Histochemical Detection of Peroxidase and Laccase Activities in Populus Secondary Xylem

Abstract

Peroxidases (PRXs) and laccases (LACs) are enzymes involved in catalyzing the oxidation of the lignin monomers to facilitate lignin polymerization. However, due to the large number of genes composing these two families of enzymes, many details regarding their specific localization are only partially understood. Here, we present a fast and easy histochemical method that makes use of the artificial substrate 3,3',5,5'-tetramethylbenzidine (TMB) to visualize PRX and LAC activities in the hybrid aspen (Populus tremula x P. tremuloides) xylem tissue. In addition, we describe a protocol that allows the detection of the PRX substrate, H₂O₂, using the nonfluorescent dye 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) in woody tissues.

Keywords: Cell wall; H2DCFDA; H2O2; Laccase; Lignin; Peroxidase; Populus; TMB; Woody tissues; Xylem.