GC-MS-Based Analysis of Chloroform Extracted Suberin-Associated Root Waxes from Arabidopsis and Other Plant Species

Materials and Reagents

1. 3.5 inch pots for Arabidopsis, bigger pots if necessary for other plants
   
2. 9 ml glass tubes with polytetrafluoroethylene (PFTE)-lined caps (Corning, catalog number: 9826-13 )
   
3. Glass Pasteur pipets
4. Glass wool (Sigma-Aldrich, catalog number: 18421 )
   
5. 2 ml GC vials and caps (Agilent Technologies, catalog number: 5190-2240 ) with 250 µl glass inserts (Agilent Technologies, catalog number: 5181-1270 )
   
6. 10 µl and 500 µl Hamilton syringes (Hamilton Company, catalog number: 80075 and 81216 , respectively)
   Note: It is advisable to rinse the glass Hamilton syringes with acetone then chloroform before each use and chloroform then acetone after each use. This will help to prevent clogging of the syringes.
   
7. Arabidopsis thaliana or other plant seeds [e.g., Brassica napus, Raphanus sativus, Beta vulgaris, see Kosma et al., (2015) for a comprehensive list of species that have been analyzed with this protocol]
   
8. Soil-less growing medium
   
   1. 2:1 (v/v) mixture of potting mix (Pro-Mix BX, Premier Tech Horticulture, Rivière-du-Loup)
      
   2. calcined clay granules (PPC Greens Grade, Profile, Buffalo Grove)
      Note: For images of these growing media please refer to https://ag.purdue.edu/hla/Hort/greenhouse/pages/101-ways-to-grow-arabidopsis.aspx
9. Distilled water
   
10. Chloroform (CHCl₃) (≥ 99.5% purity) (Sigma-Aldrich, catalog number: C2432 )
    
11. 95% Ethanol (e.g., Corning, Koptec, catalog number: V1105 )
    
12. Suggested internal standards
    
    1. Pentadecanoic acid (15:0) (Nu-Check Prep, catalog number: N-15-A ) or heptadecanoic acid (17:0) (Nu-Check Prep, catalog number: N-17-A )
       
    2. Tricosan-1-ol (23:0-OH) (Nu-Check Prep, catalog number: A-624 )
       
    3. Monoheptadecanoin (17:0 monoacylglycerol) (Nu-Check Prep, catalog number: M-159 )
       
    4. Octacosane (28:0) (Sigma-Aldrich, catalog number: O504 )
       
    5. Tridecyl (13:0) ferulate, if used need to synthesize according to Kosma et al., (2012)
       
    6. Heptadecyl (17:0) coumarate, if used need to synthesize according to Kosma et al., (2012)
       
    7. Nonadecyl (19:0) caffeate, if used need to synthesize according to Razeq et al., (2014)
13. Nitrogen gas (> 99% Purity)
    
14. Pyridine, anhydrous (C₅H₅N) (Sigma-Aldrich, catalog number: 270970 )
    
15. N, O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) (Sigma-Aldrich, catalog number: 15222 )
    
16. n-heptane, anhydrous (Sigma-Aldrich, catalog number: 246654 )
    
17. Toluene, anhydrous (Sigma-Aldrich, catalog number: 244511 )
    

Equipment

1. Controlled-environment plant growth chamber (e.g., Percival Scientific, Conviron, etc.)
   
2. Small pair of scissors or a razor blade
   
3. Temperature-controlled evaporator connected to nitrogen tank (e.g., Organomation Associates, catalog number: 11155 )
   
4. Dry heating block capable of reaching a temperature of 110 ºC and with a metal block containing 13 mm orifices
   
5. Gas chromatograph-mass spectrometer (e.g., Agilent Technologies, model: 6850/5975 GC-MS ) equipped with a HP-5MS capillary column (length 30 m, id 0.25 mm, film thickness 0.25 µm) (Agilent Technologies, J&W Scientific)
   

Software

1. ImageJ software (National Institute of Health)
   
2. Excel software (Microsoft office)
   

Procedure