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
发布: 2017年06月20日第7卷第12期 DOI: 10.21769/BioProtoc.2331 浏览次数: 10903
评审: Arsalan DaudiSibongile MafuAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Apr 2015
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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
- Petri dish (90 mm diameter) (Fisher Scientific)
- Peat moss, vermiculite and perlite (3:1:1, v/v/v; Medan S.A.)
- Paper towels
- 8 ml glass tubes (Dutscher, catalog number: 065307B ) with polytetrafluoroethylene (PTFE)-lined caps (Dutscher, catalog number: 001031 )
- Pots for Arabidopsis (Polystyrene, 9 x 9 x 9.5 cm) (SOPARCO, catalog number: 4686 )
- 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 )
- Arabidopsis seeds
- 95% ethanol
- Sodium hypochlorite (Bleach)
- Distilled water
- Isopropanol (Fisher Scientific, catalog number: 10315720 )
- Chloroform (Sigma-Aldrich, catalog number: 32211 )
- Methanol (Sigma-Aldrich, catalog number: 34885 )
- Nitrogen (H2O < 3 ppm; CnHm < 0.5 ppm; O2 < 2 ppm)
- Sulfuric acid (H2SO4) (CARLO ERBA Reagents, catalog number: E410391 )
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 )
- Methyl tert-butyl ether (MTBE) (Sigma-Aldrich, catalog number: 20256 )
- Tri(hydroxymethyl)aminomethane (Tris base) (Sigma-Aldrich, catalog number: T6066 )
- N,O-Bis(trimethylsilyl)-trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) (Sigma-Aldrich, catalog number: T6381 )
- Heptane (Sigma-Aldrich, catalog number: 32287 )
- Toluene (Sigma-Aldrich, catalog number: 32249 )
- Murashige and Skoog medium (Duchefa Biochemie, catalog number: M0222.0050 )
- Vitamins
- Plant agar (Duchefa Biochemie, catalog number: P1001.1000 )
- 2-(N-Morpholino)-ethane sulfonic acid (MES) (Euromedex, catalog number: EU0033-B )
- Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: P5958 )
- 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 ) - Murashige and Skoog (MS) medium (see Recipes)
- Stock solutions (5 mg/ml) of internal standards (see Recipes)
Equipment
- Growth chamber (Fitotron, Weiss Technik)
- Pair of scissors (Holtex) and tweezers (Hammacher)
- Dry heating block (Fisher Scientific, catalog number: FB15103 )
- Polytron (IKA, model: T 25 digital )
- Tube rotator (Cole-Parmer, Stuart, model: SB3 )
- Fume hood (Delagrave)
- Dessicator (Nalgene)
- Centrifuge (Hettich Lab Technology, model: ROTORFIX 32 A , 6000RPM max)
- Glass Pasteur pipets (e.g., VWR)
- Weighing scales (Sartorius, model: SECURA124-1S )
- Temperature-controlled evaporator connected to nitrogen tank (Meyer N-Evap Organomation)
- 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)
- pH meter (Hanna Instruments, model: Hi4222 )
- Autoclave
Procedure
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文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
植物科学 > 植物生物化学 > 脂质
植物科学 > 植物细胞生物学 > 组织分析
生物化学 > 脂质 > 胞外脂质
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