Kazumi Fukazawa

Dynamic changes in the arrangement of cortical microtubules in conifer tracheids during differentiation

The arrangement of cortical microtubules (MTs) in differentiating tracheids of Abies sachalinensis Masters was examined by confocal laser scanning microscopy after immunofluorescent staining. The arrays of MTs in the tracheids during formation of the primary wall were not well ordered and the predominant orientation changed from longitudinal to transverse. During formation of the secondary wall, the arrays of MTs were well ordered and their orientation changed progressively from a flat S-helix to a steep Z-helix and then to a flat S-helix as the differentiation of tracheids proceeded. The orientation of cellulose microfibrils (MFs) on the innermost surface of cell walls changed in a similar manner to that of the MTs. These results provide strong evidence for the co-alignment of MTs and MFs during the formation of the semi-helicoidal texture of the cell wall in conifer tracheids.

Orientation of microfibrils and microtubules in developing tension-wood fibres of Japanese ash (Fraxinus mandshurica var. japonica)

The orientation of cellulose microfibrils (MFs) and the arrangement of cortical microtubules (MTs) in the developing tension-wood fibres of Japanese ash (Fraxinus mandshurica Rupr. var. japonica Maxim.) trees were investigated by electron and immunofluorescence microscopy. The MFs were deposited at an angle of about 45° to the longitudinal axis of the fibre in an S-helical orientation at the initiation of secondary wall thickening. The MFs changed their orientation progressively, with clockwise rotation (viewed from the lumen side), from the S-helix until they were oriented approximately parallel to the fibre axis. This configuration can be considered as a semihelicoidal pattern. With arresting of rotation, a thick gelatinous (G-) layer was developed as a result of the repeated deposition of parallel MFs with a consistent texture. Two types of gelatinous fibre were identified on the basis of the orientation of MFs at the later stage of G-layer deposition. Microfibrils of type 1 were oriented parallel to the fibre axis; MFs of type 2 were laid down with counterclockwise rotation. The counterclockwise rotation of MFs was associated with a variation in the angle of MFs with respect to the fibre axis that ranged from 5° to 25° with a Z-helical orientation among the fibres. The MFs showed a high degree of parallelism at all stages of deposition during G-layer formation. No MFs with an S-helical orientation were observed in the G-layer. Based on these results, a model for the orientation and deposition of MFs in the secondary wall of tension-wood fibres with an S1 + G type of wall organization is proposed. The MT arrays changed progressively, with clockwise rotation (viewed from the lumen side), from an angle of about 35–40° in a Z-helical orientation to an angle of approximately 0° (parallel) to the fibre axis during G-layer formation. The parallelism between MTs and MFs was evident. The density of MTs in the developing tension-wood fibres during formation of the G-layer was about 17–18 per μm of wall. It appears that MTs with a high density play a significant role in regulating the orientation of nascent MFs in the secondary walls of wood fibres. It also appears that the high degree of parallelism among MFs is closely related to the parallelism of MTs that are present at a high density.

Changes in the arrangement of cellulose microfibrils associated with the cessation of cell expansion in tracheids

Abstract The relationship between the cessation of cell expansion and formation of the secondary wall was investigated in the early-wood tracheids of Abies sachalinensis Masters by image analysis and field emission scanning electron microscopy. The area of the lumen and the length of the perimeter of the lumen of differentiating tracheids increased from the cambium towards the xylem. These increases had just ceased in the case of tracheids closest to the cambium in which birefringence was first detected by observations with a polarizing light microscope. Cellulose microfibrils (MFs) deposited on the innermost surfaces of radial walls were not well ordered during the expansion of cells, but well ordered MFs were deposited at the subsequent stage of cell wall formation. The first well ordered MFs were oriented in an S-helix. The well ordered MFs had already been deposited at the tracheids where birefringence was first detected under the polarizing light microscope. These results indicate that the deposition of the well ordered MFs, namely, the formation of the secondary wall, begins before the cessation of cell expansion of tracheids. Therefore, it seems that the expansion of tracheids is restricted by the deposition of the secondary wall because the cell walls become rigid simultaneously with the development of the secondary wall and, therefore, the yield point of cell walls exceeds the turgor pressure of the cell.

Direct mapping of morphological distribution of syringyl and guaiacyl lignin in the xylem of maple by time-of-flight secondary ion mass spectrometry

Lignin, one of the main structural polymer of plant cell walls, varies in amount and monomeric composition among tissue and cell types, as well as among plant species. However, few analytical methods are available that can conveniently and accurately determine the morphological distribution of lignin units at the cellular level. In this report, we used time-of-flight secondary ion mass spectrometry (TOF-SIMS) to directly map guaiacyl (G) and syringyl (S) lignin units in several successive growth rings of the maple xylem. TOF-SIMS imaging and a semiquantitative approach revealed clear difference in the annual distribution of lignins between the fiber and vessel. While the vessel walls were constantly G-rich with varied S/G ratios through a growth ring, the fibers showed fairly regular annual distribution of lignins in which the earlywood was S-rich with an almost constant S/G ratio and the latewood was G-rich resulting from a decrease of the S unit. The reliability of TOF-SIMS results was demonstrated by its high correlation with the results of thioacidolysis on radial distribution of the S/G ratio in several contiguous tree rings and also in the latewood and earlywood of each ring. These results indicate that TOF-SIMS allows direct visualization of lignin composition in plant tissues.

Fe-Sem Observations on the Microfibrillar Orientation in the Secondary Wall of Tracheids

Field emission scanning electron microscopy was used to observe the inner surfaces of the developing secondary walls of earlywood tracheids of Abies sachalinensis Masters. Microfibrillar orientation in the secondary wall, as seen from the lumen side, changed in a clockwise direction from the outermost S1 to the middle of the S2 and from there counter-clockwise to the innermost S3. Sometimes microfibrils oriented in a steep S-helix were observed in the S3 layer. Lamellae showing different microfibrillar orientations in wall layers other than the S2 were observed beneath newly deposited microfibrils on the inner surface of the developing wall. Furthermore, on the inner surface of the wall forming the S12, S23 and S3, lamellae with microfibrils closely aligned at the same angle as one another and lacking spaces were not observed. These observations suggest that in layers other than the S2 most lamellae are not composed of closely spaced microfibrils.

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).

Microfibrillar Orientation of the Innermost Surface of Conifer Tracheid Walls

The orientation of the microfibri1s deposited on the innermost surfaces of the tracheid wall was observed in three conifer species, Larix leptolepis, Picea jezoensis, and Picea abies, using field emission scanning electron microscopy (FE-SEM). The microfibrillar orientation is different in each tracheid and exhibits either an S- or a Z-helix. The latest microfibrils deposited were normally joined into small bundles having various widths and had a different orientation from the microfibrils beneath them. When the latest deposited microfibrils on the innermost surface were oriented in an S-helix, the microfibrils beneath them were oriented in either a flatter S-helix or in a Z-helix, and when they were oriented in a Z-helix, the microfibrils beneath them were oriented in a steeper Z-helix. This is because, as seen from the lumen side, the microfibrillar orientation changes counterclockwise from the outer S23 to the innermost S3. These microfibrillar orientations varied throughout a single annual ring in each of the three species. The commonly observed angles of these microfibril were: Larix leptolepis: 70-80°, Picea jezoensis: 60-70°, and Picea abies: 40-50° in an S-helix, and the maximum range of angles was limited in extent to about 90 degrees in all species.

Histochemical study on heterogeneity of lignin in Eucalyptus species II. The distribution of lignins and polyphenols in the walls of various cell types

We examined the effects of polyphenols on the analysis of lignin by histochemical methods, namely, the Maule color reaction coupled with microspectrophotometry and ultraviolet microspectrophotometry, in wood of Eucalyptus camaldulensis and E. globulus. Thin sections and wood meals were extracted with solutions of alkali at different concentrations. The amounts of alkali-soluble extractives increased with increasing concentrations of NaOH. By contrast, there was no clear correlation between amounts of Klason lignin and the concentration of NaOH. The visible-light absorption spectra of cell walls of all woody tissues from both species changed after alkali extraction. In particular, the spectra of cell walls of vessel elements changed considerably, even when only a dilute solution of alkali was used. Ultraviolet absorption spectra did not show clear changes after extraction with alkali. These results indicate that polyphenols in cell walls affect the results of histochemical analysis. Therefore, a preliminary extraction with alkali, namely, extraction with a 1% solution of NaOH, is needed to assess the precise distribution of lignins in the cell walls of Eucalyptus wood by histochemical methods. The cell walls of wood fibers of Eucalyptus camaldulensis contained both guaiacyl and syringyl units and those of vessel walls contained mostly guaiacyl units. However, the cell walls of wood fibers in Eucalyptus globulus contained mainly syringyl units, while those of vessel elements contained both guaiacyl and syringyl units. Syringyl-type polyphenols, which have spectra similar to those of syringyl-type lignins, were found in the cell walls of wood fibers and vessel elements and in cell corners among wood fibers in both species of Eucalyptus.

Distribution of syringyl and guaiacyl lignins in hardwoods in relation to habitat and porosity form in wood

Syringyl (S) and guaiacyl (G) lignins wcrc dctcctcd by UVandVIS microspcctrophotomctry. 25 hardwood spccics in 21 gcncra and 18 familics from 3 habitats ofYunnan, China, wcrc invc.stigatcd.Thc monomcr composition of lignin in a particular arca of wood tissuc was influcnccd by thc kind of ccll clcmcnts and location wilhin a ring, vcsscl arrangcmcnt of woods, and trcc habitat.These variations and thc diffcrcnccs in G/S ratio among ccrtain cclls or trccs may suggcst thc cvolutionary stagc of trcc phylogcny.

The Occurrence and Morphology of Tyloses and Gums in the Vessels of Japanese Hardwoods

The occurrence and morphology of natural tyloses and gums in the vessels of 50 Japanese hardwoods (15 ring-, 34 diffuseand 1 radial-porous woods) were investigated using SEM. Tyloses were present exclusively or predominantly in 23 species (12 ring-, 10 diffuse- and 1 radial-porous woods) and gums in 15 species (3 ring- and 12 diffuse-porous woods). In the pore zones of most of the ringporous woods both tyloses and gums first occurred in an earlier ring number from the bark than in the diffNse- and radial-porous woods. Tyloses and gums originated from both ray and axial parenchyma cells in most species which have pit pairs connecting these cells to the vessels. Except for four species, the maximum and minimum diameters of the inner pit aperture from vessels to parenchyma cells were greater than 5 and 2 µm, respectively, in those species with tyloses, whereas the diameters were less than these values in species having gums. The forms of tylosis blockings in heartwood vessels were closely related to parenchyma patterns.