Cell wall lignin is polymerised by class III secretable plant peroxidases in Norway spruce.

Class III secretable plant peroxidases occur as a large family of genes in plants with many functions and probable redundancy. In this review we are concentrating on the evidence we have on the catalysis of lignin polymerization by class III plant peroxidases present in the apoplastic space in the xylem of trees. Some evidence exists on the specificity of peroxidase isozymes in lignin polymerization through substrate specificity studies, from antisense mutants in tobacco and poplar and from tissue and cell culture lines of Norway spruce (Picea abies) and Zinnia elegans. In addition, real time (RT-)PCR results have pointed out that many peroxidases have tissue specific expression patterns in Norway spruce. Through combining information on catalytic properties of the enzymes, on the expression patterns of the corresponding genes, and on the presence of monolignols and hydrogen peroxide in the apoplastic space, we can show that specific peroxidases catalyze lignin polymerization in the apoplastic space of Norway spruce xylem.

The role of xylem class III peroxidases in lignification.

Lignification is a cell wall fortifying process which occurs in xylem tissue in a scheduled manner during tissue differentiation. In this review, enzymes and the genes responsible for lignin biosynthesis have been studied with an emphasis on lignin polymerizing class III secretable plant peroxidases. Our aim is to understand the cell and molecular biology of the polymerization of lignin especially in tracheids and vessels of woody species but much of the experimental evidence comes from herbaceous plants. Class III peroxidases pose many problems for empirical work as their encoding genes are variable, their substrate specificities are wide and the half-life of many of the isozymes is very long. However, there is some evidence for the role of specific peroxidases in lignin polymerization through antisense mutants in tobacco and poplar and from tissue and cell culture lines of Picea abies and Zinnia elegans. Peroxidase enzyme action has been shown by substrate specificity studies and, for example, RT-PCR results have pointed out that many peroxidases have tissue-specific expression patterns. Tissue-level location of gene expression of some peroxidases has been studied by in situ hybridization and their cellular localization with antibodies and using EGFP-fusion genes. From these, it can be concluded that, although many of the xylem class III peroxidases have the potential for functioning in the synthesis of the lignin polymer, the combined information of catalytic properties, expression, and localization can reveal differences in the significance of different peroxidases in the lignification process.

The dibenzodioxocin lignin substructure is abundant in the inner part of the secondary wall in Norway spruce and silver birch xylem.

A specific condensed lignin substructure, dibenzodioxocin, was immunolocalized in differentiating cell walls of Norway spruce ( Picea abies (L.) H. Karsten) and silver birch ( Betula pendula Roth) xylem. A fluorescent probe, Alexa 488 was used as a marker on the dibenzodioxocin-specific secondary antibody. For the detection of this lignin substructure, 25-microm cross-sections of xylem were viewed with a confocal laser-scanning microscope with fluorescein isothiocyanate fluorescence filters. In mature cells, fluorescence was detected in the S3 layer of the secondary wall in both tree species, but it was more intense in Norway spruce than in silver birch. In silver birch most of the signal was detected in vessel walls and less in fiber cell walls. In very young tracheids of Norway spruce and vessels and fibers of silver birch, where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown.

Localization of dibenzodioxocin substructures in lignifying Norway spruce xylem by transmission electron microscopy-immunogold labeling.

The lignification process in mature Norway spruce [ Picea abies (L.) H. Karsten] xylem cell walls was studied using transmission electron microscopy (TEM)-immunogold detection with a polyclonal antibody raised against a specific lignin substructure, dibenzodioxocin. The study reveals for the first time the exact location of this abundant eight-ring structure in the cell wall layers of wood. Spruce wood samples were collected in Southern Finland at the time of active growth and lignification of the xylem cell walls. In very young tracheids where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown at all. During secondary cell wall thickening, the dibenzodioxocin structure was more abundant in the secondary cell wall layers than in the middle lamella. The highest number of gold particles revealing dibenzodioxocin was in the S2+S3 layer. Statistically significant differences were found in the frequency of gold particles present in various cell wall layers. For comparison, wood sections were also cut with a cryomicrotome for light and fluorescence microscopy.