Non-targeted discovery of high-value bio-products in Nicotiana glauca L: a potential renewable plant feedstock.

The evaluation of plant-based feedstocks is an important aspect of biorefining. Nicotiana glauca is a solanaceous, non-food crop that produces large amounts of biomass and is well adapted to grow in suboptimal conditions. In the present article, compatible sequential solvent extractions were applied to N. glauca leaves to enable the generation of enriched extracts containing higher metabolite content comparing to direct leaf extracts. Typically, between 60 to 100 metabolite components were identified within the fractions. The occurrence of plant fatty acids, fatty acid alcohols, alkanes, sterols and terpenoids was detected by gas liquid chromatography-mass spectrometry (GC-MS) and metabolite identification was confirmed by comparison of physico-chemical properties displayed by available authentic standards. Collectively, co-products such waxes, oils, fermentable sugars, and terpenoids were all identified and quantified. The enriched fractions of N. glauca revealed a high level of readily extractable hydrocarbons, oils and high value co-products. In addition, the saccharification yield and cell wall composition analyses in the stems revealed the potential of the residue material as a promising lignocellulosic substrate for the production of fermentable sugars. In conclusion a multifractional cascade for valuable compounds/commodities has been development, that uses N. glauca biomass. These data have enabled the evaluation of N. glauca material as a potential feedstock for biorefining.

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.

Mechanical factors contributing to the Venus flytrap's rate-dependent response to stimuli.

The sensory hairs of the Venus flytrap (Dionaea muscipula Ellis) detect mechanical stimuli imparted by their prey and fire bursts of electrical signals called action potentials (APs). APs are elicited when the hairs are sufficiently stimulated and two consecutive APs can trigger closure of the trap. Earlier experiments have identified thresholds for the relevant stimulus parameters, namely the angular displacement [Formula: see text] and angular velocity [Formula: see text]. However, these experiments could not trace the deformation of the trigger hair's sensory cells, which are known to transduce the mechanical stimulus. To understand the kinematics at the cellular level, we investigate the role of two relevant mechanical phenomena: viscoelasticity and intercellular fluid transport using a multi-scale numerical model of the sensory hair. We hypothesize that the combined influence of these two phenomena and [Formula: see text] contribute to the flytrap's rate-dependent response to stimuli. In this study, we firstly perform sustained deflection tests on the hair to estimate the viscoelastic material properties of the tissue. Thereafter, through simulations of hair deflection tests at different loading rates, we were able to establish a multi-scale kinematic link between [Formula: see text] and the cell wall stretch [Formula: see text]. Furthermore, we find that the rate at which [Formula: see text] evolves during a stimulus is also proportional to [Formula: see text]. This suggests that mechanosensitive ion channels, expected to be stretch-activated and localized in the plasma membrane of the sensory cells, could be additionally sensitive to the rate at which stretch is applied.

Microscale compressive behavior of hydrated lamellar bone at high strain rates.

The increased risk of fracture in the elderly associated with metabolic conditions like osteoporosis poses a significant strain on health care systems worldwide. Due to bone's hierarchical nature, it is necessary to study its mechanical properties and failure mechanisms at several length scales. We conducted micropillar compression experiments on ovine cortical bone to assess the anisotropic mechanical response at the lamellar scale over a wide range of strain rates (10-4 to 8·102 s-1). At the microscale, lamellar bone exhibits a strain rate sensitivity similar to what is reported at the macroscale suggesting that it is an intrinsic property of the extracellular matrix. Significant shear band thickening was observed at high strain rates by HRSEM and STEM imaging. This is likely caused by the material's inability to accommodate the imposed deformation by propagation of thin kink bands and shear cracks at high strain rates, leading to shear band thickening and nucleation. The post-yield behavior is strain rate and direction dependent: hardening was observed for transverse oriented micropillars and hardening modulus increases with strain rate by a factor of almost 2, while axially oriented micropillars showed strain softening and an increase of the softening peak width and work to ultimate stress as a function of strain rate. This suggests that for compression at the micrometer scale, energy absorption in bone increases with strain rate. This study highlights the importance of investigating bone strength and post-yield behavior at lower length scales, under hydrated conditions and at clinically relevant strain rates. STATEMENT OF SIGNIFICANCE: We performed micropillar compression experiments of ovine cortical bone at two different orientations and over seven orders of magnitude of strain rate. Experiments were performed under humid condition to mimic the natural conditions of bone in a human body using a newly developed micro-indenter setup. The strain rate sensitivity was found to be of a similar magnitude to what has been reported for higher length scales, suggesting that the strain rate sensitivity is an intrinsic property of the bone extracellular matrix. In addition, localized shear deformation in thick bands was observed for the first time at high strain rates, highlighting the importance of investigating bone under conditions representative of an accident or fall at several length scales.

Wood Derived Cellulose Scaffolds-Processing and Mechanics.

Wood-derived cellulose materials obtained by structure-retaining delignification are attracting increasing attention due to their excellent mechanical properties and great potential to serve as renewable and CO2 storing cellulose scaffolds for advanced hybrid materials with embedded functionality. Various delignification protocols and a multitude of further processing steps including polymer impregnation and densification are applied resulting in a large range of properties. However, treatment optimization requires a more comprehensive characterization of the developed materials in terms of structure, chemical composition, and mechanical properties for faster progress in the field. Herein, the current protocols for structure-retaining delignification are reviewed and the emphasis is placed on the mechanical characterization at different hierarchical levels of the cellulose scaffolds by experiments and modeling to reveal the underlying structure-property relationships.

Computational analysis of hygromorphic self-shaping wood gridshell structures.

Bi-layered composites capable of self-shaping are of increasing relevance to science and engineering. They can be made out of anisotropic materials that are responsive to changes in a state variable, e.g. wood, which swells and shrinks by changes in moisture. When extensive bending is desired, such bilayers are usually designed as cross-ply structures. However, the nature of cross-ply laminates tends to prevent changes of the Gaussian curvature so that a plate-like geometry of the composite will be partly restricted from shaping. Therefore, an effective approach for maximizing bending is to keep the composite in a narrow strip configuration so that Gaussian curvature can remain constant during shaping. This represents a fundamental limitation for many applications where self-shaped double-curved structures could be beneficial, e.g. in timber architecture. In this study, we propose to achieve double-curvature by gridshell configurations of narrow self-shaping wood bilayer strips. Using numerical mechanical simulations, we investigate a parametric phase-space of shaping. Our results show that double curvature can be achieved and that the change in Gaussian curvature is dependent on the system's geometry. Furthermore, we discuss a novel architectural application potential in the form of self-erecting timber gridshells.

Functional principles of baobab fruit pedicels - anatomy and biomechanics.

BACKGROUND AND AIMS: Fruit pedicels have to deal with increasing loads after pollination due to continuous growth of the fruits. Thus, they represent interesting tissues from a mechanical as well as a developmental point of view. However, only a few studies exist on fruit pedicels. In this study, we unravel the anatomy and structural-mechanical relationships of the pedicel of Adansonia digitata, reaching up to 90 cm in length. METHODS: Morphological and anatomical analyses included examination of stained cross-sections from various positions along the stalk as well as X-ray microtomography and scanning electron microscopy. For mechanical testing, fibre bundles derived from the mature pedicels were examined via tension tests. For establishing the structural-mechanical relationships, the density of the fibre bundles as well as their cellulose microfibril distribution and chemical composition were analysed. KEY RESULTS: While in the peduncle the vascular tissue and the fibres are arranged in a concentric ring-like way, this organization shifts to the polystelic structure of separate fibre bundles in the pedicel. The polystelic pedicel possesses five vascular strands that consist of strong bast fibre bundles. The fibre bundles have a Young's modulus of up to 5 GPa, a tensile strength of up to 400 MPa, a high density (>1 g cm-3) and a high microfibril angle of around 20°. CONCLUSIONS: The structural arrangement as well as the combination of high density and high microfibril angle of the bast fibre bundles are probably optimized for bearing considerable strain in torsion and bending while at the same time allowing for carrying high-tension loads.

A single touch can provide sufficient mechanical stimulation to trigger Venus flytrap closure.

The carnivorous Venus flytrap catches prey by an ingenious snapping mechanism. Based on work over nearly 200 years, it has become generally accepted that two touches of the trap's sensory hairs within 30 s, each one generating an action potential, are required to trigger closure of the trap. We developed an electromechanical model, which, however, suggests that under certain circumstances one touch is sufficient to generate two action potentials. Using a force-sensing microrobotic system, we precisely quantified the sensory-hair deflection parameters necessary to trigger trap closure and correlated them with the elicited action potentials in vivo. Our results confirm the model's predictions, suggesting that the Venus flytrap may be adapted to a wider range of prey movements than previously assumed.

CELLULOSE SYNTHASE INTERACTING 1 is required for wood mechanics and leaf morphology in aspen.

Cellulose microfibrils synthesized by CELLULOSE SYNTHASE COMPLEXES (CSCs) are the main load-bearing polymers in wood. CELLULOSE SYNTHASE INTERACTING1 (CSI1) connects CSCs with cortical microtubules, which align with cellulose microfibrils. Mechanical properties of wood are dependent on cellulose microfibril alignment and structure in the cell walls, but the molecular mechanism(s) defining these features is unknown. Herein, we investigated the role of CSI1 in hybrid aspen (Populus tremula × Populus tremuloides) by characterizing transgenic lines with significantly reduced CSI1 transcript abundance. Reduction in leaves (50-80%) caused leaf twisting and misshaped pavement cells, while reduction (70-90%) in developing xylem led to impaired mechanical wood properties evident as a decrease in the elastic modulus and rupture. X-ray diffraction measurements indicate that microfibril angle was not impacted by the altered CSI1 abundance in developing wood fibres. Instead, the augmented wood phenotype of the transgenic trees was associated with a reduced cellulose degree of polymerization. These findings establish a function for CSI1 in wood mechanics and in defining leaf cell shape. Furthermore, the results imply that the microfibril angle in wood is defined by CSI1 independent mechanism(s).

Cell wall structure and composition is affected by light quality in tomato seedlings.

Light affects many aspects of cell development. Tomato seedlings growing at different light qualities (white, blue, green, red, far-red) and in the dark displayed alterations in cell wall structure and composition. A strong and negative correlation was found between cell wall thickness and hypocotyl growth. Cell walls was thicker under blue and white lights and thinner under far-red light and in the dark, while intermediate values was observed for red or green lights. Additionally, the inside layer surface of cell wall presented random deposited microfibrillae angles under far-red light and in the dark. However, longitudinal transmission electron microscopy indicates a high frequency of microfibrils close to parallels related to the elongation axis in the outer layer. This was confirmed by ultra-high resolution small angle X-ray scattering. These data suggest that cellulose microfibrils would be passively reoriented in the longitudinal direction. As the cell expands, the most recently deposited layers (inside) behave differentially oriented compared to older (outer) layers in the dark or under FR lights, agreeing with the multinet growth hypothesis. High Ca and pectin levels were found in the cell wall of seedlings growing under blue and white light, also contributing to the low extensibility of the cell wall. Low Ca and pectin contents were found in the dark and under far-red light. Auxins marginally stimulated growth in thin cell wall circumstances. Hypocotyl growth was stimulated by gibberellins under blue light.

Kinematics Governing Mechanotransduction in the Sensory Hair of the Venus flytrap.

Insects fall prey to the Venus flytrap (Dionaea muscipula) when they touch the sensory hairs located on the flytrap lobes, causing sudden trap closure. The mechanical stimulus imparted by the touch produces an electrical response in the sensory cells of the trigger hair. These cells are found in a constriction near the hair base, where a notch appears around the hair's periphery. There are mechanosensitive ion channels (MSCs) in the sensory cells that open due to a change in membrane tension; however, the kinematics behind this process is unclear. In this study, we investigate how the stimulus acts on the sensory cells by building a multi-scale hair model, using morphometric data obtained from µ-CT scans. We simulated a single-touch stimulus and evaluated the resulting cell wall stretch. Interestingly, the model showed that high stretch values are diverted away from the notch periphery and, instead, localized in the interior regions of the cell wall. We repeated our simulations for different cell shape variants to elucidate how the morphology influences the location of these high-stretch regions. Our results suggest that there is likely a higher mechanotransduction activity in these 'hotspots', which may provide new insights into the arrangement and functioning of MSCs in the flytrap.

Ethylene Signaling Is Required for Fully Functional Tension Wood in Hybrid Aspen.

Tension wood (TW) in hybrid aspen trees forms on the upper side of displaced stems to generate a strain that leads to uplifting of the stem. TW is characterized by increased cambial growth, reduced vessel frequency and diameter, and the presence of gelatinous, cellulose-rich (G-)fibers with its microfibrils oriented parallel to the fiber cell axis. Knowledge remains limited about the molecular regulators required for the development of this special xylem tissue with its characteristic morphological, anatomical, and chemical features. In this study, we use transgenic, ethylene-insensitive (ETI) hybrid aspen trees together with time-lapse imaging to show that functional ethylene signaling is required for full uplifting of inclined stems. X-ray diffraction and Raman microspectroscopy of TW in ETI trees indicate that, although G-fibers form, the cellulose microfibril angle in the G-fiber S-layer is decreased, and the chemical composition of S- and G-layers is altered than in wild-type TW. The characteristic asymmetric growth and reduction of vessel density is suppressed during TW formation in ETI trees. A genome-wide transcriptome profiling reveals ethylene-dependent genes in TW, related to cell division, cell wall composition, vessel differentiation, microtubule orientation, and hormone crosstalk. Our results demonstrate that ethylene regulates transcriptional responses related to the amount of G-fiber formation and their properties (chemistry and cellulose microfibril angle) during TW formation. The quantitative and qualitative changes in G-fibers are likely to contribute to uplifting of stems that are displaced from their original position.

Analysis of hygroscopic self-shaping wood at large scale for curved mass timber structures.

The growing timber manufacturing industry faces challenges due to increasing geometric complexity of architectural designs. Complex and structurally efficient curved geometries are nowadays easily designed but still involve intensive manufacturing and excessive machining. We propose an efficient form-giving mechanism for large-scale curved mass timber by using bilayered wood structures capable of self-shaping by moisture content changes. The challenge lies in the requirement of profound material knowledge for analysis and prediction of the deformation in function of setup and boundary conditions. Using time- and moisture-dependent mechanical simulations, we demonstrate the contributions of different wood-specific deformation mechanisms on the self-shaping of large-scale elements. Our results outline how to address problems such as shape prediction, sharp moisture gradients, and natural variability in material parameters in light of an efficient industrial manufacturing.

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.

Modeling and design of thin bending wooden bilayers.

In recent architectural research, thin wooden bilayer laminates capable of self-actuation in response to humidity changes have been proposed as sustainable, programmed, and fully autonomous elements for facades or roofs for shading and climate regulation. Switches, humidistats, or motor elements represent further promising applications. Proper wood-adapted prediction models for actuation, however, are still missing. Here, a simple model that can predict bending deformation as a function of moisture content change, wood material parameters, and geometry is presented. We consider material anisotropy and moisture-dependency of elastic mechanical parameters. The model is validated using experimental data collected on bilayers made out of European beech wood. Furthermore, we present essential design aspects in view of facilitated industrial applications. Layer thickness, thickness-ratio, and growth ring angle of the wood in single layers are assessed by their effect on curvature, stored elastic energy, and generated axial stress. A sensitivity analysis is conducted to identify primary curvature-impacting model input parameters.

Downregulating aspen xylan biosynthetic GT43 genes in developing wood stimulates growth via reprograming of the transcriptome.

Xylan is one of the main compounds determining wood properties in hardwood species. The xylan backbone is thought to be synthesized by a synthase complex comprising two members of the GT43 family. We downregulated all GT43 genes in hybrid aspen (Populus tremula × tremuloides) to understand their involvement in xylan biosynthesis. All three clades of the GT43 family were targeted for downregulation using RNA interference individually or in different combinations, either constitutively or specifically in developing wood. Simultaneous downregulation in developing wood of the B (IRX9) and C (IRX14) clades resulted in reduced xylan Xyl content relative to reducing end sequence, supporting their role in xylan backbone biosynthesis. This was accompanied by a higher lignocellulose saccharification efficiency. Unexpectedly, GT43 suppression in developing wood led to an overall growth stimulation, xylem cell wall thinning and a shift in cellulose orientation. Transcriptome profiling of these transgenic lines indicated that cell cycling was stimulated and secondary wall biosynthesis was repressed. We suggest that the reduced xylan elongation is sensed by the cell wall integrity surveying mechanism in developing wood. Our results show that wood-specific suppression of xylan-biosynthetic GT43 genes activates signaling responses, leading to increased growth and improved lignocellulose saccharification.

Ethylene signaling induces gelatinous layers with typical features of tension wood in hybrid aspen.

The phytohormone ethylene impacts secondary stem growth in plants by stimulating cambial activity, xylem development and fiber over vessel formation. We report the effect of ethylene on secondary cell wall formation and the molecular connection between ethylene signaling and wood formation. We applied exogenous ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) to wild-type and ethylene-insensitive hybrid aspen trees (Populus tremula × tremuloides) and studied secondary cell wall anatomy, chemistry and ultrastructure. We furthermore analyzed the transcriptome (RNA Seq) after ACC application to wild-type and ethylene-insensitive trees. We demonstrate that ACC and ethylene induce gelatinous layers (G-layers) and alter the fiber cell wall cellulose microfibril angle. G-layers are tertiary wall layers rich in cellulose, typically found in tension wood of aspen trees. A vast majority of transcripts affected by ACC are downstream of ethylene perception and include a large number of transcription factors (TFs). Motif-analyses reveal potential connections between ethylene TFs (Ethylene Response Factors (ERFs), ETHYLENE INSENSITIVE 3/ETHYLENE INSENSITIVE3-LIKE1 (EIN3/EIL1)) and wood formation. G-layer formation upon ethylene application suggests that the increase in ethylene biosynthesis observed during tension wood formation is important for its formation. Ethylene-regulated TFs of the ERF and EIN3/EIL1 type could transmit the ethylene signal.

A close-up view of the wood cell wall ultrastructure and its mechanics at different cutting angles by atomic force microscopy.

MAIN CONCLUSION: AFM measurements on spruce sample cross-sections reveal that the structural appearance of the S2 layer changes from a network structure to a concentric lamellar texture depending on the cutting angle. The structural assembly of wood constituents within the secondary cell wall has been subject of numerous studies over the last decades, which has resulted in contradicting models on the spatial arrangement and orientation of the wood macromolecules. Here, we use multichannel atomic force microscopy by means of quantitative imaging, to gain new insights into the macromolecular assembly. Cross-sections of spruce wood, which had been cut at different angles ranging from 0° to 30° were investigated. Strikingly, depending on the cutting angle, the structural appearance of the S2 layer changed from a network-like structure to a distinct concentric lamellar texture. This makes us conclude that the often visualized lamellar organization of the secondary cell wall is not the consequence of a continuous inherent ring pattern, but rather a result of the specific surface cross-section appearance of cellulose aggregates at larger cutting angles. By analyzing the recorded force distance curves in every pixel, a nano-mechanical characterization of the secondary cell wall was conducted. Substantially lower indentation modulus values were obtained compared to nanoindentation values reported in the literature. This is potentially due to a smaller interaction volume of the probe with a by far less deep indentation.

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.