Common Mechanism of Lignification of Compression Wood in Conifers and Buxus

Woody plants develop a specialized secondary xylem known as reaction wood to enable formation of an ideal shape. Reaction wood in coniferous species is known as compression wood, and that of woody angiosperms as tension wood. However, the genus Buxus which is classified as an angiosperm, forms compression-wood-like reaction wood. We investigated the mechanism of lignification in coniferous compression wood and Buxus reaction wood: 1) Several lignin synthesis genes were upregulated in differentiating reaction wood of Buxus microphylla;2) B. microphylla possesses a specific laccase gene that is expressed specifically in differentiating reaction wood (BmLac4);3) laccase activity localization was closely related to lignification of reaction wood, and laccase activity was high in the secondary wall middle layer;4) in reaction wood cell walls, galactan was present in the outer portion of the secondary wall middle layer, and the level of xylan was reduced. These findings suggest that lignification in B. microphylla reaction wood is identical to that of coniferous compression wood. These may represent general mechanisms of increasing lignin content in various reaction woods.

Diurnal Periodicity of the Expression of Genes Involved in Monolignol Biosynthesis in Differentiating Xylem of Cryptomeria japonica

The cell wall in wood is mainly composed of three components: cellulose, hemicellulose and lignin. According to electron microscopy observations of the innermost surface of cell walls in the tracheids of Cryptomeria japonica , cellulose microfibrils are deposited during the day and a matrix containing hemicellulose is deposited at night. This indicates that the deposition of cell wall components exhibits diurnal periodicity. To gain new insights into the diurnal periodicity of lignin deposition not revealed by microscopic observations, we examined diurnal fluctuations in the expression of genes involved in monolignol biosynthesis in C. japonica saplings grown in the field and in growth chambers under a 12 h light/dark cycle. In the field experiment, two gene expression peaks were observed daily, at dusk and dawn. In the growth chamber experiment, two daily peaks were observed 0 h and 6 - 9 h after the light switched on.

Observations of Wood Cell Walls with a Scanning Probe Microscope

Scanning Probe Microscopes (SPMs) observe specimen surfaces with probes by detecting the physical amount of a material between the cantilever and the surface. SPMs have a high resolution and can measure mechanical characteristics such as stiffness, adsorptive properties, and viscoelasticity. These features make it easy to identify the surface structure of complex materials;therefore, the use of SPMs has increased in recent years. Wood cell walls are primarily composed of cellulose, hemicellulose, and lignin. It is believed that hemicellulose and lignin surround the cellulose framework;however, their detailed formation remains unknown. Therefore, we observed wood cell walls via scanning probe microscopy to try to reveal the formation of the cellulose framework. We determined that the size of the cellulose microfibril bundle and hemicellulose lignin module composite was 18.48 nm based on topography and that the size of the cellulose microfibril bundle was 15.33 nm based on phase images. In the viscoelasticity image, we found that the viscoelasticities of each cell wall of the same cell were not the same. This is because the cellulose microfibrils in each cell wall lean in different directions. The angle between the leaning of the cellulose microfibril and the cantilever affects the viscoelasticity measurement.

Immunolocalization of a Normal Wood Specific Pectin Methylesterase (CoPME) and Quantification of PME Gene Expression in Differentiating Xylem of <i>Chamaecyparis obtusa</i>

The transverse section of compression wood tracheids has a circular shape and intercellular spaces. The cause has not been determined yet;however, we hypothesized that peeling of the cell wall adhesion would cause cellular intervals, resulting in circularity of the transverse section of tracheids. Homogalacturonan, a type of pectin, functions in cell wall adhesion. Further, pectin methylesterase (PME) is involved in functionalization of homogalacturonan. We quantitated PME gene expression levels in differentiating xylem cells using different degrees of compression wood samples and examined the correlation with circularity of the transverse section of tracheids in each sample. We found that lower gene expression level of the sample corresponded with increasing circularity of the transverse section of tracheids. It is considered that the transverse section of compression wood tracheids becomes circular by suppression of PME gene expression during differentiation. Further, we observed the normal wood specific pectin methylesterase (CoPME) localization in differentiating xylem tracheids by immunolabeling. Labels localized at the entire perimeter of the compound middle lamella in normal wood, whereas sparse labeling was found in compression wood. It suggests that cell walls adhere at sites of CoPME function in differentiating xylem tracheids, but there is inadequate adhesion between cell walls where CoPME does not function. At the end of the expansion zone, the volume of the cell decreases due to a decrease in the turgor pressure of the tracheid. Further, due to moisture shrinkage of the tracheid, the adhesion begins to peel off in places of inadequate adhesion between cell walls, resulting in cell gaps and, thereby, generating a circular cell shape of cell wall formation in compression wood.

Transcriptome Analysis of Reaction Wood in Gymnosperms by Next-Generation Sequencing

Special xylem tissue called “compression wood” is formed on the lower side of inclined stems when gymnosperms grow on a slope. We investigated the molecular mechanism of compression wood formation. Transcriptome analysis by next-generation sequencing (NGS) was applied to the xylem of Chamaecyparis obtusa to develop a catalog of general gene expression in differentiating xylem during compression and normal wood formation. The sequencing output generated 234,924,605 reads and 40,602 contigs (mean size = 529 bp). Based on a sequence similarity search with known proteins, 54.2% (22,005) of the contigs showed homology with sequences in the databases. Of these annotated contigs, 19,293 contigs were assigned to Gene Ontology categories. Differential gene expression between the compression and normal wood libraries was analyzed by mapping the reads from each library to the assembled contigs. In total, 2875 contigs were identified as differentially expressed, including 1207 that were up-regulated and 1668 that were down-regulated in compression wood. We selected 30 genes and compared the transcript abundance between compression and normal wood by quantitative polymerase chain reaction analysis to validate the NGS results. We found that 27 of the 30 genes showed the same expression patterns as the original NGS results.