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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Transcriptional and Hormonal Regulation of Gravitropism of Woody Stems in Populus.

Angiosperm trees reorient their woody stems by asymmetrically producing a specialized xylem tissue, tension wood, which exerts a strong contractile force resulting in negative gravitropism of the stem. Here, we show, in Populus trees, that initial gravity perception and response occurs in specialized cells through sedimentation of starch-filled amyloplasts and relocalization of the auxin transport protein, PIN3. Gibberellic acid treatment stimulates the rate of tension wood formation and gravibending and enhances tissue-specific expression of an auxin-responsive reporter. Gravibending, maturation of contractile fibers, and gibberellic acid (GA) stimulation of tension wood formation are all sensitive to transcript levels of the Class I KNOX homeodomain transcription factor-encoding gene ARBORKNOX2 (ARK2). We generated genome-wide transcriptomes for trees in which gene expression was perturbed by gravistimulation, GA treatment, and modulation of ARK2 expression. These data were employed in computational analyses to model the transcriptional networks underlying wood formation, including identification and dissection of gene coexpression modules associated with wood phenotypes, GA response, and ARK2 binding to genes within modules. We propose a model for gravitropism in the woody stem in which the peripheral location of PIN3-expressing cells relative to the cambium results in auxin transport toward the cambium in the top of the stem, triggering tension wood formation, while transport away from the cambium in the bottom of the stem triggers opposite wood formation.

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.

Overexpression of Populus trichocarpa CYP85A3 promotes growth and biomass production in transgenic trees.

Brassinosteroids (BRs) are essential hormones that play crucial roles in plant growth, reproduction and response to abiotic and biotic stress. In Arabidopsis, AtCYP85A2 works as a bifunctional cytochrome P450 monooxygenase to catalyse the conversion of castasterone to brassinolide, a final rate-limiting step in the BR-biosynthetic pathway. Here, we report the functional characterizations of PtCYP85A3, one of the three AtCYP85A2 homologous genes from Populus trichocarpa. PtCYP85A3 shares the highest similarity with AtCYP85A2 and can rescue the retarded-growth phenotype of the Arabidopsis cyp85a2-2 and tomato dx mutants. Constitutive expression of PtCYP85A3, driven by the cauliflower mosaic virus 35S promoter, increased the endogenous BR levels and significantly promoted the growth and biomass production in both transgenic tomato and poplar. Compared to the wild type, plant height, shoot fresh weight and fruit yield increased 50%, 56% and 43%, respectively, in transgenic tomato plants. Similarly, plant height and stem diameter increased 15% and 25%, respectively, in transgenic poplar plants. Further study revealed that overexpression of PtCYP85A3 enhanced xylem formation without affecting the composition of cellulose and lignin, as well as the cell wall thickness in transgenic poplar. Our finding suggests that PtCYP85A3 could be used as a potential candidate gene for engineering fast-growing trees with improved wood production.

Cell size and wall dimensions drive distinct variability of earlywood and latewood density in Northern Hemisphere conifers.

Interannual variability of wood density - an important plant functional trait and environmental proxy - in conifers is poorly understood. We therefore explored the anatomical basis of density. We hypothesized that earlywood density is determined by tracheid size and latewood density by wall dimensions, reflecting their different functional tasks. To determine general patterns of variability, density parameters from 27 species and 349 sites across the Northern Hemisphere were correlated to tree-ring width parameters and local climate. We performed the same analyses with density and width derived from anatomical data comprising two species and eight sites. The contributions of tracheid size and wall dimensions to density were disentangled with sensitivity analyses. Notably, correlations between density and width shifted from negative to positive moving from earlywood to latewood. Temperature responses of density varied intraseasonally in strength and sign. The sensitivity analyses revealed tracheid size as the main determinant of earlywood density, while wall dimensions become more influential for latewood density. Our novel approach of integrating detailed anatomical data with large-scale tree-ring data allowed us to contribute to an improved understanding of interannual variations of conifer growth and to illustrate how conifers balance investments in the competing xylem functions of hydraulics and mechanical support.

The Contribution of Carbon and Water in Modulating Wood Formation in Black Spruce Saplings.

Nonstructural carbohydrates (NSCs) play a crucial role in xylem formation and represent, with water, the main constraint to plant growth. We assessed the relationships between xylogenesis and NSCs in order to (1) verify the variance explained by NSCs and (2) determine the influence of intrinsic (tissue supplying carbon) and extrinsic (water availability and temperature) factors. During 2 years, wood formation was monitored in saplings of black spruce (Picea mariana) subjected to a dry period of about 1 month in June and exposed to different temperature treatments in a greenhouse. In parallel, NSC concentrations were determined by extracting the sugar compounds from two tissues (cambium and inner xylem), both potentially supplying carbon for wood formation. A mixed-effect model was used to assess and quantify the potential relationships. Total xylem cells, illustrating meristematic activity, were modeled as a function of water, sucrose, and d-pinitol (conditional r(2) of 0.79). Water availability was ranked as the most important factor explaining total xylem cell production, while the contribution of carbon was lower. Cambium stopped dividing under water deficit, probably to limit the number of cells remaining in differentiation without an adequate amount of water. By contrast, carbon factors were ranked as most important in explaining the variation in living cells (conditional r(2) of 0.49), highlighting the functional needs during xylem development, followed by the tissue supplying the NSCs (cambium) and water availability. This study precisely demonstrates the role of carbon and water in structural growth expressed as meristematic activity and tissue formation.

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.

Agatharesinol biosynthesis-related changes of ray parenchyma in sapwood sticks of Cryptomeria japonica during cell death.

MAIN CONCLUSION: The work demonstrates a relationship between the biosynthesis of the secondary metabolite, agatharesinol, and cytological changes that occur in ray parenchyma during cell death in sapwood sticks of Cryptomeria japonica under humidity-regulated conditions. To characterize the death of ray parenchyma cells that accompanies the biosynthesis of secondary metabolites, we examined cell death in sapwood sticks of Cryptomeria japonica under humidity-regulated conditions. We monitored features of ray parenchyma cells, such as viability, the morphology of nuclei and vacuoles, and the amount of starch grains. In addition, we analyzed levels of agatharesinol, a heartwood norlignan, by gas chromatography-mass spectrometry in the same sapwood sticks. Dramatic changes in the amount of starch grains and in the level of agatharesinol occurred simultaneously. Therefore, the biosynthesis of agatharesinol appeared to originate from the breakdown of starch. Furthermore, we observed the expansion of vacuoles in ray parenchyma cells prior to other cytological changes at the final stage of cell death. In our experimental system, we were able to follow the process of cell death and to demonstrate relationships between cytological changes and the biosynthesis of a secondary metabolite during the death of ray parenchyma cells.

Aspen Tension Wood Fibers Contain ß-(1---> 4)-Galactans and Acidic Arabinogalactans Retained by Cellulose Microfibrils in Gelatinous Walls.

Contractile cell walls are found in various plant organs and tissues such as tendrils, contractile roots, and tension wood. The tension-generating mechanism is not known but is thought to involve special cell wall architecture. We previously postulated that tension could result from the entrapment of certain matrix polymers within cellulose microfibrils. As reported here, this hypothesis was corroborated by sequential extraction and analysis of cell wall polymers that are retained by cellulose microfibrils in tension wood and normal wood of hybrid aspen (Populus tremula × Populus tremuloides). ß-(1→4)-Galactan and type II arabinogalactan were the main large matrix polymers retained by cellulose microfibrils that were specifically found in tension wood. Xyloglucan was detected mostly in oligomeric form in the alkali-labile fraction and was enriched in tension wood. ß-(1→4)-Galactan and rhamnogalacturonan I backbone epitopes were localized in the gelatinous cell wall layer. Type II arabinogalactans retained by cellulose microfibrils had a higher content of (methyl)glucuronic acid and galactose in tension wood than in normal wood. Thus, ß-(1→4)-galactan and a specialized form of type II arabinogalactan are trapped by cellulose microfibrils specifically in tension wood and, thus, are the main candidate polymers for the generation of tensional stresses by the entrapment mechanism. We also found high ß-galactosidase activity accompanying tension wood differentiation and propose a testable hypothesis that such activity might regulate galactan entrapment and, thus, mechanical properties of cell walls in tension wood.

Genome analysis of Daldinia eschscholtzii strains UM 1400 and UM 1020, wood-decaying fungi isolated from human hosts.

BACKGROUND: Daldinia eschscholtzii is a wood-inhabiting fungus that causes wood decay under certain conditions. It has a broad host range and produces a large repertoire of potentially bioactive compounds. However, there is no extensive genome analysis on this fungal species. RESULTS: Two fungal isolates (UM 1400 and UM 1020) from human specimens were identified as Daldinia eschscholtzii by morphological features and ITS-based phylogenetic analysis. Both genomes were similar in size with 10,822 predicted genes in UM 1400 (35.8 Mb) and 11,120 predicted genes in UM 1020 (35.5 Mb). A total of 751 gene families were shared among both UM isolates, including gene families associated with fungus-host interactions. In the CAZyme comparative analysis, both genomes were found to contain arrays of CAZyme related to plant cell wall degradation. Genes encoding secreted peptidases were found in the genomes, which encode for the peptidases involved in the degradation of structural proteins in plant cell wall. In addition, arrays of secondary metabolite backbone genes were identified in both genomes, indicating of their potential to produce bioactive secondary metabolites. Both genomes also contained an abundance of gene encoding signaling components, with three proposed MAPK cascades involved in cell wall integrity, osmoregulation, and mating/filamentation. Besides genomic evidence for degrading capability, both isolates also harbored an array of genes encoding stress response proteins that are potentially significant for adaptation to living in the hostile environments. CONCLUSIONS: Our genomic studies provide further information for the biological understanding of the D. eschscholtzii and suggest that these wood-decaying fungi are also equipped for adaptation to adverse environments in the human host.