Keiji Takabe

Distribution of Guaiacyl and Syringyl Lignins in Japanese Beech (Fagus Crenata): Variation Within an Annual Ring

Microspectrometry is the most definitive technique for obtaining both ultraviolet (UV) and visible light absorption spectra from a very limited area, and this technique allows the determination of lignin distribution throughout an individual cell wall. It is generally accepted that hardwood lignin .is composed mainly of guaiacyl and syringyl moieties. Our microspectrometric investigations revealed variation of lignin distribution within an annual ring in beech (Fagus crenata).

Enzymatic saccharification of Eucalyptus bark using hydrothermal pre-treatment with carbon dioxide

In this study, saccharification of the inner bark of Eucalyptus was carried out by enzymatic hydrolysis to produce bioethanol from non-food biomass. To enhance the accessibility of the enzyme to the polysaccharides such as cellulose and holocellulose in the cell wall of the bark, the bark was subjected to hydrothermal pre-treatment with carbon dioxide. This pre-treatment considerably influenced enzymatic hydrolysis. The main component (over 90%) of the generated monosaccharide was glucose, and the yield of glucose on the basis of alpha-cellulose reaches about 80%. This result suggests that the secondary wall, whose main component is cellulose, was effectively hydrolyzed by the enzyme. Microscopic analysis revealed that after pre-treatment, the phloem parenchyma cell had a considerably swollen primary wall and the phloem fibre showed many nano-clefts within its secondary wall. These structural changes appeared to promote enzymatic hydrolysis, because of high accessibility of enzymes to cellulose in the secondary wall.

Immunolocalization and structural variations of xylan in differentiating earlywood tracheid cell walls of Cryptomeria japonica

We investigated the spatial and temporal distribution of xylans in the cell walls of differentiating earlywood tracheids of Cryptomeria japonica using two different types of monoclonal antibodies (LM10 and LM11) combined with immunomicroscopy. Xylans were first deposited in the corner of the S1 layer in the early stages of S1 formation in tracheids. Cell corner middle lamella also showed strong xylan labeling from the early stage of cell wall formation. During secondary cell wall formation, the innermost layer and the boundary between the S1 and S2 layers (S1/S2 region) showed weaker labeling than other parts of the cell wall. However, mature tracheids had an almost uniform distribution of xylans throughout the entire cell wall. Xylan localization labeled with LM10 antibody was stronger in the outer S2 layer than in the inner layer, whereas xylans labeled with LM11 antibody were almost uniformly distributed in the S2 layer. In addition, the LM10 antibody showed almost no xylan labeling in the S1/S2 region, whereas the LM11 antibody revealed strong xylan labeling in the S1/S2 region. These findings suggest that structurally different types of xylans may be deposited in the tracheid cell wall depending on the developmental stage of, or location in, the cell wall. Our study also indicates that deposition of xylans in the early stages of tracheid cell wall formation may be spatially consistent with the early stage of lignin deposition in the tracheid cell wall.

Immunolocalization of β-1-4-galactan and its relationship with lignin distribution in developing compression wood of Cryptomeria japonica

Compression wood (CW) contains higher quantities of β-1-4-galactan than does normal wood (NW). However, the physiological roles and ultrastructural distribution of β-1-4-galactan during CW formation are still not well understood. The present work investigated deposition of β-1-4-galactan in differentiating tracheids of Cryptomeria japonica during CW formation using an immunological probe (LM5) combined with immunomicroscopy. Our immunolabeling studies clearly showed that differences in the distribution of β-1-4-galactan between NW (and opposite wood, OW) and CW are initiated during the formation of the S1 layer. At this stage, CW was strongly labeled in the S1 layer, whereas no label was observed in the S1 layer of NW and OW. Immunogold labeling showed that β-1-4-galactan in the S1 layer of CW tracheids significantly decreased during the formation of the S2 layer. Most β-1-4-galactan labeling was present in the outer S2 region in mature CW tracheids, and was absent in the inner S2 layer that contained helical cavities in the cell wall. In addition, delignified CW tracheids showed significantly more labeling of β-1-4-galactan in the secondary cell wall, suggesting that lignin is likely to mask β-1-4-galactan epitopes. The study clearly showed that β-1-4-galactan in CW was mainly deposited in the outer portion of the secondary cell wall, indicating that its distribution may be spatially consistent with lignin distribution in CW tracheids of Cryptomeria japonica.

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

Abstract 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 S1 layer formation to the S2 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 1H- and 13C-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 S2 layer-forming stage and also after S3 layer formation. In contrast, coniferin was detected in the cell corner middle lamella during the S2 layer-forming stage, and the differential spectra were different from that of coniferin after S3 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 S3 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).

Occurrence of xylan and mannan polysaccharides and their spatial relationship with other cell wall components in differentiating compression wood tracheids of Cryptomeria japonica

Compression wood (CW) tracheids have different cell wall components than normal wood (NW) tracheids. However, temporal and spatial information on cell wall components in CW tracheids is poorly understood. We investigated the distribution of arabino-4-O-methylglucuronoxylans (AGXs) and O-acetyl-galactoglucomannans (GGMs) in differentiating CW tracheids. AGX labeling began to be detected in the corner of the S1 layer at the early S1 formation stage. Subsequently, the cell corner middle lamella (ccML) showed strong AGX labeling when intercellular spaces were not fully formed. AGX labeling was uniformly distributed in the S1 layer, but showed uneven distribution in the S2 layer. AGX labeling was mainly detected in the inner S2 layer after the beginning of the helical cavity formation. The outer S2 layer showed almost no labeling of low substituted AGXs. Only a very small amount of high substituted AGXs was distributed in the outer S2 layer. These patterns of AGX labeling in the S2 layer opposed the lignin and β-1-4-galactan distribution in CW tracheids. GGM labeling patterns were almost identical to AGX labeling in the early stages of CW tracheids, and GGM labeling was detected in the entire S2 layer from the early S2 formation stage of CW tracheids with some spatial differences in labeling density depending on developmental stage. Compared with NW tracheids, CW tracheids showed significantly different AGX distributions in the secondary cell wall but similar GGM labeling patterns. No significant differences were observed in labeling after delignification of CW tracheids.

Temporal and spatial immunolocalization of glucomannans in differentiating earlywood tracheid cell walls of Cryptomeria japonica.

We investigated the deposition of glucomannans (GMs) in differentiating earlywood tracheids of Cryptomeria japonica using immunocytochemical methods. GMs began to deposit at the corner of the cell wall at the early stages of S1 formation and showed uneven distribution in the cell wall during S1 formation. At the early stages of S2 formation, limited GM labeling was observed in the S2 layer, and then the labeling increased gradually. In mature tracheids, the boundary between the S1 and S2 layers and the innermost part of the cell wall showed stronger labeling than other parts of the cell wall. Deacetylation of GMs with mild alkali treatment led to a significant increase in GM labeling and a more uniform distribution of GMs in the cell wall than that observed before deacetylation, indicating that some GM epitopes may be masked by acetylation. However, the changes in GM labeling after deacetylation were not very pronounced until early stages of S2 formation, indicating that GMs deposited in the cell wall at early stages of cell-wall formation may contain fewer acetyl groups than those deposited at later stages. Additionally, the density of GM labeling increased in the cell wall in both specimens before and after GM deacetylation, even after cell-wall formation was complete. This finding suggests that some acetyl groups may be removed from GMs after cell-wall formation is complete as part one of the tracheid cell aging processes.

Temporal and spatial diversities of the immunolabeling of mannan and xylan polysaccharides in differentiating earlywood ray cells and pits of Cryptomeria japonica

Wood is composed of various types of cells and each type of cell has different structural and functional properties. However, the temporal and spatial diversities of cell wall components in the cell wall between different cell types are rarely understood. To extend our understanding of distributional diversities of cell wall components among cells, we investigated the immunolabeling of mannans (O-acetyl-galactoglucomannans, GGMs) and xylans (arabino-4-O-methylglucuronoxylans, AGXs) in ray cells and pits. The labeling of GGMs and AGXs was temporally different in ray cells. GGM labeling began to be detected in ray cells at early stages of S1 formation in tracheids, whereas AGX labeling began to be detected in ray cells at the S2 formation stage in tracheids. The occurrence of GGM and AGX labeling in ray cells was also temporally different from that of tracheids. AGX labeling began to be detected much later in ray cells than in tracheids. GGM labeling also began to be detected in ray cells either slightly earlier or later than in tracheids. In pits, GGM labeling was detected in bordered and cross-field pit membranes at early stages of pit formation, but not observed in mature pits, indicating that enzymes capable of GGM degradation may be involved in pit membrane formation. In contrast to GGMs, AGXs were not detected in pit membranes during the entire developmental process of bordered and cross-field pits. AGXs showed structural and depositional variations in pit borders depending on the developmental stage of bordered and cross-field pits.

Living wood fibers act as large-capacity ''single-use'' starch storage in black locust (Robinia pseudoacacia)

A living wood fiber (LWF) is one that retains the living protoplast. LWFs store numerous starch grains during the dormant period. In black locust (Robinia pseudoacacia), almost all wood fibers in the outer part of the annual ring are LWFs. In the outermost ring, starch is accumulated during the summer, retained in winter, and metabolized during spring. We determined the starch content of LWFs, ray parenchyma, and axial parenchyma using image analysis. More than 70% of the starch grains in the outermost ring were stored in LWFs during winter. After the breakdown of starch in spring, LWFs resulted in cell death. These results indicate that LWFs in black locust function as “single-use” large-capacity starch storage.

Diffusion pathways for heartwood substances in Acacia mangium.

The cellular distribution of heartwood substances and the structure of the pathways for their diffusion were studied in Acacia mangium Willd. Apart from ray parenchyma cells, axial parenchyma cells also are involved in the formation of heartwood substances. Heartwood substances were unevenly distributed in the heartwood. A closer inspection of interfibre pit pairs revealed that, although many pit membranes were completely covered with encrusting materials, some pit pairs had many small openings on their pit membranes. The openings possibly function as intercellular diffusion pathways for heartwood substances. The sizes of the pits varied considerably, ranging from 0.4 to 2.3 μm in diameter. These structural variations in the interfiber pits might be one of the factors contributing to the uneven distribution of the heartwood substances. A large number of blind pits were present in the ray parenchyma cells and faced the intercellular spaces, into which heartwood substances from the ray parenchyma cells were released via these blind pits. Resin-cast replicas demonstrated that the intercellular spaces and the blind pits formed a three-dimensional network that is considered to serve as an extracellular diffusion pathway for heartwood substances.