In situ FT-IR microscopic study on enzymatic treatment of poplar wood cross-sections

The feasibility of Fourier transform infrared (FT-IR) microscopy to monitor in situ the enzymatic degradation of wood was investigated. Cross-sections of poplar wood were treated with cellulase Onozuka RS within a custom-built fluidic cell. Light-optical micrographs and FT-IR spectra were acquired in situ from normal and tension wood fibers. Light-optical micrographs showed almost complete removal of the gelatinous (G) layer in tension wood. No structural and spectral changes were observed in the lignified cell walls. The accessibility of cellulose within the lignified cell wall was found to be the main limiting factor, whereas the depletion of the enzyme due to lignin adsorption could be ruled out. The fast, selective hydrolysis of the crystalline cellulose in the G-layer, even at room temperature, might be explained by the gel-like structure and the highly porous surface. Young plantation grown hardwood trees with a high proportion of G-fibers thus represent an interesting resource for bioconversion to fermentable sugars in the process to bioethanol.     

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.     

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.     

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.     

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.     

Distribution of glucomannans and xylans in poplar xylem and their changes under tension stress

Present work investigated glucomannan (GM) and xylan distribution in poplar xylem cells of normal- (NW), opposite- (OW) and tension wood (TW) with immunolocalization methods. GM labeling was mostly detected in the middle- and inner S(2) (+S(3)) layer of NW and OW fibers, while xylan labeling was observed in the whole secondary cell wall. GM labeling in vessels of NW and OW was much weaker than in fibers and mostly detected in the S(2) layer, whereas slightly stronger xylan labeling than fibers was detected in the whole secondary cell wall of vessels. Ray cells in NW and OW showed no GM labeling, but strong xylan labeling. These results indicate that GMs and xylans are spatially distributed in poplar xylem cells with different concentrations present in different cell types. Surprisingly, TW showed significant decrease of GM labeling in the normal secondary cell wall of gelatinous (G) fibers compared to NW and OW, while xylan labeling was almost identical indicating that the GM and xylan synthetic pathways in fibers have different reaction mechanisms against tension stress. Unlike fibers, no notable changes in GM labeling were detected in vessels of TW, suggesting that GM synthesis in vessels may not be affected by tension stress. GM and xylan was also detected in the G-layer with slightly stronger and much weaker labeling than the normal secondary cell wall of G-fibers. Differences in GM and xylan distribution are also discussed for the same functional cells found in hardwoods and softwoods.     

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.     

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.     

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     

Persistence of the gelatinous layer within altered tension-wood fibres of beech degraded by Ustulina deusta

The gelatinous layer (G-layer) of tension-wood fibres in reaction wood of beech showed alterations as a result of the physiological processes involved in the conversion of sapwood into false heartwood or reaction-zone tissue. Using transmitted-light, fluorescence and UV microscopy, polyphenolic compounds were found to infiltrate and encrust the cellulose microfibrils within the G-layer. Experiments with naturally infected and artificially inoculated wood showed that these processes affect the rate and mode of degradation by wood-decaying fungi. Thus, although the ascomycete Ustulina deusta was able to degrade the G-layer from within the lumina of tension-wood fibres in unaltered sapwood, it failed to do so for a prolonged period within false heartwood and reaction zones. In both situations, however, there was some degradation of the underlying secondary wall in the form of erosion troughs which can be attributed to soft rot 'type II', and internal cavity formation typical for 'type I' attack. The present study indicates that not only cell type, but also alterations in the cell wall structure, affect the activity and degradation mode of decay fungi in beech.     

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.]     

Analysis of the surfaces of wood tissues and pulp fibers using carbohydrate-binding modules specific for crystalline cellulose and mannan

Carbohydrate binding modules (CBMs) are noncatalytic substrate binding domains of many enzymes involved in carbohydrate metabolism. Here we used fluorescent labeled recombinant CBMs specific for crystalline cellulose (CBM1(HjCel7A)) and mannans (CBM27(TmMan5) and CBM35(CjMan5C)) to analyze the complex surfaces of wood tissues and pulp fibers. The crystalline cellulose CBM1(HjCel7A) was found as a reliable marker of both bacterially produced and plant G-layer cellulose, and labeling of spruce pulp fibers with CBM1(HjCel7A) revealed a signal that increased with degree of fiber damage. The mannan-specific CBM27(TmMan5) and CBM35(CjMan5C) CBMs were found to be more specific reagents than a monoclonal antibody specific for (1-->4)-beta-mannan/galacto-(1-->4)-beta-mannan for mapping carbohydrates on native substrates. We have developed a quantitative fluorometric method for analysis of crystalline cellulose accumulation on fiber surfaces and shown a quantitative difference in crystalline cellulose binding sites in differently processed pulp fibers. Our results indicated that CBMs provide useful, novel tools for monitoring changes in carbohydrate content of nonuniform substrate surfaces, for example, during wood or pulping processes and possibly fiber biosynthesis.     

Study of tension wood in the artificially inclined seedlings of <Emphasis Type="Italic">Koelreuteria henryi</Emphasis> Dummer and its biomechanical function of negative gravitropism

Key message

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.

Abstract

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.

     

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.     

Anatomy of axis contraction in seedlings from a fire prone habitat

The hypocotyls or roots of many seed plants contract during seedling growth. Anatomical evidence is here reported for the first time that G-fibers (gelatinous or tension wood fibers) may cause contraction of roots and hypocotyls in dicotyledonous seedlings long after germination. To document repositioning of seedling buds, selected perennials (20 dicotyledons and one cycad) native to the fire-prone pine rocklands of subtropical South Florida were germinated and measured for 4-5 mo. The height of cotyledonary nodes above the soil decreased because of axis contraction or bending in eight species. Anatomy suggested that two mechanisms operate: (1) previously well-documented collapse of parenchyma cells in two species (Convolvulaceae and Zamiaceae) and (2) newly documented production of G-fibers in six species (all Fabaceae). Contraction or bending of the hypocotyl and/or taproot moved the cotyledonary and later buds of the seedling closer to the soil surface or buried them. Bud repositioning by these mechanisms may protect the lateral buds from injury by fire or other environmental stresses and allow resprouting.     

Control of Negative Gravitropism and Tension Wood Formation by Gibberellic Acid and Indole Acetic Acid in Fraxinus mandshurica Rupr. var. japonica Maxim Seedlings

In the present study, we investigated the role of gibberellic acid (GA3) and indole acetic acid (IAA) in the gravity response of stems and tension wood formation using two-year-old stems of Fraxinus mandshurica Rupr. var.japonica Maxim seedlings. Forty-five seedlings were used and divided into nine groups that included five seedlings in each group. Seedlings were treated with applications of GA3 alone at concentrations of 2.89 × 10-8and 2.89 × 10-7 μmol/L, IAA alone at concentrations of 5.71×10-8 and 5.71 ×10-7 μmol/L, or their combination to the apical bud of the stem using a micropipette. Seedlings were positioned horizontally after the first treatment.The same treatments were repeated six times per week. At the end of the experiment, all seedlings were harvested. Then, stem segments were cut under a light microscope. Application of exogenous GA3 at the higher concentration stimulated the upward bending of stems, whereas exogenous IAA had no effect. A synergistic effect of GA3 and IAA on upward stem bending was observed following application of the two combinations of GA3 and IAA. Moreover, application of exogenous GA3 at the higher dose stimulated wood formation on both the upper and lower sides of the stems, whereas the mixture of GA3 and IAA had a synergistic effect on wood formation in horizontal stems. Application of exogenous IAA alone at the lower concentration (5.71×10-8 μ mol/L) or application of a mixture of the higher concentrations of GA3 (2.89 × 10-7 μmol/L) and IAA (5.71×10-7 μmol/L) inhibited the development of gelatinous fibers (the G-layer) of tension wood on the upper side of the horizontal stems. The differentiation of gelatinous fibers of tension wood was not inhibited by GA3when it was applied alone, whereas the development of the gelatinous fibers of tension wood was strongly affected by the application of IAA. The findings of the present study suggest that the development of the G-layer is not related to the dose of GA3, but needs a relatively lower concentration of IAA.     

Cellulose and lignin biosynthesis is altered by ozone in wood of hybrid poplar (Populus tremula × alba)

Wood formation in trees is a dynamic process that is strongly affected by environmental factors. However, the impact of ozone on wood is poorly documented. The objective of this study was to assess the effects of ozone on wood formation by focusing on the two major wood components, cellulose and lignin, and analysing any anatomical modifications. Young hybrid poplars (Populus tremula × alba) were cultivated under different ozone concentrations (50, 100, 200, and 300 l l(-1)). As upright poplars usually develop tension wood in a non-set pattern, the trees were bent in order to induce tension wood formation on the upper side of the stem and normal or opposite wood on the lower side. Biosynthesis of cellulose and lignin (enzymes and RNA levels), together with cambial growth, decreased in response to ozone exposure. The cellulose to lignin ratio was reduced, suggesting that cellulose biosynthesis was more affected than that of lignin. Tension wood was generally more altered than opposite wood, especially at the anatomical level. Tension wood may be more susceptible to reduced carbon allocation to the stems under ozone exposure. These results suggested a coordinated regulation of cellulose and lignin deposition to sustain mechanical strength under ozone. The modifications of the cellulose to lignin ratio and wood anatomy could allow the tree to maintain radial growth while minimizing carbon cost.