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     

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.     

Ultra-structural organisation of cell wall polymers in normal and tension wood of aspen revealed by polarisation FTIR microspectroscopy

Polarisation Fourier transform infra-red (FTIR) microspectroscopy was used to characterize the organisation and orientation of wood polymers in normal wood and tension wood from hybrid aspen (Populus tremula × Populus tremuloides). It is shown that both xylan and lignin in normal wood are highly oriented in the fibre wall. Their orientation is parallel with the cellulose microfibrils and hence in the direction of the fibre axis. In tension wood a similar orientation of lignin was found. However, in tension wood absorption peaks normally assigned to xylan exhibited a 90° change in the orientation dependence of the vibrations as compared with normal wood. The molecular origin of these vibrations are not known, but they are abundant enough to mask the orientation dependence of the xylan signal from the S₂ layer in tension wood and could possibly come from other pentose sugars present in, or associated with, the gelatinous layer of tension wood fibres.     

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.     

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.     

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.     

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 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 and the outer part of the S2 layer. The microfibril angle in the S2 layer was found to be lower in its inner part than in its outer part, especially in tension wood. In tension wood only, this decrease occurred together with an increase in cellulose lattice spacing, and this happened before the G-layer was visible. The relative increase in lattice spacing was found close to the usual value of maturation strains, strongly suggesting that microfibrils of this layer are put into tension and contribute to the generation of maturation stress.Wood cells are produced in the cambium at the periphery of the stem. The formation of the secondary wall occurs at the end of cell elongation by the deposition of successive layers made of cellulose microfibrils bounded by an amorphous polymeric matrix. Each layer has a specific chemical composition and is characterized by a particular orientation of the microfibrils relative to the cell axis (Mellerowicz and Sundberg, 2008). Microfibrils are made of crystalline cellulose and are by far the stiffest constituent of the cell wall. The microfibril angle (MFA) in each layer is determinant for cell wall architecture and wood mechanical properties.During the formation of wood cells, a mechanical stress of a large magnitude, known as “maturation stress” or “growth stress” (Archer, 1986; Fournier et al., 1991), occurs in the cell walls. This stress fulfills essential biomechanical functions for the tree. It compensates for the comparatively low compressive strength of wood and thus improves the stem resistance against bending loads. It also provides the tree with a motor system (Moulia et al., 2006), necessary to maintain the stem at a constant angle during growth (Alméras and Fournier, 2009) or to achieve adaptive reorientations. In angiosperms, a large tensile maturation stress is generated by a specialized tissue called “tension wood.” In poplar (Populus deltoides × Populus trichocarpa), as in most temperate tree species, tension wood fibers are characterized by the presence of a specific layer, called the G-layer (Jourez et al., 2001; Fang et al., 2008), where the matrix is almost devoid of lignin (Pilate et al., 2004) and the microfibrils are oriented parallel to the fiber axis (Fujita et al., 1974). This type of reaction cell is common in plant organs whose function involves the bending or contraction of axes, such as tendrils, twining vines (Bowling and Vaughn, 2009), or roots (Fisher, 2008).The mechanism at the origin of tensile maturation stress has been the subject of a lot of controversy and is still not fully understood. However, several recent publications have greatly improved our knowledge about the ultrastructure, chemical composition, molecular activity, mechanical state, and behavior of tension wood. Different models have been proposed and discussed to explain the origin of maturation stress (Boyd, 1972; Bamber, 1987, 2001; Okuyama et al., 1994, 1995; Yamamoto, 1998, 2004; Alméras et al., 2005, 2006; Bowling and Vaughn, 2008; Goswami et al., 2008; Mellerowicz et al., 2008). The specific organization of the G-layer suggests a tensile force induced in the microfibrils during the maturation process. Different hypotheses have been proposed to explain this mechanism, such as the contraction of amorphous zones within the cellulose microfibrils (Yamamoto, 2004), the action of xyloglucans during the formation of microfibril aggregates (Nishikubo et al., 2007; Mellerowicz et al., 2008), and the effect of changes in moisture content stimulated by pectin-like substances (Bowling and Vaughn, 2008). A recent work (Goswami et al., 2008) argued an alternative model, initially proposed by Münch (1938), which proposed that the maturation stress originates in the swelling of the G-layer during cell maturation and is transmitted to the adjacent secondary layers, where the larger MFAs allow an efficient conversion of lateral stress into axial tensile stress. Although the proposed mechanism is not consistent with the known hygroscopic behavior of tension wood, which shrinks when it dries and not when it takes up water (Clair and Thibaut, 2001; Fang et al., 2007; Clair et al., 2008), this hypothesis focused attention on the possible role of cell wall layers other than the G-layer. As a matter of fact, many types of wood fibers lacking a G-layer are known to produce axial tensile stress, such as normal wood of angiosperms and conifers (Archer, 1986) and the tension wood of many tropical species (Onaka, 1949; Clair et al., 2006b; Ruelle et al., 2007), so that mechanisms strictly based on an action of the G-layer cannot provide a general explanation for the origin of tensile maturation stress in wood.In order to further understanding, direct observations of the mechanical state of the different cell wall layers and their evolution during the formation of the tension wood fibers are needed. X-ray diffraction can be used to investigate the orientation of microfibrils (Cave, 1966, 1997a, 1997b; Peura et al., 2007, 2008a, 2008b) and the lattice spacing of crystalline cellulose. The axial lattice spacing d₀₀₄ is the distance between successive monomers along a cellulose microfibril and reflects its state of mechanical stress (Clair et al., 2006a; Peura et al., 2007). If cellulose microfibrils indeed support a tensile stress, they should be found in an extended state of deformation. Under this assumption, the progressive development of maturation stress during the cell wall formation should be accompanied by an increase in cellulose lattice spacing. Synchrotron radiation allows a reduction in the size of the x-ray beam to some micrometers while retaining a strong signal, whereby diffraction analysis can be performed at a very local scale (Riekel, 2000). This technique has been used to study sequences of wood cell development (Hori et al., 2000; Müller et al., 2002). In this study, we report an experiment where a microbeam was used to analyze the structural changes of cellulose in the cell wall layers of tension wood and normal wood fibers along the sequence of xylem cell differentiation extending from the cambium to mature wood (Fig. 1). The experiment was designed to make this measurement in planta, in order to minimize sources of mechanical disturbance and be as close as possible to the native mechanical state (Clair et al., 2006a). The 200 and 004 diffraction patterns of cellulose were analyzed to investigate the process of maturation stress generation in tension wood.Open in a separate windowFigure 1.Schematic of the experimental setup, showing the x-ray beam passing perpendicular to the longitudinal-radial plane of wood and the contribution of the 004 and 200 crystal planes to the diffraction pattern recorded by the camera. [See online article for color version of this figure.]     

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.     

Out-of-plane orientation of cellulose elementary fibrils on spruce tracheid wall based on imaging with high-resolution transmission electron microscopy

Main conclusion

A 3D model of the tracheid wall has been proposed based on high-resolution cryo-TEM where, in contrast to the current understanding, the cellulose elementary fibrils protrude from the cell wall plane. The ultrastructure of the tracheid walls of Picea abies was examined through imaging of ultrathin radial, tangential and transverse sections of wood by transmission electron microscopy and with digital image processing. It was found that the elementary fibrils (EFs) of cellulose were rarely deposited in the plane of the concentric cell wall layers, in contrast to the current understanding. In addition to the adopted concept of longitudinal fibril angle, EFs protruded from the cell wall plane in varying angles depending on the layer. Moreover, the out-of-plane fibril angle varied between radial and tangential walls. In the tangential S₂ layers, EFs were always out-of-plane whereas planar orientation was typical for the S₂ layer in radial walls. The pattern of protruding EFs was evident in almost all axial and transverse images of the S₁ layer. Similar out-of-plane orientation was found in the transverse sections of the S₃ layer. A new model of the tracheid wall with EF orientation is presented as a summary of this study. The outcome of this study will enhance our understanding of the elementary fibril orientation in the tracheid 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.     

Occurrence of xylan and mannan polysaccharides and their spatial relationship with other cell wall components in differentiating compression wood tracheids of <Emphasis Type="Italic">Cryptomeria japonica</Emphasis>

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.     

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.     

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.     

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.     

Cloning and expression analysis of a wood-associated xylosidase gene (PtaBXL1) in poplar tension wood

In stems of woody angiosperms responding to mechanical stress, imposed for instance by tilting the stem or formation of a branch, tension wood (TW) forms above the affected part, while anatomically distinct opposite wood (OW) forms below it. In poplar TW the S3 layer of the secondary walls is substituted by a “gelatinous layer” that is almost entirely composed of cellulose and has much lower hemicellulose contents than unstressed wood. However, changes in xylan contents (the predominant hemicelluloses), their interactions with other wall components and the mechanisms involved in TW formation have been little studied. Therefore, in the study reported here we determined the structure and distribution of xylans, cloned the genes encoding the xylan remodeling enzymes β-xylosidases (PtaBXLi), and examined their expression patterns during tension wood, normal wood and opposite wood xylogenesis in poplar. We confirm that poplar wood xylans are substituted solely by 4-O-methylglucuronic acid in both TW and OW. However, although glucuronoxylans are strongly represented in both primary and secondary layers of OW, no 4-O-methylGlcA xylan was found in G-layers of TW. Four full-length BXL cDNAs encoding putative β-xylosidases were cloned. One, PtaBXL1, for which xylosidase activity was confirmed by heterologous expression in Escherichia coli, exhibited a wood-specific expression pattern in TW. In conclusion, xylan as PtaBXL1, encoding β4-xylosidase activity, are down-regulated in TW.     

Role of GA3, GA4 and Uniconazole-P in Controlling Gravitropism and Tension Wood Formation in Fraxinus mandshurica Rupr. var. japonica Maxim. Seedlings

GA3 and GA4 (gibberellins) play an important role in controlling gravitropism and tension wood formation in woody angiosperms. In order to improve our understanding of the role of GA3 and GA4 on xylem cell formation and the G-layer, we studied the effect of GA3 and GA4 and uniconazole-P, which is an inhibitor of GA biosynthesis, on tension wood formation by gravity in Fraxinus mandshurica Rupr. var. japonica Maxim. seedlings. Forty seedlings were divided into two groups;one group was placed upright and the other tilted. Each group was further divided into four sub-groups subjected to the following treatments: 3.43 × 10-9 μmol acetone as control, 5.78 × 10-8 μmol gibberellic acid (GA3), 6.21 × 10-8 μmol GA4, and 6.86 × 10-8 μmol uniconazole-P. During the experimental period, GAs-treated seedlings exhibited negative gravitropism,whereas application of uniconazole-P inhibited negative gravitropic stem bending. GA3 and GA4 promoted wood fibers that possessed a gelatinous layer on the upper side, whereas uniconazole-P inhibited wood formation but did not inhibit the differentiation of the gelatinous layer in wood fibers on the upper side. These results suggest that: (i) both the formation of gelatinous fibers and the quantity of xylem production are important for the negative gravitropism in horizontally-positioned seedlings; (ii) GA3 and GA4 affect wood production more than differentiation of the gelatinous layer in wood fibers;G-layer development may be regulated by other hormones via the indirect-role of GA3 and GA4 in horizontally-positioned F. mandshurica seedlings rather than the direct effect of GAs; and (iii) the mechanism for upward wood stem bending is different to the newly developed shoot bending in reaction to gravity in this species.     

Distribution of tension wood like gelatinous fibres in the roots of Acacia nilotica (Lam.) Willd

Key message

The present study unravels the anatomical characteristics and distribution patterns of cell wall polymers in the G-fibres found in the roots of A. nilotica using different microscopy techniques (light, electron and immunofluorescence microscopy).

Abstract

The present study was aimed to investigate the anatomy of reaction xylem in the positively gravitropic roots of Acacia nilotica growing in compact and waterlogged soils. The roots collected from the two different sites showed occurrence of gelatinous fibres throughout xylem radii from a distance of 4 cm from the soil surface. The thickness of gelatinous layer (G-layer) increased in the root collected from the deeper soil. Further, the ultrastructural studies revealed a complete replacement of S₂ and S₃ layers in G-fibres nearer to root tip region as compared to the root portion close to upper part of the soil surface. In addition, these fibres demonstrated intense lignification in compound middle lamellae region of G-fibre walls. Moreover, the vessel density and their width increased considerably near the root tip region. The immunofluorescence analysis suggested that the β-1,4-galactans were prevalent in G-layer, whereas the xylan was restricted to only regions of lignified secondary wall. The similarities in distribution pattern and anatomical features of G-fibres in waterlogged and non-waterlogged roots suggest the occurrence of G-fibres as inherent characteristics in the roots of Acacia nilotica.     

Gene expression in Eucalyptus branch wood with marked variation in cellulose microfibril orientation and lacking G-layers

In response to gravitational stresses, angiosperm trees form tension wood in the upper sides of branches and leaning stems in which cellulose content is higher, microfibrils are typically aligned closely with the fibre axis and the fibres often have a thick inner gelatinous cell wall layer (G-layer). Gene expression was studied in Eucalyptus nitens branches oriented at 45 degrees using microarrays containing 4900 xylem cDNAs, and wood fibre characteristics revealed by X-ray diffraction, chemical and histochemical methods. Xylem fibres in tension wood (upper branch) had a low microfibril angle, contained few fibres with G-layers and had higher cellulose and decreased Klason lignin compared with lower branch wood. Expression of two closely related fasciclin-like arabinogalactan proteins and a beta-tubulin was inversely correlated with microfibril angle in upper and lower xylem from branches. Structural and chemical modifications throughout the secondary cell walls of fibres sufficient to resist tension forces in branches can occur in the absence of G-layer enriched fibres and some important genes involved in responses to gravitational stress in eucalypt xylem are identified.