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Betula platyphylla Suk (birch) is an excellent short-term hardwood species with growth and wood characteristics well suited to wood industries. To investigate the molecular mechanism of wood development in birch, a tension wood (TW) induced system was used to explore the regulatory mechanism at the protein level and identify the key proteins involved in xylem development in birch. The results of dyeing with Safranin O-Fast Green indicated that the cellulose content of TW was significantly higher than that of opposite wood (OW) or normal wood (NW), and the lignin content in TW was significantly lower than that in OW and NW after artificial bending of birch stems. Protein profile analysis of TW, NW and OW by iTRAQ revealed that there were 639 and 460 differentially expressed proteins (DEPs) between TW/OW and TW/NW, respectively. The DEPs were mainly enriched in tyrosine metabolism, glycolysis/gluconeogenesis, phenylalanine and tyrosine metabolism, phenylpropanoid and pyruvate metabolism, the pentose phosphate pathway, the citrate cycle (TCA cycle), fructose and mannose metabolism, carbon fixation in photosynthetic organisms, fatty acid biosynthesis, photosynthesis proteins and other pathways. The proteins in the citrate cycle were upregulated. The expression levels of PGI, PGM and FRK proteins related to cellulose synthesis increased and the expression levels of PAL, 4CL and COMT related to lignin synthesis decreased, leading to an increase in cellulose content and decreased lignin levels in TW. PPI analysis revealed that key DEPs interact with each other, indicating that these proteins form complexes to implement this function, which may provide important insights for wood formation at the molecular level.

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A cell wall determines the mechanical properties of a cell, serves as a barrier against plant stresses, and allows cell division and growth processes. The COBRA-Like (COBL) gene family encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein that controls cellulose deposition and cell progression in plants by contributing to the microfibril orientation of a cell wall. Despite being studied in different plant species, there is a dearth of the comprehensive global analysis of COBL genes in poplar. Poplar is employed as a model woody plant to study abiotic stresses and biomass production in tree research. Improved genome resequencing has enabled the comprehensive exploration of the evolution and functional capacities of PtrCOBLs (Poplar COBRA-Like genes) in poplar. Phylogeny analysis has discerned and classified PtrCOBLs into two groups resembling the Arabidopsis COBL family, and group I genes possess longer proteins but have fewer exons than group II. Analysis of gene structure and motifs revealed PtrCOBLs maintained a rather stable motif and exon-intron pattern across members of the same group. Synteny and collinearity analyses exhibited that the evolution of the COBL gene family was heavily influenced by gene duplication events. PtrCOBL genes have undergone both segmental duplication and tandem duplication, followed by purifying selection. Promotor analysis flaunted various phytohormone-, growth- and stress-related cis-elements (e.g., MYB, ABA, MeJA, SA, AuxR, and ATBP1). Likewise, 29 Ptr-miRNAs of 20 families were found targeting 11 PtrCOBL genes. PtrCOBLs were found localized at the plasma membrane and extracellular matrix, while gene ontology analysis showed their involvement in plant development, plant growth, stress response, cellulose biosynthesis, and cell wall biogenesis. RNA-seq datasets depicted the bulk of PtrCOBL genes expression being found in plant stem tissues and leaves, rendering mechanical strength and rejoinders to environmental cues. PtrCOBL2, 3, 10, and 11 manifested the highest expression in vasculature and abiotic stress, and resemblant expression trends were upheld by qRT-PCR. Co-expression network analysis identified PtrCOBL2 and PtrCOBL3 as hub genes across all abiotic stresses and wood developing tissues. The current study reports regulating roles of PtrCOBLs in xylem differentiating tissues, tension wood formation, and abiotic stress latency that lay the groundwork for future functional studies of the PtrCOBL genes in poplar breeding.

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BACKGROUND: To gain a better understanding of bark layer structure and function, especially of the phloem fibres and their contribution to the posture control of trees, it is important to map the structural properties of these cells. The role of bark can also be linked to the reaction wood formation and properties which are essential when it comes to studying the questions related to tree growth. To offer new insights into the role of bark in the postural control of trees, we studied the micro- and nanoscale structures of the phloem and its nearest layers. This study is the first time, in which phloem fibres in trees have been extensively examined using X-ray diffraction (XRD). We determined the orientation of cellulose microfibrils in phloem fibres of Silver birch saplings by using scanning synchrotron nanodiffraction. The samples consisted of phloem fibres extracted from tension, opposite and normal wood (TW, OW, NW). RESULTS: Using scanning XRD, we were able to obtain new information about the mean microfibril angle (MFA) in cellulose microfibrils in phloem fibres connected to reaction wood. A slight but consistent difference was detected in the average MFA values of phloem fibres between the TW and OW sides of the stem. Using scanning XRD, different contrast agents (intensity of the main cellulose reflection or calcium oxalate reflection, mean MFA value) were used to produce 2D images with 200 nm spatial resolution. CONCLUSIONS: Based on our results, the tension wood formation in the stem might be related to the structure and properties of phloem fibres. Thus, our results suggest that the nanostructure of phloem fibres is involved in the postural control of trees containing tension and opposite wood.

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To maintain or adjust posture under the challenges of gravity and increased self-weight, or the effects of light, snow, and slope, plants have the ability to develop a special type of tissue called reaction tissue. The formation of reaction tissue is a result of plant evolution and adaptation. The identification and study of plant reaction tissue are of great significance for understanding the systematics and evolution of plants, the processing and utilization of plant-based materials, and the exploration of new biomimetic materials and biological templates. Trees' reaction tissues have been studied for many years, and recently, many new findings regarding these tissues have been reported. However, reaction tissue requires further detailed exploration, particularly due to their complex and diverse nature. Moreover, the reaction tissues in gymnosperms, vines, herbs, etc., which display unique biomechanical behavior, have also garnered the attention of research. After summarizing the existing literature, this paper provides an outline of the reaction tissues in woody plants and non-woody plants, and lays emphasis on alternations in the cell wall structure of the xylem in softwood and hardwood. The purpose of this paper is to provide a reference for the further exploration and study of reaction tissues with great diversity.

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In a response to gravitropic stress, G-layers (gelatinous layers) were deposited in xylem cell walls of tilted flax plants. G-layers were produced in both tension wood (upper side) as expected but were also observed in opposite wood (lower side). Raman spectral profiles were acquired for xylem G-layers from the tension and opposite side as well as from the G-layer of bast fibers grown under non-tilted conditions. Statistical analysis by principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) clearly distinguished bast fiber G-layers from xylem G-layers. Discriminating bands were observed for cellulose (380-1150-1376 cm-1), hemicelluloses (517-1094-1126-1452 cm-1) and aromatics (1270-1599-1658 cm-1). PCA did not allow separation of G-layers from tension/opposite-wood sides. In contrast, the two types of xylem G-layers could be incompletely discriminated through PLS-DA. Overall, the results suggested that while the architecture (polymer spatial distribution) of bast fibers G-layers and xylem G-layers are similar, they should be considered as belonging to a different cell wall layer category based upon ontogenetical and chemical composition parameters.

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Gelatinous fibers (G-fibers) are specialized contractile cells found in a diversity of vascular plant tissues, where they provide mechanical support and/or facilitate plant mobility. G-fibers are distinct from typical fibers by the presence of an innermost thickened G-layer, comprised mainly of axially oriented cellulose microfibrils. Despite the disparate developmental origins-tension wood fibers from the vascular cambium or primary phloem fibers from the procambium-G-fiber development, composition, and molecular signatures are remarkably similar; however, important distinctions do exist. Here, we synthesize current knowledge of the phylogenetic diversity, compositional makeup, and the molecular profiles that characterize G-fiber development and highlight open questions for future investigation.

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BACKGROUND: Interest on the use of short rotation willow as a lignocellulose resource for liquid transport fuels has increased greatly over the last 10 years. Investigations have shown the advantages and potential of using Salix spp. for such fuels but have also emphasized the wide variations existing in the compositional structure between different species and genotypes in addition to their effects on overall yield. The present work studied the importance of tension wood (TW) as a readily available source of glucose in 2-year-old stems of four Salix clones (Tora, Björn, Jorr, Loden). Studies involved application of a novel approach whereby TW-glucose and residual sugars and lignin were quantified using stem cross sections with results correlated with HPLC analyses of milled wood. Compositional analyses were made for four points along stems and glucose derived from enzyme saccharification of TW gelatinous (G) layers (G-glucose), structural cell wall glucose (CW-glucose) remaining after saccharification and total glucose (T-glucose) determined both theoretically and from HPLC analyses. Comparisons were also made between presence of other characteristic sugars as well as acid-soluble and -insoluble lignin. RESULTS: Preliminary studies showed good agreement between using stem serial sections and milled powder from Salix stems for determining total sugar and lignin. Therefore, sections were used throughout the work. HPLC determination of T-glucose in Salix clones varied between 47.1 and 52.8%, showing a trend for higher T-glucose with increasing height (Björn, Tora and Jorr). Using histochemical/microscopy and image analysis, Tora (24.2%) and Björn (28.2%) showed greater volumes of % TW than Jorr (15.5%) and Loden (14.0%). Total G-glucose with enzyme saccharification of TW G-layers varied between 3.7 and 14.7% increasing as the total TW volume increased. CW-glucose measured after enzyme saccharification showed mean values of 41.9-49.1%. Total lignin between and within clones showed small differences with mean variations of 22.4-22.8% before and 22.4-24.3% after enzyme saccharification. Calculated theoretical and quantified values for CW-glucose at different heights for clones were similar with strong correlation: T-glucose = G-glucose + CW-glucose. Pearson's correlation displayed a strong and positive correlation between T-glucose and G-glucose, % TW and stem height, and between G-glucose with % TW and stem height. CONCLUSIONS: The use of stem cross sections to estimate TW together with enzyme saccharification represents a viable approach for determining freely available G-glucose from TW allowing comparisons between Salix clones. Using stem sections provides for discrete morphological/compositional tissue comparisons between clones with results consistent with traditional wet chemical analysis approaches where entire stems are milled and analyzed. The four clones showed variable TW and presence of total % G-glucose in the order Björn > Tora > Jorr > Loden. Calculated in terms of 1 m3, Salix stems Tora and Björn would contain ca. 0.24 and 0.28 m3 of tension wood representing a significant amount of freely available glucose.

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Tension wood (TW) is a specialized xylem tissue formed in angiosperm trees under gravitational stimulus or mechanical stresses (e.g., bending). The genetic regulation that underlies this important mechanism remains poorly understood. Here, we used laser capture microdissection of stem xylem cells coupled with full transcriptome RNA-sequencing to analyze TW formation in Populus trichocarpa. After tree bending, PtrLBD39 was the most significantly induced transcription factor gene; it has a phylogenetically paired homolog, PtrLBD22. CRISPR-based knockout of PtrLBD39/22 severely inhibited TW formation, reducing cellulose and increasing lignin content. Transcriptomic analyses of CRISPR-based PtrLBD39/22 double mutants showed that these two genes regulate a set of TW-related genes. Chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify direct targets of PtrLBD39. We integrated transcriptomic analyses and ChIP-seq assays to construct a transcriptional regulatory network (TRN) mediated by PtrLBD39. In this TRN, PtrLBD39 directly regulates 26 novel TW-responsive transcription factor genes. Our work suggests that PtrLBD39 and PtrLBD22 specifically control TW formation by mediating a TW-specific TRN in Populus.

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Lignin is a complex polymer in plant cell walls whose proportion is second only to that of cellulose and plays an important role in the mechanical properties of wood and stress resistance of plants. Here, we induced tension wood (TW) formation in Catalpa bungei by artificial bending and analyzed the lignin metabolism of the TW. LC-MS analysis showed that a significantly higher content of coniferyl aldehyde was observed in the TW cell wall than in the opposite wood (OW) and normal wood (NW) cell walls. TW had significantly lower contents of coniferyl alcohol than OW and NW. Raman spectroscopy results indicated that TW had lower total lignin than OW and NW. The transcription and translation levels of most of the differentially expressed genes (DEGs) involved in lignin monomer biosynthesis indicated upregulation in TW/OW and TW/NW. We found no significant difference in the transcription levels of three collision gases (CADs) between TW and OW or between NW, but their translation levels were significantly downregulated in TW, suggesting post-transcriptional control for CAD. We predicted and analyzed transcription factors that could target DEGs involved in lignin monomer biosynthesis in TW. Based on the analysis of the relationships of targeting and coexpression, we found that NAC (evm.model.group1.695) could potentially target 4CLs and CCoAOMT, that HD-Zip (evm.model.group7.1157) had potential targeting relationships with CCoAOMT, F5H, and CCR, and that their expression levels were significantly positive. It is speculated that the upregulation of NAC and HD-ZIP transcription factors activates the expression of downstream target genes, which leads to a significant increase in coniferyl aldehyde in TW. However, the decrease in total lignin in TW may be caused by the significant downregulation of CAD translation and the significant decrease in precursors (coniferyl alcohol). Whether the expression of CAD genes is regulated by post-transcriptional control and affects TW lignin metabolism needs further study.

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Lignin in Populus species is acylated with p-hydroxybenzoate. Monolignol p-hydroxybenzoyltransferase 1 (PHBMT1) mediates p-hydroxybenzoylation of sinapyl alcohol, eventually leading to the modification of syringyl lignin subunits. Angiosperm trees upon gravistimulation undergo the re-orientation of their growth along with the production of specialized secondary xylem, i.e., tension wood (TW), that generates tensile force to pull the inclined stem or leaning branch upward. Sporadic evidence suggests that angiosperm TW contains relatively a high percentage of syringyl lignin and lignin-bound p-hydroxybenzoate. However, whether such lignin modification plays a role in gravitropic response remains unclear. By imposing mechanical bending and/or gravitropic stimuli to the hybrid aspens in the wild type (WT), lignin p-hydroxybenzoate deficient, and p-hydroxybenzoate overproduction plants, we examined the responses of plants to gravitropic/mechanical stress and their cell wall composition changes. We revealed that mechanical bending or gravitropic stimulation not only induced the overproduction of crystalline cellulose fibers and increased the relative abundance of syringyl lignin, but also significantly induced the expression of PHBMT1 and the increased accumulation of p-hydroxybenzoates in TW. Furthermore, we found that although disturbing lignin-bound p-hydroxybenzoate accumulation in the PHBMT1 knockout and overexpression (OE) poplars did not affect the major chemical composition shifts of the cell walls in their TW as occurred in the WT plants, depletion of p-hydroxybenzoates intensified the gravitropic curving of the plantlets in response to gravistimulation, evident with the enhanced stem secant bending angle. By contrast, hyperaccumulation of p-hydroxybenzoates mitigated gravitropic response. These data suggest that PHBMT1-mediated lignin modification is involved in the regulation of poplar gravitropic response and, likely by compromising gravitropism and/or enhancing autotropism, negatively coordinates the action of TW cellulose fibers to control the poplar wood deformation and plant growth.

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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.

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Plant cellulose is synthesized by a large plasma membrane-localized cellulose synthase (CesA) complex. However, an overall functional determination of secondary cell wall (SCW) CesAs is still lacking in trees, especially one based on gene knockouts. Here, the Cas9/gRNA-induced knockouts of PtrCesA4, 7A, 7B, 8A and 8B genes were produced in Populus trichocarpa. Based on anatomical, immunohistochemical and wood composition evidence, we gained a comprehensive understanding of five SCW PtrCesAs at the genetic level. Complete loss of PtrCesA4, 7A/B or 8A/B led to similar morphological abnormalities, indicating similar and nonredundant genetic functions. The absence of the gelatinous (G) layer, one-layer-walled fibres and a 90% decrease in cellulose in these mutant woods revealed that the three classes of SCW PtrCesAs are essential for multilayered SCW structure and wood G-fibre. In addition, the mutant primary and secondary phloem fibres lost the n(G + L)- and G-layers and retained the thicker S-layers (L, lignified; S, secondary). Together with polysaccharide immunolocalization data, these findings suggest differences in the role of SCW PtrCesAs-synthesized cellulose in wood and phloem fibre wall structures. Overall, this functional understanding of the SCW PtrCesAs provides further insights into the impact of lacking cellulose biosynthesis on growth, SCW, wood G-fibre and phloem fibre wall structures in the tree.

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In response to gravistimulation under anisotropic light, tree stems showing an active cambium produce reaction wood that redirects the axis of the trees. Several studies have described transcriptomic or proteomic models of reaction wood relative to the opposite wood. However, the mechanisms leading to the formation of reaction wood are difficult to decipher because so many environmental factors can induce various signalling pathways leading to this developmental reprogramming. Using an innovative isotropic device where the phototropic response does not interfere with gravistimulation we characterized the early molecular responses occurring in the stem of poplar after gravistimulation in an isotropic environment, and without deformation of the stem. After 30 min tilting at 35° under anisotropic light, we collected the upper and lower xylems from the inclined stems. Controls were collected from vertical stems. We used a microarray approach to identify differentially expressed transcripts. High-throughput real-time PCR allowed a kinetic experiment at 0, 30, 120 and 180 min after tilting at 35°, with candidate genes. We identified 668 differentially expressed transcripts, from which we selected 153 candidates for additional Fluidigm qPCR assessment. Five candidate co-expression gene clusters have been identified after the kinetic monitoring of the expression of candidate genes. Gene ontology analyses indicate that molecular reprogramming of processes such as 'wood cell expansion', 'cell wall reorganization' and 'programmed cell death' occur as early as 30 min after gravistimulation. Of note is that the change in the expression of different genes involves a fine regulation of gibberellin and brassinosteroid pathways as well as flavonoid and phosphoinositide pathways. Our experimental set-up allowed the identification of genes regulated in early gravitropic response without the bias introduced by phototropic and stem bending responses.

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Tension wood is a type of defect of wood, however, it has some especial character and structure. In this study, cellulase lignin structures in normal and tension wood of Poplar 107 (Populus × euramericana cv. '74/76') were compared using ultraviolet (UV), Fourier transform infrared (FT-IR), Raman and magnetic resonance (NMR) spectroscopy, gel permeation chromatography (GPC) and elemental analysis. The results showed that the lignins in both normal and tension wood were dominated by syringyl units, followed by guaiacyl and p-hydroxyphenyl units. The FT-IR result presented that the relative intensity of syringyl units in normal and tension wood were 0.87 and 0.90. The contents of aliphatic hydroxyl, methoxyl, condensed phenolic hydroxyl and carboxyl of lignin in tension wood were higher than those of phenolic hydroxyl groups and ß-5. The contents of ß-O-4, ß-ß and ß-1 in tension wood were similar with those in normal wood. The average per-C9-unit formulae of the lignin in normal and tension wood of Poplar 107 were C9H7.21O1.79 (OH)1.17_x0001_(OCH3)1.55 and C9H7.23O1.76(OH)1.25_x0001_(OCH3)1.62, respectively.

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BACKGROUND: Phytohormones are the key factors regulating vascular development in plants, and they are also involved in tension wood (TW) formation. Although the theory of hormone distribution in TW formation is widely supported, the effects of endogenous hormones on TW formation have not yet been assessed. In this study, TW formation was induced in Catalpa bungei by artificial bending. The phytohormone content of TW, opposite wood (OW) and normal wood (NW) was determined using liquid chromatography-mass spectrometry (LC-MS), and transcriptome sequencing was performed. The hormone content and related gene expression data were comprehensively analyzed. RESULTS: The results of analyses of the plant hormone contents indicated significantly higher levels of cis-zeatin (cZ), indoleacetic acid (IAA) and abscisic acid (ABA) in TW than in OW. Genes involved in the IAA and ABA synthesis pathways, such as ALDH (evm. MODEL: group5.1511) and UGT (evm. MODEL: scaffold36.20), were significantly upregulated in TW. and the expression levels of ARF (evm. MODEL: group5.1332), A-ARR (evm. MODEL: group0.1600), and TCH4 (evm. MODEL: group2.745), which participate in IAA, cZ and Brassinolide (BR) signal transduction, were significantly increased in TW. In particular, ARF expression may be regulated by long noncoding RNAs (lncRNAs) and the HD-ZIP transcription factor ATHB-15. CONCLUSIONS: We constructed a multiple hormone-mediated network of C. bungei TW formation based on hormone levels and transcriptional expression profiles were identified during TW formation.

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In angiosperm trees, the gelatinous layer (G-layer) takes a great part of the fiber cell wall in the tension wood (TW). However, the mechanism underlying G-layer formation in poplar is largely unknown. In this work, we demonstrate that G-layer formation in poplar TW cells is regulated by brassinosteroid (BR) and its signaling. PtiCYP85A3, a key BR biosynthesis gene, was predominantly expressed in the xylem of TW, accompanied with a relatively higher castasterone (CS) accumulation, than in the xylem of opposite wood (OW). A wider expression zone of BZR1, a key transcriptional factor in BR singling pathway, was also observed in G-fiber cells on TW side than in wood fiber cells on the OW side, as indicated by immunohistochemistry assays. Transgenic poplar plants overexpressing PtiCYP85A3 produced thicker G-layer with higher cellulose proportion, and accumulated more BZR1 protein in the xylem of TW than did the wild type (WT) plants. Expression of most TW-associated CesAs, which were induced by 2, 4-epibrassinolide, an active BR, and inhibited by brassinazole, a BR biosynthesis inhibitor, were also up-regulated in the xylem of TW in transgenic plants compared to that in WT plants. Further studies with dual-luciferase assays demonstrated that the promoters of PtiCesAs were activated by PtiMYB128, a TW specific transcription factor, which was then regulated by BZR1. All these results indicate that BR plays a crucial role in the G-layer formation of TW fiber cells by regulating the expression of BZR1, PtiMYB128, and PtiCesAs in poplar.

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: Catalpa bungei is an economically important tree with high-quality wood and highly valuable to the study of wood formation. In this work, the xylem microstructure of C. bungei tension wood (TW) was observed, and we performed transcriptomics, proteomics and Raman spectroscopy of TW, opposite wood (OW) and normal wood (NW). The results showed that there was no obvious gelatinous layer (G-layer) in the TW of C. bungei and that the secondary wall deposition in the TW was reduced compared with that in the OW and NW. We found that most of the differentially expressed mRNAs and proteins were involved in carbohydrate polysaccharide synthesis. Raman spectroscopy results indicated that the cellulose and pectin content and pectin methylation in the TW were lower than those in the OW and NW, and many genes and proteins involved in the metabolic pathways of cellulose and pectin, such as galacturonosyltransferase (GAUT), polygalacturonase (PG), endoglucanase (CLE) and ß-glucosidase (BGLU) genes, were significantly upregulated in TW. In addition, we found that the MYB2 transcription factor may regulate the pectin degradation genes PG1 and PG3, and ARF, ERF, SBP and MYB1 may be the key transcription factors regulating the synthesis and decomposition of cellulose. In contrast to previous studies on TW with a G-layer, our results revealed a change in metabolism in TW without a G-layer, and we inferred that the change in the pectin type, esterification and cellulose characteristics in the TW of C. bungei may contribute to high tensile stress. These results will enrich the understanding of the mechanism of TW formation.

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Under the effect of disturbances, like unbalanced stem, but also during normal development, poplar trees can develop a specific secondary xylem, called "tension wood" (TW), which is easily identifiable by the presence of a gelatinous layer in the secondary cell walls (SCW) of the xylem fibers. Since TW formation was mainly performed on 2-year-old poplar models, an in vitro poplar that produces gelatinous fibers (G-fibers) while offering the same experimental advantages as herbaceous plants has been developed. Using specific cell wall staining techniques, wood structural features and lignin/cellulose distribution were both detailed in cross-sections obtained from the curved stem part of in vitro poplars. A supposed delay in the SCW lignification process in the G-fibers, along with the presence of a G-layer, could be observed in the juvenile plants. Moreover, in this G-layer, the immunolabeling of various polymers carried out in the SCW of TW has allowed detecting crystalline cellulose, arabinogalactans proteins, and rhamnogalacturonans I; however, homogalacturonans, xylans, and xyloglucans could not be found. Interestingly, extensins were detected in this typical adaptative or stress-induced structure. These observations were corroborated by a quantitation of the immunorecognized polymer distribution using gold particle labeling. In conclusion, the in vitro poplar model seems highly convenient for TW studies focusing on the implementation of wall polymers that provide the cell wall with greater plasticity in adapting to the environment.

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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.