Relative deposition of xylan and 8-5'-linked lignin structure in Chamaecyparis obtusa, as revealed by double immunolabeling by using monoclonal antibodies.

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CONCLUSION: Immunolabeling by using monoclonal antibodies showed that xylan deposition precedes the formation of 8-5'-linked structure of lignin in normal and compression woods of Chamaecyparis obtusa. Xylan deposition and formation of 8-5'-linked lignin structure in differentiating xylems from normal and compression woods in Chamaecyparis obtusa were examined by immunoelectron microscopy using monoclonal antibodies (LM10 or LM11) to detect xylan localization. The 8-5'-linked lignin structure was immunolocalized using KM1 antibody. Xylan and 8-5'-linked lignin double immunolabeling was performed using secondary antibodies labeled with colloidal gold particles of different diameters. In normal wood, KM1 labeling occurred in the compound middle lamella (CML) and S1 layer during S1 layer formation and increased as S2 and S3 layers formed, with labeling occurring at the outer part of the previous layer. In compression wood, mild KM1 labeling occurred in the CML and outer part of the S1 layer at the later S1 layer formation stage, with increased labeling as the S2 layer formed. Minor labeling occurred in the outer part of the S2 layer during helical cavity formation. Comparison between KM1 labeling and KMnO4 staining suggested that lignin other than 8-5'-linked structure was formed during early lignification, and the proportion of 8-5'-linked lignin structure increased at later stages of lignification in both normal and compression woods. LM10 and LM11 labeling occurred slightly earlier than KM1 labeling, suggesting that xylan deposition preceded the formation of 8-5'-linked lignin in normal and compression woods. Less labeling by KM1, LM10, and LM11 occurred in the outer part of the S2 layer in compression wood, which has abundant lignin. Thus, lignin in these parts is composed of lignin substructures other than the 8-5' linkage.