Oxidase activities were investigated within the cross-section of walnut trunk in relation to the brown staining of heartwood, especially in the transition zone where the colour change occurs. The distribution of peroxidase activity was investigated using 3,3'-diaminobenzidine (DAB) or guaiacol as a substrate. Generally, the highest activity was found in the cambial zone and in the middle sapwood. This activity was mainly vacuolar. However, during autumn a peak of activity was observed in the transition zone with DAB, but not with guaiacol. Immunohistolocalization of the peroxidase revealed that the protein was present in the transition zone even if the enzymatic activity was not detectable. Flavan-3-ols were abundantly localized in the transition zone and it is hypothesized that they are physiological substrates of peroxidases. By contrast, polyphenoloxidases do not seem to be implicated in heartwood formation.

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