Materials and Reagents

1. Plants grown in soil for 3 weeks
   Note: Treatment of samples is explained in the Procedure section.
   
2. Liquid N₂
   
3. Absolute ethanol
   
4. HEPES (Sigma-Aldrich, catalog number: H4034-1KG )
   
5. KOH (Panreac Applichem, catalog number: 121515 )
   
6. NaOH 50% (w/v) solution (AppliChem GmbH, catalog number: A3720, 1000 )
   
7. Milli Q grade water
   
8. 100 mM HEPES-KOH (see Recipes)
   
9. Extraction buffer 1 (EB1) (see Recipes)
   
10. 0.1 M NaOH (see Recipes)
    
11. 21.6 mM NaOH (see Recipes)
    

Equipment

1. Small mortar and pestle
   
2. 1.5 ml microfuge tubes
   
3. Automatic pipettes
   
4. Precision scale
   
5. 2 ml microcentrifuge (Eppendorf, model: 5415R )
   
6. Vacufuge concentrator 5301 (Eppendorf)
   
7. Nylon filters (Whatman, catalog number: UN203NPENYL )
   
8. Dionex HPLC system (Dionex ICS 5000)
   
9. CarboPacPA10 column (4 x 250 mm) (Thermo Fisher Scientific, catalog number: 046110 )
   
10. CarboPacPA10 Pre-column (4 x 50 mm) (Thermo Fisher Scientific, catalog number: 046115 )
    
11. Gold electrode (Dionex ICS 5000)
    
12. Computer connected to HPLC system
    

Software

1. Chromeleon software (v.7.0) (Thermo Fisher Scientific)

Procedure