Stress generation in the tension wood of poplar is based on the lateral swelling power of the G-layer

The mechanism of active stress generation in tension wood is still not fully understood. To characterize the functional interdependency between the G-layer and the secondary cell wall, nanostructural characterization and mechanical tests were performed on native tension wood tissues of poplar (Populus nigra x Populus deltoids) and on tissues in which the G-layer was removed by an enzymatic treatment. In addition to the well-known axial orientation of the cellulose fibrils in the G-layer, it was shown that the microfibril angle of the S2-layer was very large (about 36 degrees). The removal of the G-layer resulted in an axial extension and a tangential contraction of the tissues. The tensile stress-strain curves of native tension wood slices showed a jagged appearance after yield that could not be seen in the enzyme-treated samples. The behaviour of the native tissue was modelled by assuming that cells deform elastically up to a critical strain at which the G-layer slips, causing a drop in stress. The results suggest that tensile stresses in poplar are generated in the living plant by a lateral swelling of the G-layer which forces the surrounding secondary cell wall to contract in the axial direction.     

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 m 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.Abbreviations FE-SEM field emission scanning electron microscopy - G gelatinous layer - MF cellulose microfibril - MT cortical microtubule - S₁ outermost layer of the secondary wall - TEM transmission electron microscopy We thank Dr. Y. Akibayashi, Mr. Y. Sano and Mr. T. Itoh of the Faculty of Agriculture, Hokkaido University, for their experimental or technical assistance.     

GROWTH ECCENTRICITY AND REACTION ANATOMY IN BRANCHWOOD OF PSEUDOWINTERA COLORATA

The present work deals with growth eccentricity and reaction anatomy in horizontal branches of Pseudowintera colorata (Raoul) Dandy. Growth promotion is distinct and occurs on the lower (abaxial) side of inclined branches in the manner common to gymnosperms. Neither compression wood nor tension wood was found either on the upper or on the lower side of the investigated branches. The cells of the wood to the lower side show some differences from those of the upper side. The lower-side tracheids are longer, wider, possess thicker walls and have a higher microfibril angle than those of the upper side. The lower-side rays are less high but wider (because of a greater number of larger cells) than those of the upper side.     

Maturation stress generation in poplar tension wood studied by synchrotron radiation microdiffraction

Tension wood is widespread in the organs of woody plants. During its formation, it generates a large tensile mechanical stress called maturation stress. Maturation stress performs essential biomechanical functions such as optimizing the mechanical resistance of the stem, performing adaptive movements, and ensuring the long-term stability of growing plants. Although various hypotheses have recently been proposed, the mechanism generating maturation stress is not yet fully understood. In order to discriminate between these hypotheses, we investigated structural changes in cellulose microfibrils along sequences of xylem cell differentiation in tension and normal wood of poplar (Populus deltoides × Populus trichocarpa 'I45-51'). Synchrotron radiation microdiffraction was used to measure the evolution of the angle and lattice spacing of crystalline cellulose associated with the deposition of successive cell wall layers. Profiles of normal and tension wood were very similar in early development stages corresponding to the formation of the S1 layer and the outer part of the S2 layer. Subsequent layers were found with a lower microfibril angle (MFA), corresponding to the inner part of the S2 layer of normal wood (MFA approximately 10°) and the G layer of tension wood (MFA approximately 0°). In tension wood only, this steep decrease in MFA occurred together with an increase in cellulose lattice spacing. The relative increase in lattice spacing was found close to the usual value of maturation strains. Analysis showed that this increase in lattice spacing is at least partly due to mechanical stress induced in cellulose microfibrils soon after their deposition, suggesting that the G layer directly generates and supports the tensile maturation stress in poplar tension wood.     

Ultrastructural Investigation of Tension Wood Fibre in Fraxinus mandshurica Rupr. var. japonica Maxim.

The ultrastructure of the fibre wall in Fraxinus mandshuricaRupr. var. japonica Maxim. was investigated by electron microscopy.The trees had been inclined artificially at an angle of 30°to the vertical at the beginning of the initiation of cambialgrowth in early spring. The secondary walls of tension woodfibres were of the outer (S₁) layer and gelatinous (G) layertype. The microfibrils in the gelatinous (G) layer were orientedas a steep Z-helix relative to the fibre axis with a deviationthat ranged from 0° to 25° but was mainly between 5°and 10°. The cross-sectional surface of tension wood fibresrevealed the relatively strong attachment of the G-layer tothe S₁ layer. The G-layer stained weakly with potassium permanganate.The S₁ layer of tension wood fibres stained less strongly thanthat of the normal and opposite wood fibres. These results indicatethat the tension wood in F. mandshurica var. japonica is nottypical and is somewhat anomalous. The secondary walls of normaland opposite wood fibres were composed of two layers, S₁ andS₂, and lacked an S₃ layer. Microfibrils in the S₃ layer ofjuvenile stems were extremely variable in orientation and weresparsely distributed without forming a layer. By contrast, avery thin S₃ layer was present in the wood fibres of maturestems. The variations in the formation of the S₃ layer in thefibre walls were probably due to the differences in the cambialage of the stems of F. mandshurica Rupr. var. japonica.Copyright1995, 1999 Academic Press Fraxinus mandshurica Rupr. var. japonica Maxim., Japanese ash, tension wood, fibre wall, G-layer, microfibrillar orientation, normal and opposite wood, juvenile stem, field-emission scanning electron microscopy, low accelerating voltage     

Wood properties and chemical composition of the eccentric growth branch of Viburnum odoratissimum var. awabuki

To clarify the wood properties and chemical composition of branches of Viburnum odoratissimum produced by unusual eccentric growth, we investigated growth strain (GS), basic density (D _b), microfibril angle (MFA), elastic moduli (E _L and E _L/D _b), creep deformation, cellulose crystalline features, and lignin structure in upper and lower sides of the branches, and considered the correlations among these factors. In most measuring positions, the distribution of GS showed that higher tensile GS was in the upper side and compressive GS was in the lower side of the branch, which combines GS features of reaction wood. However, the generation of GS in the lower side was different from that in compression wood, because E _L/D _b and MFA had a negative correlation. The creep compliance curves show that the upper-side wood had low rigidity and high viscosity, whereas the lower-side wood had large rigidity and low viscosity. Relative creep had a negative relation with MFA in the upper side, which is unusual. The cellulose crystalline features showed no obvious difference between both sides of the branch; however, the lignin with less β-O-4 proportion and less S units but more G units seemed to exist in the lower side because of a decreased syringyl/guaiacyl (S/G) molar ratio. This suggests that cell wall could be reinforced by lignin resulting in lower viscosity in the lower side of the branch. Additionally, the S/G ratio showed a relatively high correlation with GS in the lower side. These results suggest that lignin structure plays an important role in adapting to environmental changes during eccentric growth for V. odoratissimum.     

Xylem-specific and tension stress-responsive coexpression of KORRIGAN endoglucanase and three secondary wall-associated cellulose synthase genes in aspen trees

In nature, angiosperm trees develop tension wood on the upper side of their leaning trunks and drooping branches. Development of tension wood is one of the straightening mechanisms by which trees counteract leaning or bending of stem and resume upward growth. Tension wood is characterized by the development of a highly crystalline cellulose-enriched gelatinous layer next to the lumen of the tension wood fibers. Thus experimental induction of tension wood provides a system to understand the process of cellulose biosynthesis in trees. Since KORRIGAN endoglucanases (KOR) appear to play an important role in cellulose biosynthesis in Arabidopsis, we cloned PtrKOR, a full-length KOR cDNA from aspen xylem. Using RT-PCR, in situ hybridization, and tissue-print assays, we show that PtrKOR gene expression is significantly elevated on the upper side of the bent aspen stem in response to tension stress while KOR expression is significantly suppressed on the opposite side experiencing compression stress. Moreover, three previously reported aspen cellulose synthase genes, namely, PtrCesA1, PtrCesA2, and PtrCesA3 that are closely associated with secondary cell wall development in the xylem cells exhibited similar tension stress-responsive behavior. Our results suggest that coexpression of these four proteins is important for the biosynthesis of highly crystalline cellulose typically present in tension wood fibers. Their simultaneous genetic manipulation may lead to industrially relevant improvement of cellulose in transgenic crops and trees.Suchita Bhandari and Takeshi Fujino contributed equally to this research.     

A Comparison of Two Techniques for Wood Fibre Isolation - Evaluation by Tensile Tests on Single Fibres with Different Microfibril Angle

Abstract: Fibres (tracheids) of spruce ( Picea abies [L.] Karst.) were isolated by means of two isolation techniques. On the one hand, a soft chemical treatment with Jeffrey solution was used. The isolation was carried out with a reduced time of treatment (2 h), just to achieve the state where the fibres could be separated easily with tweezers. On the other hand, fibres were directly peeled out with tweezers, taking advantage of the low shear strength between them. Following this approach, a chemical treatment could be avoided completely. In order to compare the isolation techniques, single tracheids from tissue types with different microfibril angle (earlywood, latewood, juvenile wood, opposite wood, compression wood) were tested in the dry state. The microfibril angles were determined by X-ray diffraction. Single tracheids handled with both isolation techniques were strained in microtensile tests and load-strain diagrams were obtained. The chemically treated fibres were found to have much lower strength and stiffness compared to the mechanically isolated fibres, even though the influence of microfibril angle was still obvious for both kinds of treatment. The results clearly show the importance of single fibre extraction to preserve as much as possible the original properties.     

Deposition and organisation of cell wall polymers during maturation of poplar tension wood by FTIR microspectroscopy

To advance our understanding of the formation of tension wood, we investigated the macromolecular arrangement in cell walls by Fourier transform infrared microspectroscopy (FTIR) during maturation of tension wood in poplar (Populus tremula x P. alba, clone INRA 717-1B4). The relation between changes in composition and the deposition of the G-layer in tension wood was analysed. Polarised FTIR measurements indicated that in tension wood, already before G-layer formation, a more ordered structure of carbohydrates at an angle more parallel to the fibre axis exists. This was clearly different from the behaviour of opposite wood. With the formation of the S₂ layer in opposite wood and the G-layer in tension wood, the orientation signals from the amorphous carbohydrates like hemicelluloses and pectins were different between opposite wood and tension wood. For tension wood, the orientation for these bands remains the same all along the cell wall maturation process, probably reflecting a continued deposition of xyloglucan or xylan, with an orientation different to that in the S₂ wall throughout the whole process. In tension wood, the lignin was more highly oriented in the S₂ layer than in opposite wood.     

Lignification and tension wood

Hardwood trees are able to reorient their axes owing to tension wood differentiation. Tension wood is characterised by important ultrastructural modifications, such as the occurrence in a number of species, of an extra secondary wall layer, named gelatinous layer or G-layer, mainly constituted of cellulose microfibrils oriented nearly parallel to the fibre axis. This G-layer appears directly involved in the definition of tension wood mechanical properties. This review gathers the data available in the literature about lignification during tension wood formation. Potential roles for lignin in tension wood formation are inferred from biochemical, anatomical and mechanical studies, from the hypotheses proposed to describe tension wood function and from data coming from new research areas such as functional genomics.     

Variation of cellulose microfibril angles in softwoods and hardwoods-a possible strategy of mechanical optimization

Position-resolved small-angle X-ray scattering was used to investigate the nanostructure of the wood cell wall in two softwood species (Norwegian spruce and Scots pine) and two hardwood species (pedunculate oak and copper beech). The tilt angle of the cellulose fibrils in the wood cell wall versus the longitudinal cell axis (microfibril angle) was systematically studied over a wide range of annual rings in each tree. The measured angles were correlated with the distance from the pith and the results were compared. The microfibril angle was found to decrease from pith to bark in all four trees, but was generally higher in the softwood than in the hardwood. In Norwegian spruce, the microfibril angles were higher in late wood than in early wood; in Scots pine the opposite was observed. In pedunculate oak and copper beech, low angles were found in the major part of the stem, except for the very first annual rings in pedunculate oak. The results are interpreted in terms of mechanical optimization. An attempt was made to give a quantitative estimation for the mechanical constraints imposed on a tree of given dimensions and to establish a model that could explain the general decrease of microfibril angles from pith to bark.     

Cellulose microfibril angle in the cell wall of wood fibres

The term microfibril angle (MFA) in wood science refers to the angle between the direction of the helical windings of cellulose microfibrils in the secondary cell wall of fibres and tracheids and the long axis of cell. Technologically, it is usually applied to the orientation of cellulose microfibrils in the S2 layer that makes up the greatest proportion of the wall thickness, since it is this which most affects the physical properties of wood. This review describes the organisation of the cellulose component of the secondary wall of fibres and tracheids and the various methods that have been used for the measurement of MFA. It considers the variation of MFA within the tree and the biological reason for the large differences found between juvenile (or core) wood and mature (or outer) wood. The ability of the tree to vary MFA in response to environmental stress, particularly in reaction wood, is also described. Differences in MFA have a profound effect on the properties of wood, in particular its stiffness. The large MFA in juvenile wood confers low stiffness and gives the sapling the flexibility it needs to survive high winds without breaking. It also means, however, that timber containing a high proportion of juvenile wood is unsuitable for use as high-grade structural timber. This fact has taken on increasing importance in view of the trend in forestry towards short rotation cropping of fast grown species. These trees at harvest may contain 50% or more of timber with low stiffness and therefore, low economic value. Although they are presently grown mainly for pulp, pressure for increased timber production means that ways will be sought to improve the quality of their timber by reducing juvenile wood MFA. The mechanism by which the orientation of microfibril deposition is controlled is still a matter of debate. However, the application of molecular techniques is likely to enable modification of this process. The extent to which these techniques should be used to improve timber quality by reducing MFA in juvenile wood is, however, uncertain, since care must be taken to avoid compromising the safety of the tree.     

Short-term effects of nitrogen availability on wood formation and fibre properties in hybrid poplar

The application of nitrogen-containing fertilisers is one approach used to increase growth rates and productivity of forest tree plantations. However, the effects of nitrogen fertilisation on wood properties have not been systematically assessed. The aim of this work was to document the impacts of nitrogen fertilisation on wood formation and secondary xylem fibre properties. We used three fertilisation treatments in which the level of ammonium nitrate was adjusted to 0, 1 and 10 mM in a complete nutrient solution applied daily over a period of 28 days in standardised greenhouse experiments with clonal material of Populus trichocarpa (Torr and Gray) × deltoides (Bartr. ex Marsh). We showed that there was a short-term and repeatable response in which xylem fibre morphology and secondary cell wall structure adapt to a shift in N availability. Under high-nitrogen exposure, xylem fibres were 17% wider and 18% shorter compared to the adequate nitrogen treatment. A very significant thickening of the fibre cell walls was also observed throughout the stem of trees receiving the high-N treatment. It appeared that cell wall structure was greatly affected by the high-N treatment as fibres developed a modified inner cell wall layer. Histological observations indicated that the internal cell wall layer was enriched in cellulose and chemical determinations showed that wood contained more holocellulose. Together, these results indicate that the response of poplar to nitrogen availability may involve marked effects on secondary xylem formation.     

Prior secondary cell wall formation is required for gelatinous layer deposition and posture control in gravi-stimulated aspen

Cell walls, especially secondary cell walls (SCWs), maintain cell shape and reinforce wood, but their structure and shape can be altered in response to gravity. In hardwood trees, tension wood is formed along the upper side of a bending stem and contains wood fiber cells that have a gelatinous layer (G-layer) inside the SCW. In a previous study, we generated nst/snd quadruple-knockout aspens (Populus tremula × Populus tremuloides), in which SCW formation was impaired in 99% of the wood fiber cells. In the present study, we produced nst/snd triple-knockout aspens, in which a large number of wood fibers had thinner SCWs than the wild type (WT) and some had no SCW. Because SCW layers are always formed prior to G-layer deposition, the nst/snd mutants raise interesting questions of whether the mutants can form G-layers without SCW and whether they can control their postures in response to changes in gravitational direction. The nst/snd mutants and the WT plants showed growth eccentricity and vessel frequency reduction when grown on an incline, but the triple mutants recovered their upright growth only slightly, and the quadruple mutants were unable to maintain their postures. The mutants clearly showed that the G-layers were formed in SCW-containing wood fibers but not in those lacking the SCW. Our results indicate that SCWs are essential for G-layer formation and posture control. Furthermore, each wood fiber cell may be able to recognize its cell wall developmental stage to initiate the formation of the G-layer as a response to gravistimulation.     

Immunocytochemical characterization of tension wood: Gelatinous fibers contain more than just cellulose

Gelatinous fibers (G-fibers) are the active component of tension wood. G-fibers are unlike traditional fiber cells in that they possess a thick, nonlignified gelatinous layer (G-layer) internal to the normal secondary cell wall layers. For the past several decades, the G-layer has generally been presumed to be composed nearly entirely of crystalline cellulose, although several reports have appeared that disagreed with this hypothesis. In this report, immunocytochemical techniques were used to investigate the polysaccharide composition of G-fibers in sweetgum (Liquidambar styraciflua; Hamamelidaceae) and hackberry (Celtis occidentalis; Ulmaceae) tension wood. Surprisingly, a number of antibodies that recognize arabinogalactan proteins and RG I-type pectin molecules bound to the G-layer. Because AGPs and pectic mucilages are found in other plant tissues where swelling reactions occur, we propose that these polymers may be the source of the contractile forces that act on the cellulose microfibrils to provide the tension force necessary to bend the tree trunk.