Cellular distribution of coniferin in differentiating xylem of Chamaecyparis obtusa as revealed by Raman microscopy

Cellular distribution of coniferin in differentiating xylem of Japanese cypress (Chamaecyparis obtusa) was analyzed by Raman microscopy. Small blocks were collected from differentiating xylem, frozen, cut on their surface with a sliding microtome, and then freeze-dried. Scanning electron microscopy showed numerous needle-like deposits in the tracheid lumina from the beginning of the S-1 layer formation to the S-2 layer-forming stage. The Raman spectrum of the deposits in the tracheid lumen was similar to that of coniferin. The presence of coniferin in a water extract from differentiating xylem was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy and H-1- and C-13-nuclear magnetic resonance spectra. Differential Raman spectra taken from samples before and after washing with water and dehydration in an ethanol showed that developing secondary walls contained coniferin during the S-2 layer-forming stage and also after S-3 layer formation. In contrast, coniferin was detected in the cell corner middle lamella during the S-2 layer-forming stage, and the differential spectra were different from that of coniferin after S-3 layer formation. The differential spectrum in this stage was similar to that of a dehydrogenation polymer of coniferyl alcohol prepared by the zulauf method (bulk polymerization). These results suggest that free lignin oligomers of the type bulk polymerizate might exist in the cell corner middle lamella during the S-3 layer-forming stage and can be removed from specimens during washing and dehydration. The results can be interpreted in a way that no such oligomer exists in the secondary wall during the same stage owing to endwise addition of monolignols (in analogy to a zutropf polymerization).