Lignin Extraction and Quantification, a Tool to Monitor Defense Reaction at the Plant Cell Wall Level

Materials and Reagents

1. Plant material
   
2. Flg22 (a flagellin-derived, 22 amino acid-long peptide) (QRLSTGSRINSAKDDAAGLQIA)
   
3. 80% aqueous methanol (Carl Roth, catalog number: 4627.6 )
   
4. Distilled water
   
5. Acetone (VWR International, catalog number: UN1090 )
   
6. NaOH (Carl Roth, catalog number: 6771.1 )
   
7. 86% H₃PO₄ (J.T.Baker^®, catalog number: 6024 )
   
8. Ethyl acetate (Carl Roth, catalog number: 6784.4 )
   
9. Thioglycolic acid (Sigma-Aldrich, catalog number: T3758 )
   
10. 32% HCl (Carl Roth, catalog number: P074.3 )
    
11. Lignin (alkali) (Sigma-Aldrich, catalog number: 370959 )
    
12. 1 M NaOH (see Recipes)
    
13. 2 M HCl (see Recipes)
    

Equipment

1. Plastic equipment (Grainer Falcon 50 ml tubes, catalog number: 227261 ; Grainer 6-well culture plates, catalog number: 657160 )
   
2. Freeze drying machine
   
3. Tissue Lyser2 (manufactured by Retsch, provided by QIAGEN) and stainless steel beads (QIAGEN, catalog number: 69989 )
   
4. Shaker for tube rotation (with variable temperature)
   
5. Centrifuge (with variable temperature) (Eppendorf, centrifuge number: 5417R )
   
6. Vacuum pump (Savant Systems LLC, catalog number: SC110 : 25-30 Hg)
   
7. Rotary shaker for tube rotation
   
8. Photometer (Eppendorf, BioSpectrometer basic)
   

Note: The extraction procedure includes a separation of different fractions of phenolic compounds depending on their chemical properties. The soluble phenolic fraction is extracted with 80% methanol (Day 1). The cell wall-bound phenolic fraction is hydrolyzed (alkaline hydrolysis) and solved in ethyl acetate. The lignin fractions are extracted in the later steps of the procedure by binding the to the lingo-thioglycolic acid complex (Days 2 and 3). The soluble and cell wall-bound phenols fractions can be measured according the Folin-Ciocalteau method after Strack et al. (1988), or used for further HPLC analyses. The lignin complex can be measured directly after resolving in NaOH.

Procedure