Normal Wood Fibers Research Articles

Evaluation of Wood Quality Traits in Salix viminalis Useful for Biofuels: Characterization and Method Development

Salix (willow) is a well-known coppice plant that has been used as a source for bioenergy for decades. With recent developments in changing from a fossil-based to a circular bioeconomy, greater interest has been orientated towards willow as a potential source of biomass for transport biofuels. This has created increasing interest for breeding strategies to produce interesting genotypic and phenotypic traits in different willow varieties. In the present study, 326 genetically distinct clones and several commercial varieties of S. viminalis were analyzed using complementary approaches including density, chemical, image, histochemical, and morphometric analyses. A systematic approach was adopted whereby the basal regions of harvested stems were separated and used in all studies to aid comparisons. Density analyses were performed on all clone individuals, and from the results, 20 individual plants representing 19 clones were selected for the more in-depth analyses (chemical, image analysis, histochemical, and morphometric). The absolute dry density of the clones selected varied between ca. 300 and 660 kg/m3 with less variation seen in the commercial S. viminalis varieties (ca. 450–520 kg/m3). Selected clones for chemical analysis showed the largest variation in glucose (47.3%–60.1%; i.e., cellulose) and total sugar content, which ranged between ca. 61 and 77% and only ca. 16 and 22% for lignin. Image analyses of entire basal stem sections showed presence of tension wood in variable amounts (ca. 7%–39%) with characteristic G-fibers containing cellulose-rich and non-lignified gelatinous layers. Several of the clones showing prominent tension wood also showed high glucose and total sugar content as well as low lignin levels. A morphometric approach using an optical fiber analyzer (OFA) for analyzing 1000 s (minimum 100,000 particles) of macerated fibers was evaluated as a convenient tool for determining the presence of tension wood in stem samples. Statistical analyses showed that for S. viminalis stems of the same density and thickness, the OFA approach could separate tension wood fibers from normal wood fibers by length but not fiber width. Results emphasized considerable variability between the clones in the physical and chemical approaches adopted, but that a common aspect for all clones was the occurrence of tension wood. Since tension wood with G-fibers and cellulose-rich G-layers represents an increased source of readily available non-recalcitrant cellulose for biofuels, S. viminalis breeding programs should be orientated towards determining factors for its enhancement.

Populus NST/SND orthologs are key regulators of secondary cell wall formation in wood fibers, phloem fibers and xylem ray parenchyma cells.

Wood fibers form thick secondary cell wall (SCW) in xylem tissues to give mechanical support to trees. NAC SECONDARY WALL THICKENING PROMOTING FACTOR3/SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 1 (NST3/SND1) and NST1 were identified as master regulators of SCW formation in xylem fiber cells in the model plant Arabidopsis thaliana. In Populus species, four NST/SND orthologs have been conserved and coordinately control SCW formation in wood fibers and phloem fibers. However, it remains to be elucidated whether SCW formation in other xylem cells, such as ray parenchyma cells and vessel elements, is regulated by NST/SND orthologs in poplar. We knocked out all NST/SND genes in hybrid aspen using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system and investigated the detailed histological appearance of stem tissues in the knockout mutants. Observation by light microscopy and transmission electron microscopy showed that SCW was severely suppressed in wood fibers, phloem fibers and xylem ray parenchyma cells in the knockout mutants. Although almost all wood fibers lacked SCW, some fiber cells formed thick cell walls. The irregularly cell wall-forming fibers retained primary wall and SCW, and were mainly located in the vicinity of vessel elements. Field emission-scanning electron microscope observation showed that there were no apparent differences in the structural features of pits such as the shape and size between irregularly SCW-forming wood fibers in the knockout mutants and normal wood fibers in wild-type. Cell wall components such as cellulose, hemicellulose and lignin were deposited in the cell wall of irregularly SCW-forming wood fibers in quadruple mutants. Our results indicate that four NST/SND orthologs are master switches for SCW formation in wood fibers, xylem ray parenchyma cells and phloem fibers in poplar, while SCW is still formed in limited wood fibers, which are located at the region adjacent to vessel elements in the knockout mutants.

Study of tension wood in the artificially inclined seedlings of Koelreuteria henryi Dummer and its biomechanical function of negative gravitropism

Stem reorientation is critical to tree survival. With anatomical observation and strain measurement, the tension wood formation and biomechanical behavior were studied to gain insights into tree uprighting process. Tension wood plays a role in maintaining the mechanical stability of angiosperm trees. Both biological and physical aspects of tension wood are essential in understanding the mechanism of trunk or branch reorientation. In this study, we worked on both tension wood formation and its biomechanical function in artificially inclined 2-year-old Koelreuteria henryi seedlings. The tension wood formation and reorientation process of the trunk last for about 3 months. With pinning method, we confirmed that at the beginning of inclination the cambial zone including the vascular cambium and the developing normal wood fibers on the upper side of the inclined trunk perceives the onset of mechanical change and starts to produce G-fibers that generate a strong contractile released growth strain (RGS) for gravitropic correction. Stronger contractile RGS and more tension wood were found at the trunk base than at the half-height, suggesting that the trunk base plays a key role in trunk uprighting of K. henryi seedlings. The eccentric cambial growth in the tension wood side increases the efficiency of gravitropic correction and the compressive strains measured in the opposite wood of some inclined seedlings also help the upright movement.

Evidence of the late lignification of the G-layer in Simarouba tension wood, to assist understanding how non-G-layer species produce tensile stress.

To recover verticality after disturbance, angiosperm trees produce 'tension wood' allowing them to bend actively. The driving force of the tension has been shown to take place in the G-layer, a specific unlignified layer of the cell wall observed in most temperate species. However, in tropical rain forests, the G-layer is often absent and the mechanism generating the forces to reorient trees remains unclear. A study was carried out on tilted seedlings, saplings and adult Simarouba amara Aubl. trees-a species known to not produce a G-layer. Microscopic observations were done on sections of normal and tension wood after staining or observed under UV light to assess the presence/absence of lignin. We showed that S. amara produces a cell-wall layer with all of the characteristics typical of G-layers, but that this G-layer can be observed only as a temporary stage of the cell-wall development because it is masked by a late lignification. Being thin and lignified, tension wood fibres cannot be distinguished from normal wood fibres in the mature wood of adult trees. These observations indicate that the mechanism generating the high tensile stress in tension wood is likely to be the same as that in species with a typical G-layer and also in species where the G-layer cannot be observed in mature cells.

Automatic measurement of compression wood cell attributes in fluorescence microscopy images

This paper presents a new automated method for analyzing compression wood fibers in fluorescence microscopy. Abnormal wood known as compression wood is present in almost every softwood tree harvested. Compression wood fibers show a different cell wall morphology and chemistry compared to normal wood fibers, and their mechanical and physical characteristics are considered detrimental for both construction wood and pulp and paper purposes. Currently there is the need for improved methodologies for characterization of lignin distribution in wood cell walls, such as from compression wood fibers, that will allow for a better understanding of fiber mechanical properties. Traditionally, analysis of fluorescence microscopy images of fiber cross-sections has been done manually, which is time consuming and subjective. Here, we present an automatic method, using digital image analysis, that detects and delineates softwood fibers in fluorescence microscopy images, dividing them into cell lumen, normal and highly lignified areas. It also quantifies the different areas, as well as measures cell wall thickness. The method is evaluated by comparing the automatic with a manual delineation. While the boundaries between the various fiber wall regions are detected using the automatic method with precision similar to inter and intra expert variability, the position of the boundary between lumen and the cell wall has a systematic shift that can be corrected. Our method allows for transverse structural characterization of compression wood fibers, which may allow for improved understanding of the micro-mechanical modeling of wood and pulp fibers.

Microfibril Angle Distribution of Poplar Tension Wood

Tension wood of poplar (Populus nigra) branches was studied by lightand electron microscopy. The characteristic features of tension wood such as wider growth rings, reduced vessel density and higher gross density were confirmed by our results. Based on a novel combination of transmission electron microscopy (TEM) imaging and image analysis, involving Fourier transformation, the orientation of cellulose microfibrils in the S2- and G-layer was determined. Within the G-layer microfibril angle (MFA) was parallel to the growth axis (0°). However, in the S2 it was 13° in tension wood fibres and 4° in normal wood fibres. With the exception of the relatively low fibril angle in the S2 of tension wood fibres (13°) the results are in good agreement with those of the literature.

Stem tangential strain on the tension wood side of Fagus crenata saplings

The tangential strain on the inner bark surface of Fagus crenata sapling stems was continuously measured using strain gauges. The total strain increased daily, increasing at night and decreasing during the day. When tension wood was induced by artificial inclination, the strain increased more on the upper side than on the lower side; and the increment in the strain at night was larger on the upper side than on the lower. The change in tangential strain on the inner bark surface arose from changes in the water content and the volume of differentiating cells. Differentiating tension wood fibers appear to contain more water and to expand more at night than differentiating normal wood fibers. We can determine whether tension wood is formed from the tangential strain during growth.

Understanding the role of the cytoskeleton in wood formation in angiosperm trees: hybrid aspen (Populus tremula x P. tremuloides) as the model species.

The involvement of microfilaments and microtubules in the development of the radial and axial components of secondary xylem (wood) in hybrid aspen (Populus tremula L. x P. tremuloides Michx.) was studied by indirect immunofluorescent localization techniques. In addition to cambial cells, the differentiated cell types considered were early- and late-wood vessel elements, axial parenchyma, normal-wood fibers and gelatinous fibers, and contact and isolation ray cells. Microfilaments were rare in ray cambial cells, but were abundant and axially arranged in their derivatives once cell elongation had begun, and persisted in that orientation in mature ray cells. Microfilaments were axially arranged in fusiform cambial cells and persisted in that orientation in all xylem derivatives of those cells. Microtubules were randomly oriented in ray and fusiform cells of the cambial zone. Dense arrays of parallel-aligned microtubules were oriented near axially in the developing gelatinous fibers, but at a wide range of angles in normal-wood fibers. Ellipses of microfilaments were associated with pit development in fiber cells and isolation ray cells. Rings of co-localized microtubules and microfilaments were associated with developing inter-vessel bordered pits and vessel-contact ray cell contact pits, and, in the case of bordered pits, these rings decreased in diameter as the over-arching pit border increased in size. Although only microtubules were seen at the periphery of the perforation plate of vessel elements, a prominent meshwork of microfilaments overlaid the perforation plate itself. A consensus view of the roles of the cytoskeleton during wood formation in angiosperm trees is presented.

A Survey of Buttresses and Aerial Roots of Tropical Trees for Presence of Reaction Wood

Wood from buttresses of 48 species in 20 families of tropical trees was examined microscopically for the presence of reaction wood (RW), indicated by gelatinous fibers in place of normal wood fibers. Only Eschlweilera (Lecythidaceae), Derris (Fabaceae), and three spp. of Ficus (Moraceae) had RW. All other species, including one specimen of Guarea (Meliaceae) in which the buttress was under tension due to a leaning trunk, had normal wood structure. Wood from aerial roots, including stilt roots and flying buttresses, of 18 species in six families was similarly examined. A cylinder of well-developed RW occurred in aerial roots of nine species of Ficus and in stilt roots of Cecropia (Moraceae). Reaction wood also occurred on the upper side of stilt roots of Cecropia and Pourouma (Urticaceae). Contraction of the aerial roots of Ficus is related to RW. However, although RW has been correlated wIth stresses in the wood of trunks and branches of many trees, RW cannot be related generally to a supportive role of buttresses in which stresses have been postulated previously. THE WIDESPREAD AND NOTICEABLE occurrence of buttressed trunks in tropical trees has long been interesting to biologists, and a variety of hypotheses have been offered to explain their cause and function (Richards 1952). One ccmmon assumption was that buttresses aided in the physical support of a tree, especially, when growing on shallow or wet soils, and Henwood (1973) demonstrated, on engineering principles, that buttresses do give added support to trees growing on poorly anchoring substrates and do reduce the stresses oin roolts caused by wind or uneven crown load as compared to trees with unbuttressed, cylindrical bases. Several publications are relevant to the supportive role of buttresses and suggest a survey of wood structure in buttresses and aerial roots. Reaction wood (RW) has been related to internal strains and stresses in trunk and branch wood (Trenard and Gueneau 1975, Scurfield 1973, Wilson and Archer 1977, 1979). Fayle (1976) found that secondary growth and RW formation were reduced in pine roots when plants were tied, thus preventing normal mechanical stresses. Mechanical stress has been suggested (but not widely accepted) as an inducer of RW by Boyd (1977). Since RW has been associated with stresses in branch, trunk, and root wood, and since buttresses should be subject to stresses in their supportive role, RW might be present and of importance in the buttresses of tropical trees. Chalk and Akpalu (1963) noted that several anatomical features were associated with species that produced buttresses, but they did not examine buttress wood for presence of RW nor compared trunk and buttress woods. To date, there has been only one careful study of buttress wood anatomy (Stahel 1971) in which some quantitative differences in percentages of cell types between trunk and buttress woods were found. In addition, Stahel ( 1971 ) looked for but found no RW in buttresses of the two species examined, Khaya (Meliaceae) and Piptadeniastrum

Distribution of lignin in normal and tension wood

The distribution of lignin in normal and tension wood of four hardwood species has been studied by examination in the electron microscope of the lignin skeletons remaining after removal of the polysaccharides with hydrofluoric acid. In normal wood fibers, the S1 had a higher lignin concentration than the S2 layer, which was not as highly lignified as in conifer tracheids. Vessels had a high concentration of lignin in both normal and tension wood, while the extent of lignification of the parenchyma was variable.

The xylem fibres of Salix fragilis, L

SYNOPSISGelatinous fibres of Salix fragilis L. are characterized by an inner layer of cellulose which is relatively unlignified when compared with normal wood fibres. The gelatinous layers of mature gelatinous fibres sometimes show a reaction with benzidine or with ruthenium red. Fibres may sometimes have a terminal lamella which borders the lumen. It is suggested that the irregular distribution of organelles within differentiating fusiform cells is responsible for the spasmodic histochemical reactions and presence of a terminal lamella.The Coppick and Fowler method may be used for the localization of lignin in ultra‐thin sections.Although starch grains have often been seen within fibres, their presence has not been clearly related to the presence of gelatinous fibres alone.From light, polarizing and electron microscopical evidence, the walls of normal wood fibres have been found to be composed of S1 + S2 + S3 layers or of S1 + S2 layers only. The gelatinous fibres invariably have a gelatinous layer which may replace either the S3 or both the S3 + S2 layers.Microfibrillar disorientations of the gelatinous layer as seen in carbon replicas appear to be caused by the presence of simple pits or incipient slip planes. It appears that the pits through the gelatinous layer may become distended during processing for light microscopy.The convoluted appearance of the gelatinous layer in some sections is now considered to be an artifact brought about by processing and sectioning of fibres.No evidence has been found that the gelatinous layer is formed other than by the gradual deposition of wall material, whether or not there may be periods of faster or slower wall synthesis.