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

Stress generation in the tension wood of poplar is based on

the lateral swelling power of the G-layer

Luna Goswami1, John W.C. Dunlop1, Karin Jungnikl1, Michaela Eder1, Notburga Gierlinger1, Catherine Coutand2, George Jeronimidis3, Peter Fratzl1and Ingo Burgert1,*

1

Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, 14424 Potsdam, Germany, 2

Institut National de la Recherche Agronomique (INRA) umr Physiologie Inte´grative de l’Arbre Fruitier et Forestier (PIAF), 234 av. du Bre´zet, 63100 Clermont-Ferrand, France, and

3_{Centre for Biomimetics, Composite Materials Engineering, School of Construction Management and Engineering,} University of Reading, Whiteknights, Reading RG6 2AY, UK

Recieved 23 March 2008; revised 7 June 2008; accepted 20 June 2008; published online 1 August 2008. *For correspondence (fax +49 331 567 9402; e-mail ingo.burgert@mpikg.mpg.de).

Summary

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 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). The removal of the G-S2-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. Keywords: tension wood, tensile stress generation, G-layer, poplar, cellulose microfibril orientation, enzymatic treatment.

Introduction

Trees maintain or reorientate their axes by the differentia-tion of reacdifferentia-tion wood on one side of the axis. For most angiosperm tree species, anatomical investigations show that tension wood is built up on the upper side of the organ (Wardrop, 1965) during the gravitropic phase of the tropic response and on the opposite side during the autotropic phase (Coutand et al., 2007). However, it is still not known how angiosperm trees are able to generate high tensile stresses and pull leaning stems and branches upwards. The tension wood fibres contract longitudinally during differen-tiation and generate longitudinal tensile stresses of up to about 70 MPa (Okuyama et al., 1994) which can actuate the movement, i.e. the change of curvature of the axis.

In comparison with regular fibres, tension wood fibres in many species show an additional characteristic structural feature called the G-layer, which is believed to be an operational part of the tension wood fibre (Clair et al.,

2003; Coˆte and Day, 1965). However, it is known that many angiosperm trees are able to right their organs without forming a G-layer; for instance Fisher and Stevenson (1981) and Clair et al. (2006b) found G-layers in roughly 50% of the species studied. It has been recently shown in Eucalyptus that tension wood fibres lacking G-layers possess chemi-cally and structurally modified secondary cell walls com-pared with regular fibres (Qiu et al., 2008). The focus of the present study is on the mechanical function of the G-layer, which is prominent in the poplar species examined.

Regular angiosperm fibres consist of three lignified secondary cell wall layers (S1, S2 and S3) which can be distinguished by the orientation of the parallel-arranged cellulose microfibrils and are consecutively deposited on the primary cell wall after the cell has reached its final shape and size. The G-layer of tension wood fibres is an additional layer on the lumen side which can replace the S3-layer or even the

S2-layer of the secondary cell wall (Coˆte and Day, 1965) and can even fill the whole lumen of the tension wood fibre (Coutand et al., 2004). The G-layer consists of almost pure cellulose organized in aggregates of diameter 30–40 nm (Daniel et al., 2006) accompanied by some xyloglucan and traces of monolignols or syringyl units (Gierlinger and Schwanninger, 2006; Joseleau et al., 2004; Lehringer et al., 2008; Nishikubo et al., 2007). The cellulose fibrils are oriented parallel to the axial direction [microfibril angle (MFA)0] and have a higher crystallinity than the cellulose fibrils in secondary walls (Norberg and Meier, 1966).

Although the nanostructural features of the G-layer are well described, the underlying mechanisms of stress gener-ation are not well understood. Recently, enzyme activity was reported to continue in tension wood, even in mature fibres after cell differentiation (Nishikubo et al., 2007). This per-sisting function of xyloglucan endo-transglycosylase (XET) may indicate a role in stress generation. Yamamoto (2004) developed a sophisticated model describing the deforma-tion process of a tension wood fibre regarded as a hollow cylinder consisting of four layers with different microfibril orientations. However, there is still no comprehensive mechanical model of stress generation during fibre differ-entiation in tension wood.

The fundamental question to be solved is how the length of tension wood fibres can be reduced by a G-layer consisting of axially oriented almost incontractible cellulose fibrils. One possible solution is that the contraction of the fibre is not caused by the G-layer directly, but by interaction of the G-layer with the surrounding secondary cell wall. This hypothesis was first mentioned by Mu¨nch (1938) but has not been considered further, perhaps due to a focus on the dominating G-layer or by the limited experimental accessi-bility of the complex cell wall assembly. This paper inves-tigates this hypothesis by combining studies on mechanical properties and nanostructural features of the cell wall layers with modelling of the mechanical response, as recently applied to compression wood of conifers (Burgert, 2006; Burgert et al., 2007; Keckes et al., 2003). One prerequisite for this study was to establish a technique to remove the G-layer from longitudinal tissue slices by enzymatic treatment

without mechanically altering the remaining tissue. This enabled tensile testing of tissues for a structural and mechanical characterisation of tension wood fibres with and without G-layers.

Results and discussion Structural aspects

To assess the removal of the G-layer by the enzyme, cross-sections of poplar tension wood were examined with a scanning electron microscope in the low-vacuum mode before and after treatment. The enzyme treatment resulted in the complete removal of the G-layers as illustrated in Figure 1. Alternatively, the G-layer could be separated from tension wood fibres by an ultrasound bath treatment (Nor-berg and Meier, 1966), but this technique is only applicable to thin cross-cuts which cannot be mechanically tested in the longitudinal direction.

The orientation of cellulose microfibrils in the tension wood fibres was measured by wide-angle X-ray scattering (WAXS). In the presence of the G-layer, the wide-angle scattering signal of untreated tension wood mainly originates from the axially orientated, highly crystalline cellulose fibrils of the G-layer (Figure 1c, left) which over-shadows the much weaker signal from the other cell wall layers. Removal of the G-layer facilitated the measurement of the orientation of the cellulose in the surrounding secondary cell wall. An angle of the cellulose fibrils with respect to the cell axis (the MFA) of l36 was calculated from the WAXS patterns of the treated samples (Figure 1c, right). A rather large MFA in the surrounding secondary cell wall was also found in previous studies. Mu¨ller et al. (2006) were able to measure the MFA in the secondary cell wall of poplar tension wood fibres in the presence of the G-layer using a synchrotron beamline, and reported a MFA of 20  5. Raman imaging revealed a change from straight cellulose orientation in the G-layer to a high MFA in the adjacent cell wall layer (Gierlinger and Schwanninger, 2006). To assess whether the G-layers in the fibres pre-stress the tension wood tissue, dimensional changes of tension wood

(a) (b) (c)

Figure 1. Effect of enzymatic treatment on the tension wood fibres.

Scanning electron microscopy image of (a) a cross-section of the native tension wood tissue with cell lumina almost completely filled with G-layers, (b) the same tissue after enzymatic treatment with complete degradation of the G-layers.

slices were measured upon removal of the G-layer (Fig-ure 2). The slices elongated by a strain eL= 1.60% in the longitudinal direction and shortened by eT=)1.04% in the tangential direction. Control measurements of buffer-treated tension wood slices as well as buffer- and enzyme-treated opposite wood slices showed no deformation or only minor contraction in the longitudinal direction (eL<)0.1%). Buffer-treated tension wood slices shortened slightly in the tangential direction, eT=)0.26%, whereas opposite wood slices showed slight elongation in the tangential direction for both buffer and enzyme treatments (buffer treated, eT= 0.36% elongation; enzyme treated eT= 0.17% elonga-tion). The contraction of the tension wood slices in the tangential direction upon removal of the G-layer indicates that the G-layer generates forces which result in a transverse (tangential) expansion of the fibre. The large elongation in the longitudinal direction after removal of the G-layer implies that the G-layer actively shortens the tension wood fibre as a result of the stresses generated during maturation. The ratio between the dimensional changes in both direc-tions was eL:eT 1:6. Using simple trigonometric argu-ments, and assuming very stiff cellulose microfibrils, the ratio of strains should be eL:eTffi  cot2l which is about )1.89 for a MFA l = 36 of the secondary cell wall (Fratzl et al., 2004; Keckes et al., 2003).

Mechanical aspects

To understand the mechanical role of the G-layer, tensile tests were performed on tension wood slices with and

without enzyme treatment. Figure 3(a) shows examples of the stress–strain behaviour of native poplar tension wood and enzyme-treated slices tested when wet. The buffer-treated slices showed the same behaviour as the native sli-ces, indicating that the buffer treatment had no effect on the mechanical properties (data not shown). The enzyme-treated slices (i.e. with the G-layer removed) showed a three-phase tensile behaviour that is typical for plant tissue with a high cellulose MFA in the S2-layer (Bodig and Jayne, 1993; Keckes et al., 2003; Ko¨hler and Spatz, 2002). The stiff initial portion of the stress–strain curve was followed by a region of lower slope and a final portion of increasing stiffening rate up until failure. This typical behaviour indicated that the mechanical response of the secondary cell wall was not affected by the enzymatic treatment. This was confirmed by control mea-surements of native and enzyme-treated opposite wood tis-sues which possess a normal lignified secondary cell wall without an additional G-layer. The tensile stiffness was almost the same for reference, buffer- and enzyme-treated tissue slices (Figure 3e,f). Consistently, it appears unlikely that the enzymatic treatment affected the tensile properties of the lignified secondary cell walls of the tension wood fibres. As additional Fourier transform infrared (FT-IR) mic-rospectroscopy measurements showed, the enzyme treat-ment can lead to changes in the biochemistry of the lignified secondary cell wall. Minor changes in peak height after 2 days’ treatment and increased changes in peak height after 7 days’ treatment time were found (data not shown). But due to limits on sample thickness with this technique the mea-surements were done on thin cross sections. Therefore, the detected changes could be related to surface degradation of the open cell wall structure in the cross sections and do not have to be directly related to possible degradation of the comparably large longitudinal samples. Hence, we assume that the changes in peak height are due to surface modifi-cation, because lignin is known to act as a physical barrier against enzyme penetration (Huang, 1975) and no measur-able influence on the tensile properties was seen. Moreover, it should be highlighted that such modifications are not rel-evant to the mechanical model presented in the following.

The general shape of the stress–strain curve of the native slices (with a G-layer) was the same as that of the enzyme-treated slices (stiff initial region followed by a region of lower slope and a portion of increasing stiffening). However, the initial stiffness was significantly higher (native tension wood2  0.241 GPa; enzyme treated 0.5  0.164 GPa). Density measurements indicated that the tissue density in tension wood fibres with a G-layer was about two and a half times higher than after removal of the G-layer (data not shown). Hence a factor of about four between the initial stiffness of both samples cannot be explained by the difference in density of the materials alone. This implies an intrinsically higher stiffness of the G-layer compared with the surrounding cell wall layers.

Elongation (%) –1.5 –0.5 0.5 1.5 –2 –1 0 1 2 Longitudinal Tangential

Figure 2. Change of dimensions in the longitudinal and tangential direction of tension wood tissue slices as calculated from length both before and after removal of the G-layer due to enzyme treatment. Bar graphs show arithmetic means SD (n = 10).

A particularly informative feature is the jagged appear-ance of the stress–strain curves of untreated tension wood (Figure 3a) which can be seen in more detail when the curve of the enzymatically treated tissue is subtracted from the curve of the native tension wood (Figure 3b). The tissue does not deform smoothly but in ‘stops and starts’. Fourier analysis of the stress–strain curves showed that the drops in stress are not periodic, suggesting a random slip or defor-mation process. Defordefor-mation is linear everywhere along the curve except at local discontinuities, at which the stress drops show roughly constant strain. This is very similar to the phenomenon of ‘jerky flow’ seen in certain metallic alloys (Lebyodkin et al., 2000).

Mechanical deformation analysis

The jagged stress–strain curves of native tension wood slices indicate that stored elastic energy is being released

locally somewhere in the tissue before building up again. The loss of this behaviour upon enzyme treatment (but not upon buffer treatment) and its absence in opposite wood (Figure 3e) indicate that this mechanical response is due to the G-layer and its interactions with the surrounding secondary cell wall. In addition, the recoverable stiffness upon cyclic loading (Figure 3c,d), measured by the slope of the stress–strain curve during reloading, shows that the underlying behaviour of the material is not damaged during plastic deformation, which can be explained either by slipping events at the interface of the G-layer and the S2-layer or by self-repair mechanisms.

The jagged deformation behaviour is similar to the slipping between surfaces of objects under intermittent frictional forces (the so-called slip–stick mechanism). A high static friction between the layers is responsible for the initial phase of stress build-up. Once a critical stress/strain is reached the G-layer slips with respect to the secondary cell 0 500 1000 1500 2000 2500 Reference Reference Buffer Buffer Enzym e Enzym e

Tension wood Opposite wood

Tensile stiffness [MPa]

0.00 0.05 0.10 0.15 0.20 0 10 20 30 40 50 60 70 80 Stress [MPa] Strain [–] R E 0.00 0.05 0.10 0.15 0.20 0 10 20 30 40 50 60 70 80 Stress [MPa] Strain [–] 0.00 0.05 0.10 0.15 0.20 0 10 20 30 40 50 60 70 80 Stress [MPa] Strain [–] R E 0.00 0.05 0.10 0.15 0.20 0 10 20 30 40 50 60 70 80 Stress [MPa] Strain [–] (a) (b) (c) (d) (e) (f) 0.00 0.05 0.10 0.15 0.20 0 10 20 30 40 50 60 70 80 Stress [MPa] Strain [–] Figure 3.

Mechanical response of tension wood and opposite wood with and without enzymatic treatment.

(a) Representative stress–strain curves of wet tissue slices of native tension wood (R) and after removal of the G-layer (E).

(b) Difference in the stress–strain response cal-culated by subtracting the curve of the tissue without a G-layer from the native one. (c) Cyclic loading curve of wet native tension wood tissue.

(d) Cyclic loading curve of wet tension wood tissue after removal of the G-layer.

(e) Representative stress–strain curves of wet tissue slices of native opposite wood (R) and after enzyme treatment (E).

(f) Arithmetic means and standard SD of the tensile tests on tissue slides of tension wood (reference, n = 10), buffer-treated (n = 8), enzyme-treated (n = 9) and opposite wood (reference, n = 10), buffer-treated (n = 10), enzyme-treated (n = 10).

wall and the stress drops. Since the stress state is now below the critical stress, the G-layer sticks once more to the secondary cell wall, and the stress starts to increase again.

Modelling of this deformation pattern for a set of independent tension wood fibres can clarify the contribution of the G-layer to the overall stress–strain curve (for details see Figure S2). Assuming that a slip event of a G-layer within a fibre causes a stress drop in the tissue, the larger stress drop events in the jagged curve can only be explained by a slip of the G-layers in a number of fibres. If every G-layer had the same stiffness and critical strain (or stress) then all would slip simultaneously and a jagged curve, as shown in Figure 4(a), would result. This simulated stress–strain curve periodically oscillates with strain, because no standard deviation in the critical strain distribution is calculated. However, if G-layers slip at different strain levels a distribu-tion of critical strains for a multitude of fibres has to be assumed. Then the tissue would be expected to behave like Figure 4(b). As the spread in critical strain becomes larger, the stress–strain curve is superimposed by random oscilla-tions, resulting in a jagged appearance which corresponds

well with the experimental results for tension wood slices in Figure 3(b).

Both experiments and modelling point towards localized slip events within the tension wood fibres. This can be explained by short intervals of slippage between the G-layer and the surrounding secondary cell wall when friction forces are temporarily exceeded. Indeed, the contact between the G-layer and the secondary cell wall cannot result from strong bonds, as indicated by the removal of the G-layer by ultrasonic treatment (Norberg and Meier, 1966). However, efficient transfer of stresses between the G-layer and the surrounding secondary cell wall must exist to explain the generation of tensile stress as measured in the living plant (Okuyama et al., 1994) and the stress–strain behaviour shown in the present study.

This interfacial bonding between the G-layer and the S2-layer, which provides a strong interconnection, and efficient stress transfer might be mediated by enzyme activity. Enhanced XET activity was observed at the interface between the primary wall and the S1-layer during secondary cell wall formation, which was interpreted as a potential

(b) 0.00 0.02 0.04 0.06 0.08 0.10 0 5 10 15 20 25 30 35 40 45

Stress [MPa]

Strain [–]

Stress [MPa]

Strain [–]

Simulated stress-strain curves of the mechanical contribution of the G-layer.

(a) Simulated stress–strain curve of the contri-bution of the G-layer to the response of native tension wood without any variation in critical strain between the fibres.

(b) Simulated stress–strain curve of the contri-bution of the G-layer to the response of native tension wood (i.e. Figure 3b) with a Gaussian distribution (SD = 0.009) in critical strain between the fibres (see also Figure S3).

reinforcement of cellulose microfibrils of both layers (Bour-quin et al., 2002). In tension wood high XET activity was detected in the G-layer during differentiation and in the surrounding S2-layer after maturation, which may have a function in connecting the G-layer to the S2-layer and in repairing cross-links in the course of stress generation (Nishikubo et al., 2007).

However, physiological control of the interfacial bonding between both layers cannot be the sole explanation for the stress generation mechanism. For this, the mechanical interplay of the G-layer and the surrounding secondary cell wall layer has to be taken into account, as already suggested by Mu¨nch (1938). The dimensional changes after removal of the G-layer (see Figure 2), the slip events upon straining (see Figures 3 and 4) and the axial orientation of the cellulose in the G-layer (see Figure 1) point towards a high lateral swelling capacity of the water-saturated G-layer in the living tree. A very high hygroscopicity and water content of the G-layer have been consistently reported (Abe and Yamamoto, 2007; Gierlinger and Schwanninger, 2006) despite the high degree of crystallinity of the cellulose.

Model on stress generation

The swelling power of the G-layer is likely to be strongest in the radial direction (MFA0), perpendicular to the cellulose fibrils, and generates an internal pressure p onto the other cell walls, as sketched in Figure 5(a). This pressure is con-verted into a circumferential hoop stress which can simply be written rr= pR/t, where R is the radius of the lumen and t is the thickness of the cell wall (assumed to be small compared with R). Based on a simple mechanical model for the defor-mation of the cell wall (Fratzl et al., 2008), whereby cellulose fibrils reinforce an otherwise isotropic matrix (with Young’s modulus E and Poisson ratio m) in the direction of their length only, it is possible to relate cell wall stress in the circumfer-ential direction, rr, to the stress in the axial direction, ra:

ra¼

mþ m2_n2_f

1þ n4_f rr¼ F rr; where f = (1–m2_)E

F/E is a parameter describing the extra stiffness EF introduced by the fibres, m = cos l and n = sin l, l being the MFA. When f = 0, the cell wall is iso-tropic and the stress enhancement factor F (i.e. the ratio between ra and rr) is just equal to the Poisson ratio m. In Figure 5(d), we have plotted this factor for several values of f (and m = 1/3), and it turns out that a MFA of 36, as observed in the cell wall of stress fibres, gives the largest enhance-ment when f» 2 (that is, when the cell wall is three times stiffer in the direction of the cellulose fibrils than perpen-dicular to them). Finally, ra, which is the stress within the cell wall, may be converted to an actual longitudinal tensile stress rG(that is, tensile force per unit surface of tissue) with rG= 2pRtra/pR2. The final result is that rG= 2Fp with the

stress enhancement factor F, which depends on the MFA (Figure 5d). Hence, it appears that a highly hydrated G-layer and a suitable choice of the MFA in the secondary cell wall is needed to yield the highest maturation stress for a given swelling pressure of the G-layer.

As long as the G-layer is hydrated, the system can generate tensile stresses, but a slight drying of the G-layer may result in a complete breakdown of stresses. Mattheck and Burkhardt (1991) found that in dry summer seasons curved branches tend to split longitudinally due to radial stresses. They argued that the limited water supply could cause a reduction in stress generation in the tension wood. The model for the generation of tensile stresses in tension wood is based on experimental results obtained from samples in which the internal stresses were already released. Therefore, the influence of the load conditions under which the tension wood fibres are formed in leaning stems or branches could not be taken into account. Hints could be given by the work of Clair et al. (2006a), who showed that the lattice spacing of crystalline cellulose fibrils was reduced during the release of the internal tensile stresses and that the nanostructural strain was of the same order of magnitude as

Microfibril angle [°]

0 20 40 60 80

Stress enhancement factor

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 (d) (a) (b) (c) f = 2 1 0

Figure 5. Model for the generation of tensile stresses in a tension wood cell. (a) The pressure p generated by the swelling of the G-layer is converted into a circumferential hoop stress rrwithin the cell wall.

(b) This circumferential tensile stress is converted into an axial tensile stress rawithin the cell wall.

(c) The ratio of rato rr(stress enhancement factor, F) is governed by the

direction of the stiff cellulose fibrils within the cell wall. The microfibril angle is schematically indicated by inclined lines in the cell wall.

(d) Calculation of the stress enhancement factor F within a simple mechanical model of the cell wall (Fratzl et al., 2008) plotted as a function of microfibril angle for three different values of f. The coefficient f is a parameter describing the extra stiffness of the cell wall in the direction of the cellulose fibril which quantifies the anisotropy of the cell wall (f = 0 corresponds to an isotropic cell wall).

the macrostructural strain. Therefore, in situ measurements are needed in the living system to understand the effect of load on the tension wood fibre during stress generation.

The ability of an angiosperm tree to change the form and orientation of its axes depends on the generation of sufficiently high tensile stresses in the newly formed tissue. These high tensile stresses are generated by the tendency of differentiating cells to contract. The contraction is resisted, however, as the fibres are forming within a bulk tissue. The magnitude of the developed stresses depends on the stiffness of the newly formed fibres. Sufficient contractibility and high stiffness appear to be contradictory, as a stiff material is by definition a material which is difficult to deform. The proposed mechanism explains how a tree can generate tensile stresses with a material which is stiff in one direction and sufficiently contractible in another. The G-layer itself, although very stiff in the direction of the cellulose microfibrils, cannot be expected to lead to a substantial shortening in the longitudinal direction as it consists of axially oriented cellulose fibrils. However, the S2-layer with its large MFA has a low stiffness in the longitudinal direction and can be easily deformed. The stresses developed from the radial swelling of the G-layer are transformed into a longitudinal contraction of the S2-layer of the cell wall and thereby the fibre. Therefore it is the combination of the G-layer and the surrounding secondary cell wall which together produce a stiff composite structure with sufficient contractibility.

Experimental procedures Sample preparation

The material was taken from the second growth ring of a 3-year-old poplar tree (Populus nigra· Populus deltoides) that was tilted by 35 from the vertical during the second growth season at the end of May to artificially introduce tension wood formation. The tree was about 3 m tall and the material was taken from the base of the trunk. Small wooden blocks were cut from the area where the G-layer was prominently visible and from opposite wood blocks. The material was stored at 4C until testing. Longitudinal–tangential (LT) slices were obtained from the wooden block using a rotary microtome. Samples were kept wet during slice preparation to avoid drying effects. The slices were cut into three segments along the fibre direction, resulting in samples with a length of 20 mm, a width of 1.5 mm and a thickness of 0.150 mm. These samples were used as reference slice, enzyme-treated slice and buffer-treated slice, respectively.

For the enzymatic treatment ‘Cellulase Onozuka RS’ from Tricho-derma viride (E.C.3.2.1.4, Yakult Pharmaceutical Industry and Co. Ltd, http://www.yakult.co.jp/ypi/en) which acts on cellulose and xylan was used to remove the G-layer. The tissue slices were incubated in the enzyme solution (0.1 g ml)1dissolved in working buffer 50 mMNa2HPO4titrated with citric acid, pH 5) at 50C for

9 days under constant shaking. The enzyme solutions were changed twice to avoid microbial contamination. To visualize the degradation of the G-layer after enzyme treatment, tissue slices were cut at different positions and examined by scanning electron

microscopy. Opposite wood slices consisting of normal secondary cell walls without a G-layer were subjected to the same enzyme treatment, to investigate a possible influence on the mechanical behaviour of the secondary cell wall.

Deformation upon removal of the G-layer

Longitudinal and transverse dimensions of the longitudinal– tangential (LT) slices were measured before and after the buffer or enzyme treatment. The wet tissue slices had dimensions of about 20· 2.3 · 0.150 mm3_{. Ten specimens each were treated with}

enzyme/buffer or buffer solution, respectively, for 9 days. Due to the large size, the entire slices were imaged in several consecutive images. The entire length of one tissue slice was reconstructed by recombining all images in one frame (Burgert et al., 2007). Additional control measurements with the same protocol were carried out on slices of opposite wood tissue of the same dimen-sions. Five specimens were examined for each buffer and enzyme treatment for opposite wood.

Microtensile tests

For the mechanical tests, ten untreated, eight buffer-treated and nine enzyme-treated tissue slices of tension wood were successfully tested in the wet condition using a microtensile tester with a 22-N load cell. For opposite wood 10 tissue slices of each category were successfully tested. The samples were clamped by pressure bars, with a free test length of 12 mm. The slices were strained until failure with a displacement rate of 15 lm sec)1. Stress calculations for the slices were based on their cross-sectional areas.

Slightly unsteady initial stages of the stress–strain curves of the wood tissues due to the alignment of the slices upon loading were eliminated by determining the point of origin via extrapolation of the slope of the linear range. Additionally, seven reference slices, three buffer-treated slices and five enzyme-treated slices were successfully strained in cyclic loading tests. The effect of the buffer was tested using the same treatment without enzyme.

WAXS measurement

Wide-angle X-ray scattering experiments were performed on a Nanostar (Bruker AXS, http://www.bruker-axs.com/) instrument with a sample–detector distance of 5 cm using CuKa radiation (wavelength 1.54 A˚). The diffraction patterns were collected with a two-dimensional (2D) position-sensitive (Hi-star) detector, with a measuring time of 1 h. The 2D patterns were corrected for back-ground, radially averaged from scattering angle 2h 21–24, corre-sponding to the 002 reflection, and the intensity was plotted against the azimuthal angle. The MFA was calculated according to the method described by Lichtenegger et al. (1998). The calculated value represents an average MFA for all fibres (including vessels) in the area hit by the beam (diameter600 lm).

Acknowledgements

Financial support by the FWF (Austrian Science Fund) is gratefully acknowledged. John Dunlop thanks the Alexander von Humboldt Foundation and Notburga Gierlinger the Austrian Academy of Sci-ence (APART-scholarship). We thank Susann Weichold, Annemarie Martins and Petra Leibner for their technical assistance and Dr Staffan Persson for helpful comments on the manuscript.

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Figure S1. Schematic of tension wood structure illustrating the ‘parallel’ structure.

Figure S2. Idealised model in which n-tension wood fibres deform in parallel. The tissue stress is related to the sum of the stresses in each fibre.

Figure S3. Schematic stress–strain response of the contribution of the G-layer/secondary cell wall interface to the mechanical response of a single wood fibre.

Appendix S1.

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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