Lignin distribution across tracheid cell walls of poorly lignified wood from deformed copper deficient Pinus radiata (D. Don)

Summary Lignin topochemistry of tracheid walls from a deformed, copper deficient Pinus radiata (D. Don) tree was examined by linescan and point analyses using a Scanning Electron Microscope and Energy Dispersive Spectrometry. Both opposite and compression wood had abnormal lignin distributions compared to those observed in normal wood from a straight tree. Lignin contents in the compound middle lamella were lower than lignin contents in the secondary wall in both opposite and compression wood tracheids.One of us (G. D.) held a Commonwealth Forestry Postgraduate Research Award during this study. The research was supported in part by a grant from the Reserve Bank of Australia Rural Credits Development Fund, the Pine Fund, and members of the forest industry     

The supply of matrix containing glucomannans to the innermost surface of S2 layers is associated with changes in turgor pressure of differentiating tracheids in Cryptomeria japonica

We investigated the relationship between turgor pressure and diurnal differences in secondary wall formation of differentiating tracheids. Saplings of Cryptomeria japonica were grown in a growth chamber with 12-h light:12-h dark cycles, and the tangential strain on the inner bark surface was measured as an indicator of the volumetric changes of differentiating cells. The innermost surface of developing secondary walls was then observed using field emission scanning electron microscopy at 1-h intervals after both light and dark periods. Dramatic changes in the aspects of the innermost surface of developing secondary walls occurred 3h after the light was switched on and 4h after the light was switched off. The amorphous material containing glucomannans became evident when the differentiating cells became fully turgid during the dark period. Conversely, cellulose microfibrils became clearly visible when the cell volume was low during the light period. These results suggest that the diurnal periodicity in the supply of hemicellulose-containing matrix to developing secondary walls is associated with the changes in turgor pressure of differentiating tracheids that result from the change in light conditions during the photoperiodic cycle.     

The effect of day length on diurnal differences in the innermost surface of the S2 layer in differentiating tracheids

This article describes the effect of day length during the photoperiodic cycle on the diurnal differences in the innermost surface of developing secondary walls. Saplings of Cryptomeria japonica D. Don. were grown in growth chambers at constant temperature and relative humidity, but with different photoperiods. Samples of differentiating xylem were collected during the light and dark periods. The innermost surface of developing secondary walls in differentiating tracheids were observed using field emission scanning electron microscopy, and observations made during the light and dark periods were compared. In the saplings grown under long-day or short-day conditions, diurnal differences in aspects of the innermost surface of developing secondary walls were observed. Cellulose microfibrils were observed on the innermost surface of developing secondary walls during the light period when the volumes of differentiating cells were low, and amorphous material was observed during the dark period, when differentiating tracheids were turgid. The amorphous material was labeled with antiglucomannan antiserum. These results suggest that the range of day-length conditions set in this study does not affect the diurnal periodicity in the supply of cell wall components to the innermost surface of developing secondary walls.     

Ultrastructure of compression wood in Ginkgo biloba

Summary Compression wood in the ancient Ginkgo biloba differs from that in most of the younger gymnosperms in the more angular outline of its tracheids, their thinner walls, and their lack of helical cavities. Both normal and compression woods of Ginkgo contain two types of tracheids, one wide, with a thin wall, and another, narrow, with a thicker wall. In all other respects the compression wood tracheids in Ginkgo are ultrastructurally similar to those in other gymnosperms. Helical cavities probably developed relatively late in the evolution of compression wood, since they are missing not only in Ginkgo but also in the Taxales and the Araucariaceae. The occurrence of compression wood in Ginkgo biloba indicates that this tissue probably has existed since the Devonean period. Very likely, the arborescent habit of the gymnosperms has always been dependent on their ability to form compression wood.This investigation was carried out under the McIntire-Stennis Program, Cooperative State Research Service. I am indebted to Mr. A. C. Day of this College and to Mr. A. Rezanowich of the Pulp and Paper Research Institute of Canada for kindly providing the scanning electron micrographs.     

Morphological changes in the cytoskeleton, nuclei, and vacuoles during cell death of short-lived ray tracheids in the conifer Pinus densiflora

Morphological changes in the cytoskeleton, nuclei, and vacuoles were monitored during the cell death of short-lived ray tracheids in the conifer Pinus densiflora. After formation of the dentate thickenings that occurred at the final stage of formation of cell walls, organelles started to disappear in differentiating ray tracheids. First, the microtubules and vacuoles disappeared. Then actin filaments disappeared in the differentiating ray tracheids adjacent to ray tracheids that lacked nuclei, and, finally, the nuclei disappeared. These features indicate that cell death in ray tracheids might differ from the programmed cell death of tracheary elements that has been studied in vitro in the Zinnia culture system. This study was presented at the 57th Annual Meeting of the Japan Wood Research Society, August 8–10, 2007, Hiroshima, Japan     

Formation and structure of reaction wood in Buxus microphylla var. insularis Nakai

Summary Anatomical differences in xylem between the upper and lower sides formed in the inclined stems of Buxus microphylla with different angular displacement from the vertical were examined microscopically. B. microphylla exhibited a pronounced growth promotion on the lower side of the inclined stems. Formation of tension wood (gelatinous fibers) was not observed. Xylem formed on the lower side showed some interesting features resembling the compression wood formed in gymnosperms. The reaction wood tracheids and vessels showed an excessive lignification in their secondary walls but lacked both helical cavities and an S₃ layer, features that were almost the same as those of primitive gymnosperms. These results indicate that B. microphylla has an ability to form compression wood, suggesting that in the genus Buxus a different mechanism in the conducting elements was developed in the phylogenetic evolution.The first author would like to express his sincere thanks to Dr. T. E. Timell, College of Environmental Science and Forestry, State University of New York, Syracuse, New York, for his invaluable suggestions in connection with this research     

Anatomy and lignin distribution of reaction wood in two Magnolia species

Summary Anatomical features of reaction wood formed in two Magnolia species, M. obovata Thunb. and M. kobus DC. which are considered to be among the primitive angiosperms, were observed. In addition, the distribution of guaiacyl and syringyl units of lignins in the cell walls of normal and reaction wood was examined using ultraviolet (UV)- and visible light (VL)- microspectrophotometry coupled with the Wiesner and M?ule reactions. The two Magnolia species formed a tension-like reaction wood without possessing the typical gelatinous layer (G-layer) on the upper side of the inclined stem or branch, in which a radial growth promotion occurred. Compared with the normal wood, the reaction wood had the following anatomical features: (1) the secondary walls of fiber tracheids lacked the S₃ layer, (2) the innermost layer of fiber-tracheid walls showed a small microfibril angle, a fact being similar to the orientation of the microfibril angle of the G-layer in tension wood, and (3) the amounts of lignin decreased in the cell walls of fiber tracheids, especially with great decrease in proportion of guaiacyl units in lignins. In addition, VL-microspectrophotometry coupled with the Wiesner and M?ule reactions adopted in the present study showed potential to estimate the lignin contents in the cell walls and the proportion of guaiacyl and syringyl units in lignins. Received: 15 July 1998     

Seasonal dynamics of wood formation: a comparison between pinning,microcoring and dendrometer measurements

Three different methods were evaluated for analysing wood formation of Norway spruce [Picea abies (L.) Karst.] and Scots pine (Pinus sylvestris L.) in Finland. During two growing seasons, wood formation dynamics were determined both by wounding the cambium with a needle followed by localisation of the wound-associated tissue modification after the growing season (pinning), and by extracting small increment cores during the growing season (microcoring). Stem radius was additionally monitored with band dendrometers. For Norway spruce, pinning and microcoring yielded similar dates for the onset of wood formation. The timing of wood production during the growing season was also similar for pinning and microcoring. For Scots pine, the onset of wood formation was recorded from microcores almost 2 weeks later than from pinning samples. In Scots pine, microcore measurements also produced somewhat later cessation dates for tracheid formation than the pinning samples. For both tree species, the total number of tracheids formed during the growing season was, however, about the same for pinning and microcoring. Dendrometer results clearly differed from those of pinning and microcoring. In particular, the dendrometers showed an increase of stem radius considerably earlier in spring, when the other methods did not detect wood formation. Thus, pinning and microcoring currently represent the most reliable techniques for detailed monitoring of wood formation.     

Distribution of lignin in normal and compression wood of Pinus taeda L.

Summary The distribution of lignin in normal and compression wood of loblolly pine (Pinus taeda L.) has been studied by the technique of lignin skeletonizing. Hydrolysis of the wood carbohydrates with hydrofluoric acid left normal wood tracheids with a uniform distribution of lignin in the S1 and S2 cell wall layers. However, the S3 region of both earlywood and latewood tracheids consistently retained a dense network of unhydrolyzable material throughout, perhaps lignin.Lignin content in compression wood averaged about 7% more than in normal wood and appears to be concentrated in the outer zone of the S2 layer. The inner S2 region, despite helical checking, is also heavily lignified. The S1 layer, although thicker than normal in compression wood tracheids, contains relatively little lignin.Ray cells, at least in normal wood, appear to be lignified to the same extent, if not more so in certain cases, than the longitudinal tracheids. Other locations where lignin may be concentrated include initial pit border regions and the membranes of bordered pits.This report is a detailed excerpt from the Ph. D. dissertation of R. A. P. Financial support provided by the College of Forestry at Syracuse University and the National Defense Education Act is hereby gratefully acknowledged.     

Effect of light intensity on diurnal differences in the supply of cell wall components to the innermost surface of developing S<Subscript>2</Subscript> layers of tracheids in <Emphasis Type="Italic">Cryptomeria japonica</Emphasis>

We investigated the effect of light intensity on diurnal differences in secondary wall formation of tracheids. Saplings of Cryptomeria japonica were grown in growth chambers with light intensity cycles set for 12-h high light: 12-h low light by combining two of four light intensity levels: 1.5, 2.8, 4.3, and 10.0 klx. Volumetric changes of differentiating cells were monitored by measuring the tangential strain on the inner bark surface, and the innermost surface of developing secondary walls of differentiating tracheids during the high-light and low-light periods was observed by field-emission scanning electron microscopy. Changes in the aspects of the innermost surface of developing secondary walls and the tangential strain corresponded to changes in the light intensity level. Cellulose microfibrils were clearly observed when the light intensity was high (10.0 or 4.3 klx) and the volume of differentiating cells was low, while abundant amorphous material was observed when the light intensity was lowest (1.5 klx) and the cells were turgid, regardless of the light intensity cycle. These results suggest that the diurnal periodicity in the supply of cell wall components to developing secondary walls is associated with changes in light intensity during the photoperiodic cycle.     

Peroxidase activity, isoenzymes and histological localisation in sapwood and heartwood of Scots pine (Pinus sylvestris L.)

Peroxidase activity and isoenzymes of fresh wood samples of the third shoot of 12-year old trees and from the sapwood, transition zone and heartwood of c. 60-year old stems of Scots pine (Pinus sylvestris L.) were investigated. Wood samples were ground at −30°C, extracted, and the extracts concentrated c. 20-fold for peroxidase activity assays (guaiacol method) and for IEF-PAGE. At least 11 major isoenzymes could be found in the gels. Even the heartwood contained some peroxidase isoenzymes. Isoenzyme patterns of the juvenile wood did not change with the season. However, juvenile wood showed the highest peroxidase activity at the end of the growing season. Peroxidase activity decreased from the outer sapwood towards the heartwood. Thin sections of different wood zones stained for peroxidase revealed activity in ray parenchyma and resin canal epithelial cells. Intensive staining was localised in the bordered pits of vertical and ray tracheids, and in the end walls of ray parenchyma cells.     

Ray tracheids in Pinus radiata are more highly resistant to soft rot as compared to axial tracheids: relationship to lignin concentration

A visual decay assessment of Pinus radiata wood, which was part of a framing timber in a house in the North Island of New Zealand, indicated the presence of surface decay. Microscopic observations, employing confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM), provided evidence of decay by cavity-forming soft rot (SR) fungi. A comparison of ray tracheids (RT) with axial tracheids (AT) indicated that RT were considerably more resistant to SR than AT. In the heavily degraded regions of wood, where axial tracheid walls contained abundant SR cavities, the walls of RT contained only a few or no cavities. An assessment of lignin concentration in the cell walls by a combination of TEM, confocal fluorescence and UV microscopy provided evidence of greater lignin concentration in the secondary wall of RT as compared to AT, which may explain the observed greater resistance of RT to soft rot. Dedicated to Professor Dr. Dr. h.c. mult. Walter Liese on the occasion of his 80th birthday.     

Tangential pitting in black spruce tracheids

Tangential pit features were studied in a 55-year old black spruce (Picea mariana (Mill) B.S.P.) tree by means of light and electron microscopy.It was found that tangential pitting is lacking from the greatest part of the growth ring, except for the last four tangential rows of latewood tracheids and the first row of early wood tracheids. The average number of pits per tangential wall of a 3.55-mm-long tracheid is 234, 144, 28, 4 and zero, respectively, in the last 5 tangential rows of latewood tracheids, starting at the growth-ring boundary.On the average, tangential pits measure 5.4 m in diameter, possess oval to elliptical apertures, and are randomly distributed uniformly over the tangential tracheid wall. All tangential intertracheid pits are bordered and in that respect are similar to those in the radial walls. Although most of the pits contain membranes with tori, some at the growth-ring boundary lack tori and exhibit randomly oriented microfibrillar structure.     

Heterogeneity in formation of lignin

Summary The formation of lignin in the cell wall of compression wood of Pinus thunbergii was examined by selective radio-labeling of specific structural units in the lignin and visualization of the label in the different morphological regions by microautoradiography. Deposition of lignin in the tracheid cell wall of compression wood occurred in the order: p-hydroxyphenyl, guaiacyl and syringyl lignin, which is the same order as observed in normal wood. However, the period of lignification in the compression wood was quite different from those of normal and opposite woods. The p-hydroxyphenyl units were deposited mainly in the early stage of cell wall formation in compound middle lamella in normal and opposite woods, while in compression wood, they were formed in both the compound middle lamella and the secondary wall. The most intensive lignification was observed during the formation of the S₂ layer, proceeding from the outer to inner S₂ layers for a long period in compression wood. In the normal or opposite woods, in contrast, the lignification became active after formation of S₃ had begun, then proceeded uniformly in the secondary wall and ended after a short period.A part of this report was originally presented at the 1989 International Symposium on Wood and Pulping Chemistry at Raleigh, NC, U.S.A.     

Reaction wood in Pseudowintera colorata — A vessel-less dicotyledon

Summary Inclined branches of Pseudowintera colorata exhibit pronounced growth promotion to the lower (abaxial) side similar to that found in gymnosperms. The only other significant difference between the anatomy of the upper and lower regions is that the tracheids on the lower side have a larger microfibril angle. Other microscopic features normally associated with compression wood or tension wood are completely absent. The longitudinal shrinkage of samples from the upper and lower regions is shown to be related to the mean microfibril angle in a highly non-linear way, and a relatively small change in microfibril angle is associated with a large change in longitudinal shrinkage. This result is in agreement with the hypothesis that compression wood force generation arises during the lignification phase of secondary wall deposition and is critically dependent on mean microfibril angle.The author is indebted to Mr R. R. Exley of this laboratory who prepared the samples and made all the measurements in this project     

Microfibril angle non-uniformities within normal and compression wood tracheids

The pattern and extent of variation of microfibril angle (MFA) in normal and compression tracheids of softwood were investigated by using confocal laser scanning microscopy technique. All measurements support the idea that the orientation of microfibrils in single wood tracheids is not uniform. MFA of the radial wall of earlywood tracheids was highly non-uniform and had an approximately circular form of arrangement around the bordered pits (inside the border). Between the bordered pits the measured MFAs were less than the other parts of the tracheid. In the latewood tracheids MFA was less variable. The average orientation of simple pits in the crossfield region was consistent with the mean MFA of the tracheids; however some of the measurements showed a highly variable arrangement in the areas between the simple pits. In many cases the local measured MFAs of compression wood tracheids agreed with the orientation of natural helical cavities of compression wood. Comparing the measured results in different growth rings showed that MFAs in juvenile wood are generally larger than in perfect wood.     

Study of the transverse liquid flow paths in pine and spruce using scanning electron microscopy

Samples of pine (Pinus sylvestris) and spruce (Picea abies) were impregnated with a low-viscous epoxy resin using a vacuum process. The epoxy was cured in situ and the specimens sectioned. Deposits of the cured epoxy was then observed in the wood cavities using a scanning electron microscope. The investigation concentrated on tracing the transverse movements of a viscous liquid in the wood, and special attention was therefore given to the cross-field area between ray cells and longitudinal tracheids. A damage hypothesis is proposed based on the results obtained in the present investigation in combination with those from earlier studies on linseed oil-impregnated pine: In addition to the morphology of the bordered pits, viscous liquid flow in wood is dependent on damage that occurs during the impregnation procedure. For pine sapwood, liquid flow is enabled through disrupted window pit membranes, which divide the longitudinal tracheids and the ray parenchyma cells. A mechanism accounting for the reduced permeability of pine heartwood is believed to be deposits of higher-molecular-weight substances (extractives) in the ray parenchyma cells and on the cell walls. In spruce the thicker ray cells in combination with the smaller pits, which are connected to the longitudinal tracheids, reduce permeability considerably.     

Seasonal changes in lignin distribution during tracheid development in Pinus radiata D. Don

Summary Lignin distribution in developing tracheids of Pinus radiata was studied throughout the growth' season using quantitative interference microscopy. The pattern of lignification remained constant although the number of lignifying cells varied reaching a maximum in summer. Lignification of the secondary wall of latewood tracheids was incomplete at the onset of winter. Each stage of lignification was preceded by deposition of carbohydrates with lignification of the middle lamella starting after S1 formation and lignification of the secondary wall starting after S3 formation. Lignification of the middle lamella was completed before the start of lignin deposition in the secondary wall. In one of the trees examined, the secondary wall lignified concurrently with the middle lamella and this was associated with a low lignin concentration in the middle lamella of mature cells. The secondary wall reached a mature lignin concentration of 21–22% v/v except in one specimen containing severe compression wood which reached 28% v/v. The cell corner middle lamella reached a mature lignin concentration of 74–87% v/v.     

Structure and function of flexure wood in Abies fraseri

Wood produced during flexure in one-year-old leaders of Abies fraseri (Pursh) Poir. (Fraser fir) was analyzed anatomically and radio-densitometrically. More xylem cells were produced in stems subjected to flexing than in stems that were not flexed. The lumens of tracheids produced in response to flexure were smaller than the lumens of tracheids in normal wood. This was manifest as an increase in the cell wall area/cell lumen area ratio. Microfibril orientation in flexure-induced wood approached the less extreme values found in compression wood. The growth ring composed of flexure-induced wood also had a greater density than normal wood. Compression wood, as defined by cellular characteristics observed in transverse section, was absent in flexed stems. Detailed analysis of the anatomical structure, wood density and biomechanical properties of flexure-induced wood indicated that it has more in common with compression wood than with normal wood.     

Helical thickenings and helical cavities in normal and compression woods of Taxus baccata

Summary The longitudinal tracheids in compression wood of Taxus baccata contain helical thickenings but no helical cavities. The thickenings are as frequent and well developed and have the same ropelike appearance as in normal wood of this species. They are an integral part of the S₃ in normal and of the S₂ in compression wood and have the same orientation as the innermost microfibrils in these layers. Except for the absence of cavities and presence of thickenings, compression wood tracheids of Taxus baccata possess all the anatomical features typical of such cells, including a rounded outline, intercellular spaces, a thick S₁ layer, a highly lignified S₂ (L) layer, and no S₃ layer. Pronounced compression wood of Pseudotsuga menziesii contains helical cavities but no helical thickenings. Thickenings and cavities seem to be mutually exclusive in Pseudotsuga and Taxus.This investigation was carried out under the McIntire-Stennis Program, Cooperative State Research Service. I am indebted to Mr. A. Rezanowich of the Pulp and Paper Research Institute of Canada for kindly providing the scanning electron micrographs.