Significant influence of lignin on axial elastic modulus of poplar wood at low microfibril angles under wet conditions.

Wood is extensively used as a construction material. Despite increasing knowledge of its mechanical properties, the contribution of the cell-wall matrix polymers to wood mechanics is still not well understood. Previous studies have shown that axial stiffness correlates with lignin content only for cellulose microfibril angles larger than around 20°, while no influence is found for smaller angles. Here, by analysing the wood of poplar with reduced lignin content due to down-regulation of CAFFEOYL SHIKIMATE ESTERASE, we show that lignin content also influences axial stiffness at smaller angles. Micro-tensile tests of the xylem revealed that axial stiffness was strongly reduced in the low-lignin transgenic lines. Strikingly, microfibril angles were around 15° for both wild-type and transgenic poplars, suggesting that cellulose orientation is not responsible for the observed changes in mechanical behavior. Multiple linear regression analysis showed that the decrease in stiffness was almost completely related to the variation in both density and lignin content. We suggest that the influence of lignin content on axial stiffness may gradually increase as a function of the microfibril angle. Our results may help in building up comprehensive models of the cell wall that can unravel the individual roles of the matrix polymers.

Unravelling the impact of lignin on cell wall mechanics: a comprehensive study on young poplar trees downregulated for CINNAMYL ALCOHOL DEHYDROGENASE (CAD).

Lignin engineering is a promising tool to reduce the energy input and the need of chemical pre-treatments for the efficient conversion of plant biomass into fermentable sugars for downstream applications. At the same time, lignin engineering can offer new insight into the structure-function relationships of plant cell walls by combined mechanical, structural and chemical analyses. Here, this comprehensive approach was applied to poplar trees (Populus tremula × Populus alba) downregulated for CINNAMYL ALCOHOL DEHYDROGENASE (CAD) in order to gain insight into the impact of lignin reduction on mechanical properties. The downregulation of CAD resulted in a significant decrease in both elastic modulus and yield stress. As wood density and cellulose microfibril angle (MFA) did not show any significant differences between the wild type and the transgenic lines, these structural features could be excluded as influencing factors. Fourier transform infrared spectroscopy (FTIR) and Raman imaging were performed to elucidate changes in the chemical composition directly on the mechanically tested tissue sections. Lignin content was identified as a mechanically relevant factor, as a correlation with a coefficient of determination (r²) of 0.65 between lignin absorbance (as an indicator of lignin content) and tensile stiffness was found. A comparison of the present results with those of previous investigations shows that the mechanical impact of lignin alteration under tensile stress depends on certain structural conditions, such as a high cellulose MFA, which emphasizes the complex relationship between the chemistry and mechanical properties in plant cell walls.

The effect of altered lignin composition on mechanical properties of CINNAMYL ALCOHOL DEHYDROGENASE (CAD) deficient poplars.

MAIN CONCLUSION: CAD-deficient poplars enabled studying the influence of altered lignin composition on mechanical properties. Severe alterations in lignin composition did not influence the mechanical properties. Wood represents a hierarchical fiber-composite material with excellent mechanical properties. Despite its wide use and versatility, its mechanical behavior has not been entirely understood. It has especially been challenging to unravel the mechanical function of the cell wall matrix. Lignin engineering has been a useful tool to increase the knowledge on the mechanical function of lignin as it allows for modifications of lignin content and composition and the subsequent studying of the mechanical properties of these transgenics. Hereby, in most cases, both lignin composition and content are altered and the specific influence of lignin composition has hardly been revealed. Here, we have performed a comprehensive micromechanical, structural, and spectroscopic analysis on xylem strips of transgenic poplar plants, which are downregulated for cinnamyl alcohol dehydrogenase (CAD) by a hairpin-RNA-mediated silencing approach. All parameters were evaluated on the same samples. Raman microscopy revealed that the lignin of the hpCAD poplars was significantly enriched in aldehydes and reduced in the (relative) amount of G-units. FTIR spectra indicated pronounced changes in lignin composition, whereas lignin content was not significantly changed between WT and the hpCAD poplars. Microfibril angles were in the range of 18°-24° and were not significantly different between WT and transgenics. No significant changes were observed in mechanical properties, such as tensile stiffness, ultimate stress, and yield stress. The specific findings on hpCAD poplar allowed studying the specific influence of lignin composition on mechanics. It can be concluded that the changes in lignin composition in hpCAD poplars did not affect the micromechanical tensile properties.

Chitinase-like1/pom-pom1 and its homolog CTL2 are glucan-interacting proteins important for cellulose biosynthesis in Arabidopsis.

Plant cells are encased by a cellulose-containing wall that is essential for plant morphogenesis. Cellulose consists of ß-1,4-linked glucan chains assembled into paracrystalline microfibrils that are synthesized by plasma membrane-located cellulose synthase (CESA) complexes. Associations with hemicelluloses are important for microfibril spacing and for maintaining cell wall tensile strength. Several components associated with cellulose synthesis have been identified; however, the biological functions for many of them remain elusive. We show that the chitinase-like (CTL) proteins, CTL1/POM1 and CTL2, are functionally equivalent, affect cellulose biosynthesis, and are likely to play a key role in establishing interactions between cellulose microfibrils and hemicelluloses. CTL1/POM1 coincided with CESAs in the endomembrane system and was secreted to the apoplast. The movement of CESAs was compromised in ctl1/pom1 mutant seedlings, and the cellulose content and xyloglucan structures were altered. X-ray analysis revealed reduced crystalline cellulose content in ctl1 ctl2 double mutants, suggesting that the CTLs cooperatively affect assembly of the glucan chains, which may affect interactions between hemicelluloses and cellulose. Consistent with this hypothesis, both CTLs bound glucan-based polymers in vitro. We propose that the apoplastic CTLs regulate cellulose assembly and interaction with hemicelluloses via binding to emerging cellulose microfibrils.

Enhanced plastic deformations of nanofibrillated cellulose film by adsorbed moisture and protein-mediated interactions.

Biological composites are typically based on an adhesive matrix that interlocks rigid reinforcing elements in fiber composite or brick-and-mortar assemblies. In nature, the adhesive matrix is often made up of proteins, which are also interesting model systems, as they are unique among polymers in that we know how to engineer their structures with atomic detail and to select protein elements for specific interactions with other components. Here we studied how fusion proteins that consist of cellulose binding proteins linked to proteins that show a natural tendency to form multimer complexes act as an adhesive matrix in combination with nanofibrillated cellulose. We found that the fusion proteins are retained with the cellulose and that the proteins mainly affect the plastic yield behavior of the cellulose material as a function of water content. Interestingly, the proteins increased the moisture absorption of the composite, but the well-known plastifying effect of water was clearly decreased. The work helps to understand the functional basis of nanocellulose composites as materials and aims toward building model systems for molecular biomimetic materials.

Deficient sucrose synthase activity in developing wood does not specifically affect cellulose biosynthesis, but causes an overall decrease in cell wall polymers.

The biosynthesis of wood in aspen (Populus) depends on the metabolism of sucrose, which is the main transported form of carbon from source tissues. The largest fraction of the wood biomass is cellulose, which is synthesized from UDP-glucose. Sucrose synthase (SUS) has been proposed previously to interact directly with cellulose synthase complexes and specifically supply UDP-glucose for cellulose biosynthesis. To investigate the role of SUS in wood biosynthesis, we characterized transgenic lines of hybrid aspen with strongly reduced SUS activity in developing wood. No dramatic growth phenotypes in glasshouse-grown trees were observed, but chemical fingerprinting with pyrolysis-GC/MS, together with micromechanical analysis, showed notable changes in chemistry and ultrastructure of the wood in the transgenic lines. Wet chemical analysis showed that the dry weight percentage composition of wood polymers was not changed significantly. However, a decrease in wood density was observed and, consequently, the content of lignin, hemicellulose and cellulose was decreased per wood volume. The decrease in density was explained by a looser structure of fibre cell walls as shown by increased wall shrinkage on drying. The results show that SUS is not essential for cellulose biosynthesis, but plays a role in defining the total carbon incorporation to wood cell walls.

Plant material features responsible for bamboo's excellent mechanical performance: a comparison of tensile properties of bamboo and spruce at the tissue, fibre and cell wall levels.

BACKGROUND AND AIMS: Bamboo is well known for its fast growth and excellent mechanical performance, but the underlying relationships between its structure and properties are only partially known. Since it lacks secondary thickening, bamboo cannot use adaptive growth in the same way as a tree would in order to modify the geometry of the stem and increase its moment of inertia to cope with bending stresses caused by wind loads. Consequently, mechanical adaptation can only be achieved at the tissue level, and this study aims to examine how this is achieved by comparison with a softwood tree species at the tissue, fibre and cell wall levels. METHODS: The mechanical properties of single fibres and tissue slices of stems of mature moso bamboo (Phyllostachys pubescens) and spruce (Picea abies) latewood were investigated in microtensile tests. Cell parameters, cellulose microfibril angles and chemical composition were determined using light and electron microscopy, wide-angle X-ray scattering and confocal Raman microscopy. KEY RESULTS: Pronounced differences in tensile stiffness and strength were found at the tissue and fibre levels, but not at the cell wall level. Thus, under tensile loads, the differing wall structures of bamboo (multilayered) and spruce (sandwich-like) appear to be of minor relevance. CONCLUSIONS: The superior tensile properties of bamboo fibres and fibre bundles are mainly a result of amplified cell wall formation, leading to a densely packed tissue, rather than being based on specific cell wall properties. The material optimization towards extremely compact fibres with a multi-lamellar cell wall in bamboo might be a result of a plant growth strategy that compensates for the lack of secondary thickening growth at the tissue level, which is not only favourable for the biomechanics of the plant but is also increasingly utilized in terms of engineering products made from bamboo culms.

Enhanced cellulose orientation analysis in complex model plant tissues.

The orientation distribution of cellulose microfibrils in the plant cell wall is a key parameter for understanding anisotropic plant growth and mechanical behavior. However, precisely visualizing cellulose orientation in the plant cell wall has ever been a challenge due to the small size of the cellulose microfibrils and the complex network of polymers in the plant cell wall. X-ray diffraction is one of the most frequently used methods for analyzing cellulose orientation in single cells and plant tissues, but the interpretation of the diffraction images is complex. Traditionally, circular or square cells and Gaussian orientation of the cellulose microfibrils have been assumed to elucidate cellulose orientation from the diffraction images. However, the complex tissue structures of common model plant systems such as Arabidopsis or aspen (Populus) require a more sophisticated approach. We present an evaluation procedure which takes into account the precise cell geometry and is able to deal with complex microfibril orientation distributions. The evaluation procedure reveals the entire orientation distribution of the cellulose microfibrils, reflecting different orientations within the multi-layered cell wall. By analyzing aspen wood and Arabidopsis stems we demonstrate the versatility of this method and show that simplifying assumptions on geometry and orientation distributions can lead to errors in the calculated microfibril orientation pattern. The simulation routine is intended to be used as a valuable tool for nanostructural analysis of plant cell walls and is freely available from the authors on request.

Plant micro- and nanomechanics: experimental techniques for plant cell-wall analysis.

In the last few decades, micro- and nanomechanical methods have become increasingly important analytical techniques to gain deeper insight into the nanostructure and mechanical design of plant cell walls. The objective of this article is to review the most common micro- and nanomechanical approaches that are utilized to study primary and secondary cell walls from a biomechanics perspective. In light of their quite disparate functions, the common and opposing structural features of primary and secondary cell walls are reviewed briefly. A significant part of the article is devoted to an overview of the methodological aspects of the mechanical characterization techniques with a particular focus on new developments and advancements in the field of nanomechanics. This is followed and complemented by a review of numerous studies on the mechanical role of cellulose fibrils and the various matrix components as well as the polymer interactions in the context of primary and secondary cell-wall function.

Ectopic callose deposition into woody biomass modulates the nano-architecture of macrofibrils.

Plant biomass plays an increasingly important role in the circular bioeconomy, replacing non-renewable fossil resources. Genetic engineering of this lignocellulosic biomass could benefit biorefinery transformation chains by lowering economic and technological barriers to industrial processing. However, previous efforts have mostly targeted the major constituents of woody biomass: cellulose, hemicellulose and lignin. Here we report the engineering of wood structure through the introduction of callose, a polysaccharide novel to most secondary cell walls. Our multiscale analysis of genetically engineered poplar trees shows that callose deposition modulates cell wall porosity, water and lignin contents and increases the lignin-cellulose distance, ultimately resulting in substantially decreased biomass recalcitrance. We provide a model of the wood cell wall nano-architecture engineered to accommodate the hydrated callose inclusions. Ectopic polymer introduction into biomass manifests in new physico-chemical properties and offers new avenues when considering lignocellulose engineering.

Influence of over-expression of the Flowering Promoting Factor 1 gene (FPF1) from Arabidopsis on wood formation in hybrid poplar (Populus tremula L. × P. tremuloides Michx.).

Constitutive expression of the FPF1 gene in hybrid aspen (Populus tremula L. × P. tremuloides Michx.) showed a strong effect on wood formation but no effect on flowering time. Gene expression studies showed that activity of flowering time genes PtFT1, PtCO2, and PtFUL was not increased in FPF1 transgenic plants. However, the SOC1/TM3 class gene PTM5, which has been related to wood formation and flowering time, showed a strong activity in stems of all transgenic lines studied. Wood density was lower in transgenic plants, despite significantly reduced vessel frequency which was overcompensated by thinner fibre cell walls. Chemical screening of the wood by pyrolysis GC/MS showed that FPF1 transgenics have higher fractions of cellulose and glucomannan products as well as lower lignin content. The latter observation was confirmed by UV microspectrophotometry on a cellular level. Topochemical lignin distribution revealed a slower increase of lignin incorporation in the developing xylem of the transgenics when compared with the wild-type plants. In line with the reduced wood density, micromechanical wood properties such as stiffness and ultimate stress were also significantly reduced in all transgenic lines. Thus, we provide evidence that FPF1 class genes may play a regulatory role in both wood formation and flowering in poplar.

Reorientation of cellulose nanowhiskers in agarose hydrogels under tensile loading.

Agarose hydrogels filled with cellulose nanowhiskers were strained in uniaxial stretching under different humidity conditions. The orientation of the cellulose whiskers was examined before and after testing with an X-ray laboratory source and monitored in situ during loading by synchrotron X-ray diffraction. The aim of this approach was to determine the process parameters for reorienting the cellulose nanowhiskers toward a preferential direction. Results show that a controlled drying of the hydrogel is essential to establish interactions between the matrix and the cellulose nanowhiskers which allow for a stress transfer during stretching and thereby promote their alignment. Rewetting of the sample after reorientation of the cellulose nanowhiskers circumvents a critical increase of stress. This improves the extensibility of the hydrogel and is accompanied by a further moderate alignment of the cellulose nanowhiskers. Following this protocol, cellulose nanowhiskers with an initial random distribution can be reoriented toward a preferential direction, creating anisotropic nanocomposites.

Intercellular Matrix Infiltration Improves the Wet Strength of Delignified Wood Composites.

Delignified wood (DW) represents a promising bio-based fibrous material as a reinforcing component in high-performance composites. These cellulose composites possess excellent strength and stiffness in the dry state, which are significantly higher than for natural wood. However, in the wet state, a penetrating water layer enters the intercellular regions and disrupts the stress transfer mechanisms between cell fibers in fully DW. This water layer initially facilitates complex shaping of the material but imparts DW composites with very low wet stiffness and strength. Therefore, a sufficient stress transfer in the wet state necessitates a resin impregnation of these intercellular regions, establishing bonding mechanisms between adjacent fibers. Here, we utilize a water-based dimethyloldihydroxyethylene urea thermosetting matrix (DMDHEU) and compare it with a non-water-based epoxy matrix. We infiltrate these resins into DW and investigate their spatial distribution by scanning electron microscopy, atomic force microscopy, and confocal Raman spectroscopy. The water-based resin impregnates the intercellular areas and generates an artificial compound middle lamella, while the epoxy infiltrates only the cell lumina of the dry DW. Tensile tests in the dry and wet states show that the DMDHEU matrix infiltration of the intercellular areas and the cell wall results in a higher tensile strength and stiffness compared to the epoxy resin. Here, the artificial compound middle lamella made of DMDHEU bonds adjacent fibers together and substantially increases the composites' wet strength. This study elucidates the importance of the interaction and spatial distribution of the resin system within the DW structure to improve mechanical properties, particularly in the wet state.

Cellulose lattice strains and stress transfer in native and delignified wood.

Small specimens of spruce wood with different degrees of delignification were studied using in-situ tensile tests and simultaneous synchrotron X-ray diffraction to reveal the effect of delignification and densification on their tensile properties at relative humidities of 70-80 %. In addition to mechanical properties, these analyses yield the ratio of strains in the cellulose crystals and in the bulk, which reflects the stress-transfer to crystalline cellulose. While the specific modulus of elasticity slightly increases from native wood by partial or complete delignification, the lattice strain ratio does not show a significant change. This could indicate a compensatory effect from the decomposition of the amorphous matrix by delignification and from a tighter packing of cellulose crystals that would increase the stress transfer. The reduced strain to failure and maximum lattice strain of delignified specimens suggests that the removal of lignin affects the stress-strain behavior with fracture at lower strain levels.

G-fibres in storage roots of Trifolium pratense (Fabaceae): tensile stress generators for contraction.

Root contraction has been described for many species within the plant kingdom for over a century, and many suggestions have been made for mechanisms behind these contractions. To move the foliage buds deeper into the soil, the proximal part of the storage root of Trifolium pratense contracts by up to 30%. Anatomical studies have shown undeformed fibres next to strongly deformed tissues. Raman imaging revealed that these fibres are chemically and structurally very similar to poplar (Populus) tension wood fibres, which are known to generate high tensile stresses and bend leaning stems or branches upright. Analogously, an almost pure cellulosic layer is laid down in the lumen of certain root fibres, on a thin lignified secondary cell wall layer. To reveal its stress generation capacities, the thick cellulosic layer, reminiscent of a gelatinous layer (G-layer) in tension wood, was selectively removed by enzymatic treatment. A substantial change in the dimensions of the isolated wood fibre bundles was observed. This high stress relaxation indicates the presence of high tensile stress for root contraction. These findings indicate a mechanism of root contraction in T. pratense (red clover) actuated via tension wood fibres, which follows the same principle known for poplar tension wood.

Wood and the Activity of Dead Tissue.

Wood is a prototypical biological material, which adapts to mechanical requirements. The microarchitecture of cellulose fibrils determines the mechanical properties of woody materials, as well as their actuation properties, based on absorption and desorption of water. Herein it is argued that cellulose fiber orientation corresponds to an analog code that determines the response of wood to humidity as an active material. Examples for the harvesting of wood activity, as well as bioinspiration, are given.

Ultrastructure and mechanical properties of populus wood with reduced lignin content caused by transgenic down-regulation of cinnamate 4-hydroxylase.

Several key enzymes in lignin biosynthesis of Populus have been down-regulated by transgenic approaches to investigate their role in wood lignification and to explore their potential for lignin modification. Cinnamate 4-hydroxylase is an enzyme in the early phenylpropanoid pathway that has not yet been functionally analyzed in Populus . This study shows that down-regulation of cinnamate 4-hydroxylase reduced Klason lignin content by 30% with no significant change in syringyl to guaiacyl ratio. The lignin reduction resulted in ultrastructural differences of the wood and a 10% decrease in wood density. Mechanical properties investigated by tensile tests and dynamic mechanical analysis showed a decrease in stiffness, which could be explained by the lower density. The study demonstrates that a large modification in lignin content only has minor influences on tensile properties of wood in its axial direction and highlights the usefulness of wood modified beyond its natural variation by transgene technology in exploring the impact of wood biopolymer composition and ultrastructure on its material properties.

Structure-function relationships of different vascular bundle types in the stem of the Mexican fanpalm (Washingtonia robusta).

Structure and mechanics of fibre caps of different types of vascular bundles were studied at a certain height in the trunk of the palm Washingtonia robusta. By correlating these features with the distribution of the different bundle types across the trunk, possible strategies to cope with mechanical loads were elucidated. Micromechanical properties, cell parameters, microfibril orientation and lignification were studied using micromechanical testing, image analysis, synchrotron X-ray diffraction and UV-microspectrophotometry. The adjustment of stiffness followed a common principle in the fibre caps of all bundle types. Gradients in stiffness appeared across the caps in the centre of the trunk whereas stiffness remained high across the caps in the periphery of the trunk. The difference in stiffness profiles was interpreted as an adaptation to different mechanical constraints arising across the trunk. The gradual transition in stiffness prevents high local stress discontinuities between cap fibres and parenchyma and might be beneficial for trunk damping. At the periphery, the fibre caps without stiffness gradients contribute to a high flexural stiffness of the trunk as this has to be exclusively maintained by the material properties because geometric adjustments through increasing trunk diameter hardly occur in monocotyledonous palms.

Facilitated delignification in CAD deficient transgenic poplar studied by confocal Raman spectroscopy imaging.

Lignocellulosic biomass represents the only renewable carbon resource which is available in sufficient amounts to be considered as an alternative for our fossil-based carbon economy. However, an efficient biochemical conversion of lignocellulosic feedstocks is hindered by the natural recalcitrance of the biomass as a result of a dense network of cellulose, hemicelluloses, and lignin. These polymeric interconnections make a pretreatment of the biomass necessary in order to enhance the susceptibility of the polysaccharides. Here, we report on a detailed analysis of the favourable influence of genetic engineering for two common delignification protocols for lignocellulosic biomass, namely acidic bleaching and soda pulping, on the example of CAD deficient poplar. The altered lignin structure of the transgenic poplar results in a significantly accelerated and more complete lignin removal at lower temperatures and shorter reaction times compared to wildtype poplar. To monitor the induced chemical and structural alterations at the tissue level, confocal Raman spectroscopy imaging, FT-IR spectroscopy, and X-ray diffraction were used.

Fabrication and Design of Wood-Based High-Performance Composites.

Delignified densified wood is a new promising and sustainable material that possesses the potential to replace synthetic materials, such as glass fiber reinforced composites, due to its excellent mechanical properties. Delignified wood, however, is rather fragile in a wet state, which makes handling and shaping challenging. Here we present two fabrication processes, closed-mold densification and vacuum densification, to produce high-performance cellulose composites based on delignified wood, including an assessment of their advantages and limitations. Further, we suggest strategies for how the composites can be re-used or decomposed at the end-of-life cycle. Closed-mold densification has the advantage that no elaborate lab equipment is needed. Simple screw clamps or a press can be used for densification. We recommend this method for small parts with simple geometries and large radii of curvature. Vacuum densification in an open-mold process is suitable for larger objects and complex geometries, including small radii of curvature. Compared to the closed-mold process, the open-mold vacuum approach only needs the manufacture of a single mold cavity.