total) is indicated on the left side table in the order of elution. X:Y stands for fatty acids with X carbon atoms and Y unsaturations, X:Y-OH stands for fatty alcohols, X:Y-ωOH stands for ω-hydroxy fatty acids, X:Y-DCA stands for dicarboxylic fatty acids and 2OH-X:Y stands for 2-hydroxy fatty acids. IS stands for internal standards:pentadecanol (C15OH), heptadecanoic acid (C17) and 15-hydroxy-pentadecanoic acid (C15ωOH). Question marks and star indicate unidentified compounds and a contaminant, respectively.

  2. Quantification of monomers is based on peak areas in the GC-MS chromatograms, identified using their retention time, and using the peak area of the respective internal standards (C17:0 for fatty acids and dicarboxylic acids, ω-OH-C15:0 for ω-hydroxyacids, and C15:0-OH for fatty alcohols). Constituent amounts (in μg) are calculated by multiplying the internal standard amount (5 μg) with the peak area of the constituent of interest and dividing by the peak area of the internal standard. Divide the constituent amounts by the fresh weight (FW) or dry residue (DR) of roots to obtain values in µg/mg FW or DR for non-extraction and solvent-extraction method, respectively.
  3. The global acyl-chain composition of suberin (including polymerized as well as soluble acyl-chains, principally fatty alcohols, in the form of suberin associated waxes) is then calculated as follows:
    1. From the non-extraction method, suberin comprises all dicarboxylic acids, ω-hydroxyacids, and fatty alcohols, but only about 3% of the C16 and C18 fatty acids (which are mainly coming from the lipids making membranes), 50% of 20:0 and 22:0 fatty acids, and 25% of very-long chain fatty acids above C22 (since suberin is enriched in very-long chain fatty acids, especially in 20:0 and 22:0) as previously described (Delude et al., 2016).
    2. From the solvent-extraction method, suberin comprises all dicarboxylic acids, ω-hydroxyacids, and fatty alcohols present in both polymerized and soluble fraction, but only the polymerized fatty acids.
    The global acyl-chain composition of the suberin from roots of 4 week-old seedlings grown in tissue culture according to both methods is shown in Figure 2.


    Figure 2. Global acyl-chain composition of the suberin from roots of 4 week-old seedlings were harvested and the suberin aliphatic composition was analyzed according to the solvent- and non-extraction methods. X:Y stands for fatty acids with X carbon atoms and Y unsaturations. Roots from 4 week-old seedlings were harvested and the suberin aliphatic composition was analyzed according to the solvent- and non-extraction methods. In the solvent-extraction method, root fresh weight (FW) and dry residue weight (DR) were measured, indicating that DR was about 14% of FW. This correlation was used to calculate the suberin composition in μg/mg DR in the non-extraction method.

Notes

  1. Plasticware should be not be used at any point in the protocol or else plastic contamination may obscure peaks in the GC traces. Pre-rinse all glassware and Hamilton syringes three times with chloroform. New glass Pasteur pipettes do not need to be solvent rinsed.

Recipes

  1. Murashige and Skoog (MS) medium (400 ml)
    Mix 1.8 g MS with Vitamins, 0.2 g MES, 2.8 g agar in 400 ml distilled water
    Adjust pH to 5.7 with 1 N KOH (5.61 g in 100 ml water)
    Autoclave for 20 min at 120 °C, cool, and then pour into the Petri dishes (20 to 25 ml per dish)
  2. Stock solutions (5 mg/ml) of internal standards
    Stock solutions (5 mg/ml) of internal standards (heptadecanoic acid, C17:0; pentadecanol, C15:0-OH; C15-hydroxypentadecanoic acid, ω-OH-C15:0) should be prepared in methanol and stored in glass tube with polytetrafluoroethylene (PTFE)-lined caps at -20 °C until use

Acknowledgments

The solvent extraction method described here is adapted from Delude et al., 2016. This work was supported by the French Ministère de l’Enseignement Supérieur et de la Recherche (doctoral fellowship to C.D.), by the Natural Sciences and Engineering Research Council of Canada (grant to O.R.), and by grant no. MetaboHUB–ANR–11–INBS–0010 to the Functional Genomic Center of the Bordeaux-Metabolome/Lipidome platform.

References

  1. Andersen, T. G., Barberon, M. and Geldner, N. (2015). Suberization - the second life of an endodermal cell. Curr Opin Plant Biol 28: 9-15.
  2. Barberon, M., Vermeer, J. E., De Bellis, D., Wang, P., Naseer, S., Andersen, T. G., Humbel, B. M., Nawrath, C., Takano, J., Salt, D. E. and Geldner, N. (2016). Adaptation of root function by nutrient-induced plasticity of endodermal differentiation. Cell 164(3): 447-459.
  3. Bernards, M. A. (2002). Demystifying suberin. Can J Bot 80: 227-240.
  4. Delude, C., Fouillen, L., Bhar, P., Cardinal, M. J., Pascal, S., Santos, P., Kosma, D. K., Joubès, J., Rowland, O. and Domergue, F. (2016). Primary fatty alcohols are major components of suberized root tissues of Arabidopsis in the form of alkyl hydroxycinnamates. Plant Physiol 171(3): 1934-1950.
  5. Domergue, F., Vishwanath, S. J., Joubes, J., Ono, J., Lee, J. A., Bourdon, M., Alhattab, R., Lowe, C., Pascal, S., Lessire, R. and Rowland, O. (2010). Three Arabidopsis fatty acyl-coenzyme A reductases, FAR1, FAR4, and FAR5, generate primary fatty alcohols associated with suberin deposition. Plant Physiol 153(4): 1539-1554.
  6. Kosma, D. K., Molina, I., Ohlrogge, J. B. and Pollard, M. (2012). Identification of an Arabidopsis fatty alcohol:caffeoyl-Coenzyme A acyltransferase required for the synthesis of alkyl hydroxycinnamates in root waxes. Plant Physiol 160(1): 237-248.
  7. Ranathunge, K., Schreiber, L. and Franke, R. (2011). Suberin research in the genomics era--new interest for an old polymer. Plant Sci 180(3): 399-413.
  8. Vishwanath, S. J., Delude, C., Domergue, F. and Rowland, O. (2015). Suberin: biosynthesis, regulation, and polymer assembly of a protective extracellular barrier. Plant Cell Rep 34(4): 573-586.
  9. Vishwanath, S. J., Kosma, D. K., Pulsifer, I. P., Scandola, S., Pascal, S., Joubes, J., Dittrich-Domergue, F., Lessire, R., Rowland, O. and Domergue, F. (2013). Suberin-associated fatty alcohols in Arabidopsis: distributions in roots and contributions to seed coat barrier properties. Plant Physiol 163(3): 1118-1132.

简介

在这里我们描述了非提取和溶剂萃取方法的根脂肪族苏维林分析。 非提取方法快速,因为根使用酸性转甲基化直接解聚。 然而,苏维林脂族组分与构成根中存在的脂质(例如膜)的所有其它酰基链一起分离。 对于溶剂萃取方法,首先在转甲基化之前使根脱皮。 该方法较长,但可分离可溶性和聚合的根脂质。 该方案针对组织培养或土壤种植的拟南芥植物进行了优化,但可与其他植物的根一起使用。
【背景】Suberin是沉积在各种组织的细胞壁中的细胞外植物脂质聚合物,例如根内皮,外皮和根周围。 Suberin作为控制水和溶质通量并限制病原体感染的屏障(Ranathunge et al。,2011; Andersen等,2015; Vishwanath等,2015; Barberon等,2016)。 Suberin是由脂类,酚类和甘油组成的复杂杂聚物,与溶剂萃取蜡相关(Bernards,2002)。在拟南芥模拟植物中,苏维林聚合物主要由ω-羟基酸和α,ω-二羧酸制成,但也含有未取代的脂肪酸和伯醇脂肪醇(Domergue等,2010; Vishwanath et al。 2013),而相关的蜡是羟基肉桂酸烷基酯(AHC; Kosma等,2012; Delude等,2016)的形式。这里描述的非提取方法允许对ubberin组合物的高通量和快速分析,这在筛选大量植物系(例如突变体,过表达转基因品系或天然变体)时是特别有利的。包括可溶性和聚合脂质(即苏维林聚酯)需要单独分析的更传统和准确的溶剂萃取方法,以供比较。

关键字:木栓质, 脂质聚合物, 细胞壁, 根, 脂质提取, 气相色谱法, 拟南芥

材料和试剂

  1. 培养皿(直径90mm)(Fisher Scientific)
  2. 泥炭苔,蛭石和珍珠岩(3:1:1,v/v/v;棉兰S.A.)
  3. 纸巾
  4. 使用聚四氟乙烯(PTFE)线帽(Dutscher,目录号:001031)的8ml玻璃管(Dutscher,目录号:065307B)
  5. 拟南芥花盆(聚苯乙烯,9 x 9 x 9.5厘米)(SOPARCO,目录号:4686)
  6. 带有盖帽的2 ml GC小瓶(Agilent Technologies,目录号:5182-0557)和400μl平底玻璃插件(Agilent Technologies,目录号:5181-3377)
  7. 拟南芥种子
  8. 95%乙醇
  9. 次氯酸钠(漂白剂)
  10. 蒸馏水
  11. 异丙醇(Fisher Scientific,目录号:10315720)
  12. 氯仿(Sigma-Aldrich,目录号:32211)
  13. 甲醇(Sigma-Aldrich,目录号:34885)
  14. 氮(H 2 O 3 O <3ppm; C H <0.5ppm; O 2< 3< ; 2ppm)
  15. 硫酸(H 2 SO 3 SO 4)(CARLO ERBA试剂,目录号:E410391)
  16. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S5886)
  17. 甲基叔丁基醚(MTBE)(Sigma-Aldrich,目录号:20256)
  18. 三(羟甲基)氨基甲烷(Tris碱)(Sigma-Aldrich,目录号:T6066)
  19. 具有1%三甲基氯硅烷(TMCS)的N,O - 双(三甲基甲硅烷基) - 三氟乙酰胺(BSTFA)(Sigma-Aldrich,目录号:T6381)
  20. 庚烷(Sigma-Aldrich,目录号:32287)
  21. 甲苯(Sigma-Aldrich,目录号:32249)
  22. Murashige和Skoog培养基(Duchefa Biochemie,目录号:M0222.0050)
  23. 维生素
  24. 植物琼脂(Duchefa Biochemie,目录号:P1001.1000)
  25. 2-(N-吗啉代) - 乙磺酸(MES)(Euromedex,目录号:EU0033-B)
  26. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:P5958)
  27. 内部标准:
    十七烷酸(C17:0)(Sigma-Aldrich,目录号:H3500)
    十五醇(C15:0-OH)(Sigma-Aldrich,目录号:412228)
    ω-十五内酯(产生ω-OH-C15:0)(Sigma-Aldrich,目录号:W284009)
  28. Murashige和Skoog(MS)培养基(见食谱)
  29. 库存溶液(5 mg/ml)内标(见配方)

设备

  1. 生长室(Fitotron,Weiss Technik)
  2. 一对剪刀(Holtex)和镊子(Hammacher)
  3. 干燥加热块(Fisher Scientific,目录号:FB15103)
  4. Polytron(IKA,型号:T 25数字)
  5. 管旋转器(Cole-Parmer,Stuart,型号:SB3)
  6. 通风柜(Delagrave)
  7. Dessicator(Nalgene)
  8. 离心机(Hettich Lab Technology,型号:ROTORFIX 32 A,最大6000RPM)
  9. 玻璃巴斯德移液管(例如,,VWR)
  10. 称重秤(Sartorius,型号:SECUR A124-1S)
  11. 连接氮气罐的温度控制蒸发器(Meyer N-Evap Organomation)
  12. Agilent 6850(Agilent Technologies,型号:Agilent 6850)气相色谱仪配有HP-5MS色谱柱(长度30 m,标称为0.25 mm,膜厚为0.25μm)和Agilent 5975质谱检测仪(70 eV,质量均匀比例50-750)(或等效GC-MS)
  13. pH计(Hanna Instruments,型号:Hi4222)
  14. 高压灭菌器

程序

  1. 植物生长和根系收获
    1. 在Murishige和Skoog(MS)培养基的培养皿上,将拟南芥灭活的种子(95%乙醇中1分钟,漂白剂[6-14%次氯酸钠中15分钟,用无菌水洗涤6次)补充了0.7%琼脂和2.5mM MES,pH 5.7(参见食谱)。
    2. 在4℃下将种子在黑暗中分层3至4天,并在22℃的温度下在长时间条件(16小时光照和8小时黑暗)下将板转移到受控环境生长室。种植幼苗长达4周。
    3. 对于土壤种植的植物,将在MS培养基板上生长的2周龄的幼苗转移到含有预先润湿的泥炭苔藓 - 蛭石 - 珍珠岩(3:1:1,v/v/v)生长培养基(约700cm 3 每盆)。五盆五盆。将盆在22°C放置在受环境温度控制的生长室中,环境湿度在长时间的条件下
    4. 为了从组织培养生长的幼苗收集根,使用一把剪刀来消除幼苗的空中部分,从培养皿中取出整个琼脂培养基盘,并将其倒置在盘盖中。用镊子从琼脂中收集根。从琼脂培养基中收集后,不需要清洗根。
    5. 为了从土壤种植的植物收集根,通过浸泡在水中小心地除去土壤生长介质,用一把剪刀将植物的其他部分的根分离,并用蒸馏水彻底冲洗根,尽可能地除去剩余的生长培养基。

  2. 非提取方法
    1. 池根形成束根。进行4到5次重复。
    2. 通过在纸巾之间按压材料(即,直到纸巾保持干燥),尽可能干燥根部。
    3. 测量鲜重(FW)。对于拟南芥,每次重复需要5至25mg的FW根。

  3. 溶剂萃取法
    1. 池根形成束根。
    2. 用纸巾干根,测量鲜重(FW)。对于拟南芥,每次重复需要70至100毫克的FW根
    3. 将新鲜收集的根放在含有4ml热(85℃)异丙醇的玻璃提取管中,并在85℃下在干燥加热块中孵育30分钟。冷却至室温后,当分析大根或大量材料时,用Polytron还原成细粉,以便有效脱脂。
    4. 在残渣沉淀后,将异丙醇相转移到新的玻璃管中,并通过以下方式提取可溶性脂质进一步去除根部:
      1. 4ml氯仿:甲醇(2:1,v/v)
      2. 4毫升氯仿:甲醇(1:1,v/v)
      3. 4ml氯仿:甲醇(1:2,v/v)
      4. 4 ml 100%甲醇
    5. 在轮旋转40rpm的管旋转器上,在室温下进行所有去脂步骤24小时。在每个脱脂步骤之后,在相同的9ml玻璃管中收集溶剂级分,每次在温和的氮气流下每次还原新收集的溶剂,使得没有溶剂从管中溅出(但不完全干燥,从而最小化脂质的氧化)。最后,完全干燥所得到的合并的提取物,其对应于可溶性脂质部分,将其重悬于1ml氯仿/甲醇(1:1; [v:v])中,并将其储存在4℃直至进一步分析如脂肪酰基链分析部分所述
    6. 在用甲醇进行最终脱脂步骤后,将得到的溶剂提取的根材料在通风橱中在室温下干燥2-3天。这种脱脂的干燥残余物将含有苏木精聚合物。将含有脱脂根(无盖)的管放置在干燥器中另外2天,然后测量干燥残留物(DR)重量(通常为FW的10至15%)。

  4. 通过GC-MS进行脂肪酰基链分析
    1. 加入1ml 5%(v/v)硫酸的甲醇,其中含有十五烷酸(C17:0),十五醇(C15:0-OH)和C15-羟基十五烷酸(ω-OH-C15:0)作为新鲜收集的根(非提取方法)的内标,或溶剂萃取的干燥残留物,或溶解脂质部分的五分之一(200μl)(溶剂萃取法)。
    2. 紧闭管并在85℃下在干热加热块中孵育3小时,无需搅拌。该转甲基化步骤解聚苏木精聚合物,同时释放酰基链使其成为所有可溶性脂质。
    3. 在室温下冷却后,向每个管中加入1ml NaCl(2.5%,w/v)和2.2ml甲基叔丁基醚(MTBE),并通过剧烈振荡混合。 >
    4. 在室温下800g离心5分钟以允许相分离,并使用玻璃巴斯德移液管将上MTBE相转移到干净的玻璃管上。
    5. 向MTBE相中加入1ml含有0.09%(w/v)NaCl的100mM Tris碱性pH 8.0,通过剧烈振荡混合,并在室温下以800xg离心5分钟以允许相分离。
    6. 使用玻璃巴斯德吸管在干净的玻璃管中收集上MTBE相,避免任何水相(较低)相,并在温和的氮气流下蒸发MTBE。留下一些较高的MTBE相,比任何水相(下)相污染更好。
    7. 加入100μl99%BSTFA(N,O-双(三甲基甲硅烷基) - 三氟乙酰胺),用1%TMCS,密闭管子,并在干燥加热块中在110℃温育15分钟,搅动。该步骤使游离羟基三甲基甲硅烷基化,使得含有这些基团的化合物更适合于气相色谱分离
    8. 在室温下冷却后,在温和的氮气流下蒸发溶剂,并将产物溶于250μl庚烷:甲苯(1:1,v/v)中。
    9. 将每个样品转移到含有玻璃插入物的GC小瓶中
    10. 对于GC-MS分析,将1μl样品溶于庚烷:甲苯(1:1)中的样品以不分流模式注入。注射器的温度保持在250°C。柱温箱在50℃下保持1分钟,然后以每分钟25℃的速度从50℃升至200℃,然后保持1分钟,然后以一定速度再次升高为10℃/分钟,最终温度为320℃,保持8分钟。总运行时间为28分钟。高纯氦气用作载气,流速为每分钟1.5毫升

数据分析

  1. 总共(非提取方法),可溶性和聚合(溶剂萃取方法)在组织培养中生长的4周龄野生型幼苗的根的酰基链的典型色谱图如图1所示。


    图1.组织培养中生长的4周龄野生型幼苗的根,总可溶性和聚合酰基链的代表性色谱图。所有主要单体的同一性(代表超过1%总量)按照洗脱顺序在左侧表格中显示。 X:Y代表X碳原子和Y不饱和的脂肪酸,X:Y-OH代表脂肪醇,X:Y-ωOH代表ω-羟基脂肪酸,X:Y-DCA代表二元脂肪酸和2OH -X:Y代表2-羟基脂肪酸。 IS代表内标:十五烷醇(C15OH),十七烷酸(C17)和15-羟基 - 十五烷酸(C15ωOH)。问号和星号分别表示不明的化合物和污染物
  2. 单体的定量是基于GC-MS色谱图中的峰面积,使用其保留时间确定,并使用各自内标的峰面积(脂肪酸和二羧酸为C17:0,ω-OH-C15:0为ω-羟基酸,C15:0-OH)。通过将内标(5μg)与感兴趣成分的峰面积乘以内标峰面积来计算成分量(μg)。将组分量除以根的鲜重(FW)或干残渣(DR),以分别获得非萃取和溶剂萃取方法中μg/mg FW或DR的值。
  3. ubberin(包括聚合的以及可溶性酰基链,主要是脂质醇,以桧烯相关蜡的形式)的全球酰基链组成然后计算如下:
    1. 从非提取方法,苏维林含有所有二羧酸,ω-羟基酸和脂肪醇,但只有约3%的C16和C18脂肪酸(主要来自制备膜的脂质),20% 0和22:0脂肪酸和25%超过C22的非常长链脂肪酸(因为ubberin富含非常长链脂肪酸,特别是20:0和22:0),如前所述(Delude > et al。,2016)。
    2. 从溶剂萃取法中,苏维林包含存在于聚合和可溶部分中的所有二羧酸,ω-羟基酸和脂肪醇,但仅包含聚合的脂肪酸。
    根据两种方法在组织培养中生长的4周龄幼苗的根的全部酰基链组成如图2所示。


    图2.收获了来自4周龄幼苗根的全部酰基链组成,并根据溶剂和非提取方法分析桧木脂肪组合物。 X:Y对于具有X碳原子和Y不饱和度的脂肪酸。收获来自4周龄幼苗的根,并根据溶剂和非提取方法分析桧木脂肪族组合物。在溶剂萃取方法中,测量根鲜重(FW)和干残渣重量(DR),表明DR为FW的约14%。这种相关性用于在非提取方法中以μg/mg DR计算ubberin组成

笔记

  1. 塑料软件不应在协议中的任何一点使用,否则塑料污染物可能会掩盖GC痕迹中的峰值。用氯仿预先冲洗所有玻璃器皿和汉密尔顿注射器三次。新型玻璃巴斯德移液器不需要进行溶剂冲洗。

食谱

  1. Murashige和Skoog(MS)培养基(400 ml)
    将1.8克MS与维生素,0.2克MES,2.8克琼脂混合在400毫升蒸馏水中 用1N KOH(5.61g,在100ml水中)调节pH至5.7 在120℃高压灭菌20分钟,冷却,然后倒入培养皿(每碟20至25毫升)
  2. 库存溶液(5 mg/ml)的内部标准
    储存溶液(5 mg/ml)内标(十七烷酸,C17:0;十五醇,C15:0-OH; C15-羟基十五酸,ω-OH-C15:0)应在甲醇中制备,并储存在玻璃管与聚四氟乙烯(PTFE)线帽在-20°C直到使用

致谢

这里描述的溶剂萃取方法改编自2016年11月的Delude等人。这项工作得到了法国自然资源研究所(CD)的博士研究生资助,由Natural加拿大科学与工程研究理事会(授予OR),授权号MetaboHUB-ANR-11-INBS-0010到波尔多代谢组织/脂质体平台的功能基因组中心。

参考

  1. Andersen,TG,Barberon,M.和Geldner,N。(2015)。 Curr Opin Plant Biol 28:9-15。
  2. Barberon,M.,Vermeer,JE,De Bellis,D.,Wang,P.,Naseer,S.,Andersen,TG,Humbel,BM,Nawrath,C.,Takano,J.,Salt,DE和Geldner,N 。(2016)。根据营养 - 诱导内胚分化的可塑性。细胞 164(3):447-459。
  3. Bernards,MA(2002)。  神秘的苏维林。 Can J Bot 80:227-240。
  4. Delude,C.,Fouillen,L.,Bhar,P.,Cardinal,MJ,Pascal,S.,Santos,P.,Kosma,DK,Joubès,J.,Rowland,O.and Domergue, 。主要脂肪醇是腰果根组织的主要成分拟南芥以羟基肉桂酸烷基酯的形式。植物生理学171(3):1934-1950。
  5. Domergue,F.,Vishwanath,SJ,Joubes,J.,Ono,J.,Lee,JA,Bourdon,M.,Alhattab,R.,Lowe,C.,Pascal,S.,Lessire,R.and Rowland, O.(2010)。三种拟南芥<脂肪酰辅酶A还原酶FAR1,FAR4和FAR5产生与ubberin沉积相关的原发性脂肪醇。植物生理学153(4):1539-1554。 />
  6. Kosma,DK,Molina,I.,Ohlrogge,JB和Pollard,M。(2012)。拟南芥的鉴定脂肪醇:咖啡酰辅酶A在根蜡中合成烷基羟基肉桂酸酯所需的酰基转移酶。植物生理学 em> 160(1):237-248。
  7. Ranathunge,K.,Schreiber,L.and Franke,R。(2011)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/21421386"目标="_ blank">基因组学时代的Suberin研究 - 对旧聚合物的新兴趣植物科学 180(3):399-413。
  8. Vishwanath,SJ,Delude,C.,Domergue,F.and Rowland,O.(2015)。  Suberin:保护性细胞外屏障的生物合成,调节和聚合物组装。植物细胞代谢34(4):573-586。 />
  9. Vishwanath,SJ,Kosma,DK,Pulsifer,IP,Scandola,S.,Pascal,S.,Joubes,J.,Dittrich-Domergue,F.,Lessire,R.,Rowland,O.and Domergue, )。拟南芥中的Suberin相关脂肪醇:根系分布和对种皮阻隔性质的贡献。植物生理学163(3):1118-1132。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Delude, C., Vishwanath, S. J., Rowland, O. and Domergue, F. (2017). Root Aliphatic Suberin Analysis Using Non-extraction or Solvent-extraction Methods. Bio-protocol 7(12): e2331. DOI: 10.21769/BioProtoc.2331.
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