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.

Anatomical characterization and technological properties of Pterogyne nitens wood, a very interesting species of the Brazilian Caatinga biome.

Pterogyne nitens is commonly known in northeastern Brazil as a lesser-known fast-growing species in the Caatinga biome, which is a difficult place for tree development due to the low natural fertility soils and low availability of water. Due to the importance of expanding information about the anatomical wood properties of Caatinga native species, the aim of this work was to characterize the anatomical elements, to macroscopically describe the wood and make inferences about its possible end-uses. Maceration was performed which enabled measuring fiber dimensions, pore frequency and the following technological indexes: cell wall fraction, slenderness ratio, Runkel index and flexibility coefficient. Histological sections enabled describing the arrangements of the cellular elements in different observation sections and to determine the pore diameter. P. nitens wood has anatomical arrangements characterized by confluent axial parenchyma, being diffuse-porous with the presence of tylosis and heterogeneous/stratified rays (biseriate). The fibers were classified as very short (length 0.81 mm), not flexible and Runkel index 0.82. The pores were few in number with a frequency of 32.9 pores/mm2, distributed in a diffuse format and many were obstructed by tylosis. Based on the anatomical results and considering other technological studies, P. nitens wood is most suitable for charcoal production.

In-situ quantification of microscopic contributions of individual cells to macroscopic wood deformation with synchrotron computed tomography.

Wood-based composites hold the promise of sustainable construction. Understanding the influence on wood cellular microstructure in the macroscopic mechanical behavior is key for engineering high-performance composites. In this work, we report a novel Individual Cell Tracking (ICT) approach for in-situ quantification of nanometer-scale deformations of individual wood cells during mechanical loading of macroscopic millimeter-scale wood samples. Softwood samples containing > 104 cells were subjected to controlled radial tensile and longitudinal compressive load in a synchrotron radiation micro-computed tomography (SRµCT) setup. Tracheid and wood ray cells were automatically segmented, and their geometric variations were tracked during load. Finally, interactions between microstructure deformations (lumen geometry, cell wall thickness), cellular arrangement (annual growth rings, anisotropy, wood ray presence) with the macroscopic deformation response were investigated. The results provide cellular insight into macroscopic relations, such as anisotropic Poisson effects, and allow direct observation of previously suspected wood ray reinforcing effects. The method is also appropriate for investigation of non-linear deformation effects, such as buckling and deformation recovery after failure, and gives insight into less studied aspects, such as changes in lumen diameter and cell wall thickness during uniaxial load. ICT provides an experimental tool for direct validation of hierarchical mechanical models on real biological composites.

The Structural Origins of Wood Cell Wall Toughness.

The remarkable mechanical stability of wood is primarily attributed to the hierarchical fibrous arrangement of the polymeric components. While the mechanisms by which fibrous cell structure and cellulose microfibril arrangements lend stiffness and strength to wood have been intensively studied, the structural origins of the relatively high splitting fracture toughness remain unclear. This study relates cellulose microfibril arrangements to splitting fracture toughness in pine wood cell walls using in situ electron microscopy and reveals a previously unknown toughening mechanism: the specific arrangement of cellulose microfibrils in the cell wall deflects cracks from the S2 layer to the S1/S2 interface, and, once there, causes the crack to be repetitively arrested and shunted along the interface in a zig-zag path. It is suggested that this natural adaptation of wood to achieve tough interfaces and then deflect and trap cracks at them can be generalized to provide design guidelines to improve toughness of high-performance and renewable engineering materials.

Rhamnogalacturonan-I is a determinant of cell-cell adhesion in poplar wood.

The molecular basis of cell-cell adhesion in woody tissues is not known. Xylem cells in wood particles of hybrid poplar (Populus tremula × P. alba cv. INRA 717-1B4) were separated by oxidation of lignin with acidic sodium chlorite when combined with extraction of xylan and rhamnogalacturonan-I (RG-I) using either dilute alkali or a combination of xylanase and RG-lyase. Acidic chlorite followed by dilute alkali treatment enables cell-cell separation by removing material from the compound middle lamellae between the primary walls. Although lignin is known to contribute to adhesion between wood cells, we found that removing lignin is a necessary but not sufficient condition to effect complete cell-cell separation in poplar lines with various ratios of syringyl:guaiacyl lignin. Transgenic poplar lines expressing an Arabidopsis thaliana gene encoding an RG-lyase (AtRGIL6) showed enhanced cell-cell separation, increased accessibility of cellulose and xylan to hydrolytic enzyme activities, and increased fragmentation of intact wood particles into small cell clusters and single cells under mechanical stress. Our results indicate a novel function for RG-I, and also for xylan, as determinants of cell-cell adhesion in poplar wood cell walls. Genetic control of RG-I content provides a new strategy to increase catalyst accessibility and saccharification yields from woody biomass for biofuels and industrial chemicals.

An AP2/ERF transcription factor ERF139 coordinates xylem cell expansion and secondary cell wall deposition.

Differentiation of xylem elements involves cell expansion, secondary cell wall (SCW) deposition and programmed cell death. Transitions between these phases require strict spatiotemporal control. The function of Populus ERF139 (Potri.013G101100) in xylem differentiation was characterized in transgenic overexpression and dominant repressor lines of ERF139 in hybrid aspen (Populus tremula × tremuloides). Xylem properties, SCW chemistry and downstream targets were analyzed in both types of transgenic trees using microscopy techniques, Fourier transform-infrared spectroscopy, pyrolysis-GC/MS, wet chemistry methods and RNA sequencing. Opposite phenotypes were observed in the secondary xylem vessel sizes and SCW chemistry in the two different types of transgenic trees, supporting the function of ERF139 in suppressing the radial expansion of vessel elements and stimulating accumulation of guaiacyl-type lignin and possibly also xylan. Comparative transcriptomics identified genes related to SCW biosynthesis (LAC5, LBD15, MYB86) and salt and drought stress-responsive genes (ANAC002, ABA1) as potential direct targets of ERF139. The phenotypes of the transgenic trees and the stem expression profiles of ERF139 potential target genes support the role of ERF139 as a transcriptional regulator of xylem cell expansion and SCW formation, possibly in response to osmotic changes of the cells.

A study on the tubular composite with tunable compression mechanical behavior inspired by wood cell.

Biological materials have fascinating mechanical properties built up from simple basic material blocks. It is worthwhile to learn how biological materials are constructed, and to apply the knowledge in advanced manufacturing, and to realize new materials by design. In this study, we chose the tubular cell in the soft wood as a biological prototype, and tried to mimic its intelligent construction principle to regulate the compression mechanical behavior through the helical structure. First, by using the multi-material three-dimensional printing technology, we fabricated a series of tubular composites with the helix fibers of a rigid plastic embedded into an elastomeric matrix. Then, through the uniaxial compression tests, we characterized the mechanical behavior of the specimens, having different fiber angle from 0 to 50 deg at constant volume fraction. The results showed that both stiffness and fracture toughness of the printed composite could be regulated effectively by adjusting the fiber angle of the helical structure. Moreover, the helical structure with high fiber angle is able to improve the compression stability of the tubular composite with big lumen. In addition, for the biomimetic composites, the volume fraction of the reinforcements should exceed 40%. Finally, we proposed a new structural design method by combining the reinforcements of different architectures into a double-layered configuration. The intelligent strategy is proven to balance the conflict between the stiffness and toughness of the composites to some extent, and without changing in the building constituents.

Couplings in cell differentiation kinetics mitigate air temperature influence on conifer wood anatomy.

Conifer trees possess a typical anatomical tree-ring structure characterized by a transition from large and thin-walled earlywood tracheids to narrow and thick-walled latewood tracheids. However, little is known on how this characteristic structure is maintained across contrasting environmental conditions, due to its crucial role to ensure sap ascent and mechanical support. In this study, we monitored weekly wood cell formation for up to 7 years in two temperate conifer species (i.e., Picea abies (L.) Karst and Larix decidua Mill.) across an 8°C thermal gradient from 800 to 2,200 m a.s.l. in central Europe to investigate the impact of air temperature on rate and duration of wood cell formation. Results indicated that towards colder sites, forming tracheids compensate a decreased rate of differentiation (cell enlarging and wall thickening) by an extended duration, except for the last cells of the latewood in the wall-thickening phase. This compensation allows conifer trees to mitigate the influence of air temperature on the final tree-ring structure, with important implications for the functioning and resilience of the xylem to varying environmental conditions. The disappearing compensation in the thickening latewood cells might also explain the higher climatic sensitivity usually found in maximum latewood density.

Comparison of Micropore Distribution in Cell Walls of Softwood and Hardwood Xylem.

The porosity of wood cell walls is of interest for both understanding xylem functionality and from a wood materials perspective. The movement of water in xylem generally occurs through the macroporous networks formed in softwood by bordered pits and in hardwood by the intervessel pits and open conduits created by vessels and perforation plates. In some situations, such as cavitated xylem, water can only move through the micropores that occur in lignified tracheid and fiber cell walls; however, these micropore networks are poorly understood. Here, we used molecular microscopy analysis of radiata pine (Pinus radiata) and red beech (Nothofagus fusca) to determine the distribution of micropores in the secondary walls and middle lamellae of tracheids and fibers in relation to cell wall composition. Using two different types of probe, we identified a greater porosity of secondary cell walls and a reduced porosity of the middle lamella. Areas of reduced porosity were observed in the outer regions of the secondary cell wall of both tracheids and fibers that appear unrelated to lignification or the distribution of cellulose, mannan, and xylan. Hardwood fiber cell walls were less lignified than those of softwood tracheids and showed greater accessibility to porosity probes. Vessel cell walls were comparable to those of fibers in terms of both porosity and lignification. Lignification is probably the primary determinant of cell wall porosity in xylem. The highly lignified middle lamella, and lumen surface, act as a barrier to probe movement and, therefore, water movement in both softwood and hardwood.

Preliminary study of Cell Wall Structure and its Mechanical Properties of C3H and HCT RNAi Transgenic Poplar Sapling.

This research focused on the cell wall structure and its mechanical properties of down-regulated Coumaroyl shikimate 3-hydroxylase (C3H) transgenic poplar and down-regulated hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) transgenic poplar (Populus alba × P. glandulosa cv '84 k'). The wood samples with respect to microstructure, the longitudinal elastic modulus (MOE) and hardness of wood fiber secondary cell wall were investigated. The results show that the lignin contents in the two transgenic poplar woods were lower than non-modified wood. The C3H transgenic poplar and HCT transgenic poplar have more than 18.5% and 16.1% cellulose crystalline regions than non-modified poplar respectively. The diameter of the fiber cell and the vessel element of transgenic poplars are smaller. Double radial vessel cell wall thicknesses of both transgenic poplars were smaller than non-modified poplar. Cell wall ratios for the transgenic poplar were higher than non-modified poplar and cell wall density was significantly lower in both C3H and HCT transgenic poplar. The cell wall MOEs of C3H and HCT transgenic poplar was 5.8% and 7.0% higher than non-modified poplar. HCT can be more effective than C3H to modify the trees by considerably increasing mechanical properties of the cell wall.

Antifungal stilbene impregnation: transport and distribution on the micron-level.

The transition from the living water-transporting sapwood to heartwood involves in many tree species impregnation with extractives. These differ in amount and composition, and enhance resistance against bacteria, insects or fungi. To understand the synthesis, transport and impregnation processes new insights into the biochemical processes are needed by in-situ methods. Here we show the extractive distribution in pine (Pinus sylvestris) microsections with a high lateral resolution sampled in a non-destructive manner using Confocal Raman Microscopy. Integrating marker bands of stilbenes and lipids enables to clearly track the rapid change from sapwood to heartwood within one tree ring. The higher impregnation of the cell corner, compound middle lamella, the S3 layer and pits reveals the optimization of decay resistance on the micron-level. Furthermore, deposits with changing chemical composition are elucidated in the rays and lumen of the tracheids. The spectral signature of these deposits shows the co-location of lipids and pinosylvins with changing ratios from the living to the dead tissue. The results demonstrate that the extractive impregnation on the micro- and nano-level is optimized by a symbiotic relationship of lipids and pinosylvins to enhance the tree's resistance and lifetime.

Spatial specificity of auxin responses coordinates wood formation.

Spatial organization of signalling events of the phytohormone auxin is fundamental for maintaining a dynamic transition from plant stem cells to differentiated descendants. The cambium, the stem cell niche mediating wood formation, fundamentally depends on auxin signalling but its exact role and spatial organization is obscure. Here we show that, while auxin signalling levels increase in differentiating cambium descendants, a moderate level of signalling in cambial stem cells is essential for cambium activity. We identify the auxin-dependent transcription factor ARF5/MONOPTEROS to cell-autonomously restrict the number of stem cells by directly attenuating the activity of the stem cell-promoting WOX4 gene. In contrast, ARF3 and ARF4 function as cambium activators in a redundant fashion from outside of WOX4-expressing cells. Our results reveal an influence of auxin signalling on distinct cambium features by specific signalling components and allow the conceptual integration of plant stem cell systems with distinct anatomies.

Water uptake can occur through woody portions of roots and facilitates localized embolism repair in grapevine.

Water acquisition is thought to be limited to the unsuberized surface located close to root tips. However, there are recurring periods when the unsuberized surfaces are limited in woody root systems, and radial water uptake across the bark of woody roots might play an important physiological role in hydraulic functioning. Using X-ray microcomputed tomography (microCT) and hydraulic conductivity measurements (Lpr ), we examined water uptake capacity of suberized woody roots in vivo and in excised samples. Bark hydration in grapevine woody roots occurred quickly upon exposure to water (c. 4 h). Lpr measurements through the bark of woody roots showed that it is permeable to water and becomes more so upon wetting. After bark hydration, microCT analysis showed that absorbed water was utilized to remove embolism locally, where c. 20% of root xylem vessels refilled completely within 15 h. Embolism removal did not occur in control roots without water. Water uptake through the bark of woody roots probably plays an important role when unsuberized tissue is scarce/absent, and would be particularly relevant following large irrigation events or in late winter when soils are saturated, re-establishing hydraulic functionality before bud break.

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.

Regular Motifs in Xylan Modulate Molecular Flexibility and Interactions with Cellulose Surfaces.

Xylan is tightly associated with cellulose and lignin in secondary plant cell walls, contributing to its rigidity and structural integrity in vascular plants. However, the molecular features and the nanoscale forces that control the interactions among cellulose microfibrils, hemicelluloses, and lignin are still not well understood. Here, we combine comprehensive mass spectrometric glycan sequencing and molecular dynamics simulations to elucidate the substitution pattern in softwood xylans and to investigate the effect of distinct intramolecular motifs on xylan conformation and on the interaction with cellulose surfaces in Norway spruce (Picea abies). We confirm the presence of motifs with evenly spaced glycosyl decorations on the xylan backbone, together with minor motifs with consecutive glucuronation. These domains are differently enriched in xylan fractions extracted by alkali and subcritical water, which indicates their preferential positioning in the secondary plant cell wall ultrastructure. The flexibility of the 3-fold screw conformation of xylan in solution is enhanced by the presence of arabinofuranosyl decorations. Additionally, molecular dynamic simulations suggest that the glycosyl substitutions in xylan are not only sterically tolerated by the cellulose surfaces but that they increase the affinity for cellulose and favor the stabilization of the 2-fold screw conformation. This effect is more significant for the hydrophobic surface compared with the hydrophilic ones, which demonstrates the importance of nonpolar driving forces on the structural integrity of secondary plant cell walls. These novel molecular insights contribute to an improved understanding of the supramolecular architecture of plant secondary cell walls and have fundamental implications for overcoming lignocellulose recalcitrance and for the design of advanced wood-based materials.

Non-cellulosic polysaccharide distribution during G-layer formation in poplar tension wood fibers: abundance of rhamnogalacturonan I and arabinogalactan proteins but no evidence of xyloglucan.

MAIN CONCLUSION: RG-I and AGP, but not XG, are associated to the building of the peculiar mechanical properties of tension wood. Hardwood trees produce tension wood (TW) with specific mechanical properties to cope with environmental cues. Poplar TW fibers have an additional cell wall layer, the G-layer responsible for TW mechanical properties. We investigated, in two poplar hybrid species, the molecules potentially involved in the building of TW mechanical properties. First, we evaluated the distribution of the different classes of non-cellulosic polysaccharides during xylem fiber differentiation, using immunolocalization. In parallel, G-layers were isolated and their polysaccharide composition determined. These complementary approaches provided information on the occurrence of non-cellulosic polysaccharides during G-fiber differentiation. We found no evidence of the presence of xyloglucan (XG) in poplar G-layers, whereas arabinogalactan proteins (AGP) and rhamnogalacturonan type I pectins (RG-I) were abundant, with an apparent progressive loss of RG-I side chains during G-layer maturation. Similarly, the intensity of immunolabeling signals specific for glucomannans and glucuronoxylans varies during G-layer maturation. RG-I and AGP are best candidate matrix components to be responsible for TW mechanical properties.

Bordered pits in xylem of vesselless angiosperms and their possible misinterpretation as perforation plates.

Vesselless wood represents a rare phenomenon within the angiosperms, characterizing Amborellaceae, Trochodendraceae and Winteraceae. Anatomical observations of bordered pits and their pit membranes based on light, scanning and transmission electron microscopy (SEM and TEM) are required to understand functional questions surrounding vesselless angiosperms and the potential occurrence of cryptic vessels. Interconduit pit membranes in 11 vesselless species showed a similar ultrastructure as mesophytic vessel-bearing angiosperms, with a mean thickness of 245 nm (± 53, SD; n = six species). Shrunken, damaged and aspirated pit membranes, which were 52% thinner than pit membranes in fresh samples (n = four species), occurred in all dried-and-rehydrated samples, and in fresh latewood of Tetracentron sinense and Trochodendron aralioides. SEM demonstrated that shrunken pit membranes showed artificially enlarged, > 100 nm wide pores. Moreover, perfusion experiments with stem segments of Drimys winteri showed that 20 and 50 nm colloidal gold particles only passed through 2 cm long dried-and-rehydrated segments, but not through similar sized fresh ones. These results indicate that pit membrane shrinkage is irreversible and associated with a considerable increase in pore size. Moreover, our findings suggest that pit membrane damage, which may occur in planta, could explain earlier records of vessels in vesselless angiosperms.

AspWood: High-Spatial-Resolution Transcriptome Profiles Reveal Uncharacterized Modularity of Wood Formation in Populus tremula.

Trees represent the largest terrestrial carbon sink and a renewable source of ligno-cellulose. There is significant scope for yield and quality improvement in these largely undomesticated species, and efforts to engineer elite varieties will benefit from improved understanding of the transcriptional network underlying cambial growth and wood formation. We generated high-spatial-resolution RNA sequencing data spanning the secondary phloem, vascular cambium, and wood-forming tissues of Populus tremula The transcriptome comprised 28,294 expressed, annotated genes, 78 novel protein-coding genes, and 567 putative long intergenic noncoding RNAs. Most paralogs originating from the Salicaceae whole-genome duplication had diverged expression, with the exception of those highly expressed during secondary cell wall deposition. Coexpression network analyses revealed that regulation of the transcriptome underlying cambial growth and wood formation comprises numerous modules forming a continuum of active processes across the tissues. A comparative analysis revealed that a majority of these modules are conserved in Picea abies The high spatial resolution of our data enabled identification of novel roles for characterized genes involved in xylan and cellulose biosynthesis, regulators of xylem vessel and fiber differentiation and lignification. An associated web resource (AspWood, http://aspwood.popgenie.org) provides interactive tools for exploring the expression profiles and coexpression network.

Transfer of Biomatrix/Wood Cell Interactions to Hemicellulose-Based Materials to Control Water Interaction.

The family of hemicelluloses stands out as a very promising natural resource that can be utilized as a biobased materials feedstock. An in-depth understanding of the hemicellulose inherent structural and property features as well as the structure-property relationships induced by the specific supramolecular hierarchical organization of lignocellulosic biopolymers will be a key enabling technology in the emerging biorefinery sector. This Review aims to give a perspective on these issues and demonstrate how the transfer of molecular wood cell interactions into hemicellulose-based materials may offer new design principles for material formulations.