Abstract
Lignin is a complex polymer of phenolic compounds (monolignins), which contributes to the rigidity of the plant cell wall. Lignification is essential for plant development, however it is also one of the mechanisms of plant defense. Accumulation of lignin and the polymerization of monolignins at sides of pathogen attack protect the cell wall against cell wall-degrading enzymes and prevent therefore the pathogen’s penetration. In addition to cross-linkage of phenolic compounds, this resistance mechanism includes also callose and cellulose appositions on the cell wall. This results in structures called papillae, which provide the necessary resistance to the mechanical pressure exercised by fungal appressorium. Lignin accumulation in cell walls is therefore a part of plant defense responses. Here we describe a quantification method for lignin and cell wall phenolic compounds, which is based on an acid-catalyzed reaction resulting in a colored and soluble lignin-thioglycolate complex suitable for photometric measurements.
Keywords: Plant Defense, Cell Wall, Priming, Lignin
Materials and Reagents
Equipment
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
Representative data
Notes
Working under a laboratory fume hood during the whole extraction procedure is recommended. The metal beads used to crush the plant material should be removed in the first steps of extraction procedure. For reproducibility dry plant material should be weighed for adequate calculation, the content of roots and leave tissue should be the same in all samples, and the dry plant material should be homogenized via TissueLyser to an appropriated powder.
Recipes
Acknowledgments
The protocol was modified after Bruce and West (1989), Eynck et al. (2009) and Strack et al. (1988). This work was supported by the Federal Office for Agriculture and Food (BLE), Germany.
References
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