Abstract text: Dirigent proteins (DIRs) are frequently observed in lignifying plant cells and tissues. Certain DIRs have been found to be essential in lignin formation in Casparian strips of roots, providing an indication of their involvement in lignin synthesis. Some DIRs are known to guide the stereoselective coupling of coniferyl alcohol radicals during dimeric lignan synthesis by stabilizing radicals prior to their coupling during e.g. (-) pinoresinol formation.

We have observed that several DIRs are bound to extracellular lignin of Norway spruce. We produced two Norway spruce DIRs heterologously and used them for synthetic lignin (DHP) polymerization assays with horseradish peroxidase and a fungal Trametes hirsuta laccase as oxidative enzymes, to determine whether the DIRs affect linkage proportions in the lignin polymer. HSQC₀ 2D NMR analyses showed no change in the linkage type proportions compared to DHP produced without DIR supplementation. A chirality analysis of dimeric pinoresinol isolated from the DHP production mixture showed it to be a racemic mixture of equal amounts of (+) and (–) forms. These data suggest that the two spruce DIRs do not guide linkage formation in lignin polymer nor stereoselective coupling of coniferyl alcohol radicals in pinoresinol formation.