Wood Formation Research Articles

Sample preparation protocol for wood and phloem formation analyses

This technical note and its corresponding video show the procedure for optimal sample preparation to perform wood and phloem formation analyses.

Cell Fate Decisions Within the Vascular Cambium-Initiating Wood and Bast Formation.

Precise coordination of cell fate decisions is a hallmark of multicellular organisms. Especially in tissues with non-stereotypic anatomies, dynamic communication between developing cells is vital for ensuring functional tissue organization. Radial plant growth is driven by a plant stem cell niche known as vascular cambium, usually strictly producing secondary xylem (wood) inward and secondary phloem (bast) outward, two important structures serving as much-needed CO2 depositories and building materials. Because of its bidirectional nature and its developmental plasticity, the vascular cambium serves as an instructive paradigm for investigating principles of tissue patterning. Although genes and hormones involved in xylem and phloem formation have been identified, we have a yet incomplete picture of the initial steps of cell fate transitions of stem cell daughters into xylem and phloem progenitors. In this mini-review perspective, we describe two possible scenarios of cell fate decisions based on the current knowledge about gene regulatory networks and how cellular environments are established. In addition, we point out further possible research directions.

Integrated mRNA and Small RNA Sequencing Reveals microRNAs Associated With Xylem Development in Dalbergia odorifera.

Dalbergia odorifera is a rare and precious rosewood specie, whose wood is a very high-quality material for valuable furniture and carving crafts. However, limited information is available about the process of wood formation in D. odorifera. To determine genes that might be closely associated with the xylem differentiation process, we analyzed the differentially expressed genes (DEGs) and microRNAs (miRNAs) from specific xylem tissues of D. odorifera by RNA sequencing (RNA-seq) and small RNA sequencing (small RNA-seq). In total, we obtained 134,221,955 clean reads from RNA-seq and 90,940,761 clean reads from small RNA-seq. By comparing the transition zone (Dotz) and sapwood (Dosw) samples, a total of 395 DEGs were identified. Further analysis revealed that DEGs encoded for WRKY transcription factors (eight genes), lignin synthesis (PER47, COMT, CCR2), cell wall composition (UXS2), gibberellin synthesis (KAO2, GA20OX1), jasmonic acid synthesis (OPR2, CYP74A), and synthesis of flavonoids (PAL2) and terpenoids (CYP71A1). Subsequently, a preliminary analysis by small RNA-seq showed that the expressions of 14 miRNAs (such as miR168a-5p, miR167f-5p, miR167h-5p, miR167e, miR390a, miR156g, novel_52, and novel_9) were significantly different between Dotz and Dosw. Further analysis revealed that the target genes of these differentially expressed miRNAs were enriched in the GO terms “amino acid binding,” “cellulase activity,” and “DNA beta-glucosyltransferase activity”. Further, KEGG pathway annotation showed significant enrichment in “fatty acid elongation” and “biosynthesis of unsaturated fatty acids”. These processes might be participating in the xylem differentiation of D. odorifera. Next, expression correlation analysis showed that nine differentially expressed miRNAs were significantly negatively associated with 21 target genes, which encoded for proteins such as pyrH, SPL6, SPL12, GCS1, and ARF8. Overall, this is the first study on miRNAs and their potential functions in the xylem development of D. odorifera, which provides a stepping stone for a detailed functional investigation of D. odorifera miRNAs.

Variations in tension wood characteristics of Populus alba under alternate bending, nitrogen fertilization, and gibberellin treatments

Summary Reaction wood formation (tension wood) in trees such as poplar is a response to stress and environmental factors. Tension wood is a rich source of cellulose that can be used for products including paper or biofuels and is thus a target product in forestry. This study aimed to evaluate the formation of tension wood in two-year-old saplings of Populus alba by using alternate bending, nitrogen fertilization, and gibberellin hormone. Saplings were bent alternately in one or another direction every month during the growing season, fertilized twice at the beginning and in the middle of the growing season, and treated with gibberellin early in the growing season. The physical and anatomical characteristics of the wood were studied after the end of the growing season. Evaluation of transverse sections of specimens stained with safranin/Astra-blue showed that, compared with straight saplings, alternate bending saplings had a wider tension wood area in the growth ring and clear formation of a gelatinous layer. The wood of alternate bending saplings with nitrogen fertilization and gibberellin hormone had a higher wood density, greater longitudinal shrinkage, and less radial and tangential shrinkage than saplings with other treatments. Moreover, the alternate bending saplings treated with nitrogen fertilization and gibberellin hormone had tension wood with the largest vessels, the lowest vessel density, and the smallest total vessel lumen area than saplings with other treatments. Wood fibers of treated saplings also had the thickest wall with the smallest fiber and lumen diameters. Overall, the bending treatment with the addition of nitrogen fertilization and gibberellin hormone was the most effective for the stimulation of tension wood formation in terms of volume and intensity.

Integrated Degradome and Srna Sequencing Revealed miRNA-mRNA Regulatory Networks between the Phloem and Developing Xylem of Poplar.

Lignin and cellulose are the most abundant natural organic polymers in nature. MiRNAs are a class of regulatory RNAs discovered in mammals, plants, viruses, and bacteria. Studies have shown that miRNAs play a role in lignin and cellulose biosynthesis by targeting key enzymes. However, the specific miRNAs functioning in the phloem and developing xylem of Populus deltoides are still unknown. In this study, a total of 134 miRNAs were identified via high-throughput small RNA sequencing, including 132 known and two novel miRNAs, six of which were only expressed in the phloem. A total of 58 differentially expressed miRNAs (DEmiRNAs) were identified between the developing xylem and the phloem. Among these miRNAs, 21 were significantly upregulated in the developing xylem in contrast to the phloem and 37 were significantly downregulated. A total of 2431 target genes of 134 miRNAs were obtained via high-throughput degradome sequencing. Most target genes of these miRNAs were transcription factors, including AP2, ARF, bHLH, bZIP, GRAS, GRF, MYB, NAC, TCP, and WRKY genes. Furthermore, 13 and nine miRNAs were involved in lignin and cellulose biosynthesis, respectively, and we validated the miRNAs via qRT-PCR. Our study explores these miRNAs and their regulatory networks in the phloem and developing xylem of P. deltoides and provides new insight into wood formation.

Induction of compression wood inhibits development of spiral grain in radiata pine

Summary Spiral grain refers to the helical patterns formed by the wood grain in the trunks of many tree species. In most gymnosperms, grain near the pith is vertical but wood formed after several years of growth has a slight to pronounced left-handed twist. Grain changes presumably involve the slow rotation of cells within the vascular cambium, but the mechanisms that allow this reorientation to occur remain unclear. Understanding this process is, however, important as the presence of strong spiral grain within the corewood of gymnosperms is a major wood quality issue devaluing cut timber. In this study, we measured wood grain in stems of Pinus radiata (radiata pine) saplings through reconstructions of resin canals that follow the grain, visualised by serial sectioning and scanning with circularly polarised light, and through X-ray computed microtomography (μCT) and image analysis in ImageJ. Vertical trees retained a symmetrical grain pattern that was weakly right-handed near the pith, but which became progressively more left-handed during the first eight months of growth. In tilted trees, however, the development of left-handed grain was inhibited by the formation of compression wood on the lower side of the tree whereas the wood on the upper side of the tree developed increasingly more left-handed grain as in the vertical controls. These results demonstrate that a previously unidentified link exists between compression wood formation and the inhibition of grain development.

Wood Formation under Changing Environment: Omics Approaches to Elucidate the Mechanisms Driving the Early-to-Latewood Transition in Conifers

The global change scenarios highlight the urgency of clarifying the mechanisms driving the determination of wood traits in forest trees. Coniferous xylem is characterized by the alternation between earlywood (EW) and latewood (LW), on which proportions the wood density depend, one of the most important mechanical xylem qualities. However, the molecular mechanisms triggering the transition between the production of cells with the typical features of EW to the LW are still far from being completely elucidated. The increasing availability of omics resources for conifers, e.g., genomes and transcriptomes, would lay the basis for the comprehension of wood formation dynamics, boosting both breeding and gene-editing approaches. This review is intended to introduce the importance of wood formation dynamics and xylem traits of conifers in a changing environment. Then, an up-to-date overview of the omics resources available for conifers was reported, focusing on both genomes and transcriptomes. Later, an analysis of wood formation studies using omics approaches was conducted, with the aim of elucidating the main metabolic pathways involved in EW and LW determination. Finally, the future perspectives and the urgent needs on this research topic were highlighted.

Modern mannan: a hemicellulose's journey.

Hemicellulosic polysaccharides built of β-1,4-linked mannose units have been found throughout the plant kingdom and have numerous industrial applications. Here, I review recent advances in the biosynthesis and modification of plant β-mannans. These matrix polymers can associate with cellulose bundles to impact the mechanical properties of plant fibers or biocomposites. In certain algae, mannan microfibrils even replace cellulose as the dominant structural component of the cell wall. Conversely, patterned galactoglucomannan found in Arabidopsis thaliana seed mucilage significantly modulates cell wall architecture and abiotic stress tolerance despite its relatively low content. I also discuss the subcellular requirements for β-mannan biosynthesis, the increasing number of carbohydrate-active enzymes involved in this process, and the players that continue to be puzzling. I discuss how cellulose synthase-like enzymes elongate (gluco)mannans in orthogonal hosts and highlight the discoveries of plant enzymes that add specific galactosyl or acetyl decorations. Hydrolytic enzymes such as endo-β-1,4-mannanases have recently been involved in a wide range of biological contexts including seed germination, wood formation, heavy metal tolerance, and defense responses. Synthetic biology tools now provide faster tracks to modulate the increasingly-relevant mannan structures for improved plant traits and bioproducts.

Genomics-Based Systems and Multi-disciplinary Approaches to Unlock Complex Gene Networks Underlying Wood Formation

Genomics-Based Systems and Multi-disciplinary Approaches to Unlock Complex Gene Networks Underlying Wood Formation

Control of cambium initiation and activity in Arabidopsis by the transcriptional regulator AHL15

Plant secondary growth, which is the basis of wood formation, includes the production of secondary xylem, which is derived from meristematic cambium cells embedded in vascular tissue. Here, we identified an important role for the Arabidopsis thaliana (Arabidopsis) AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED 15 (AHL15) transcriptional regulator in controlling vascular cambium activity. The limited secondary xylem development in inflorescence stems of herbaceous Arabidopsis plants was significantly reduced in ahl15 loss-of-function mutants, whereas constitutive or vascular meristem-specific AHL15 overexpression produced woody inflorescence stems. AHL15 was required for enhanced secondary xylem formation in the woody suppressor of overexpression of constans 1 (soc1) fruitfull (ful) double loss-of-function mutant. Moreover, we found that AHL15 induces vascular cambium activity downstream of the repressing SOC1 and FUL transcription factors, most likely similar to how it enhances lateral branching by promoting biosynthesis of the hormone cytokinin. Our results uncover a novel pathway driving cambium development, in which AHL15 expression levels act in parallel to and are dependent on the well-established TDIF-PXY-WOX pathway to differentiate between herbaceous and woody stem growth.

Ethylene regulates miRNA-mediated lignin biosynthesis and leaf serration in Arabidopsis thaliana

microRNAs (miRNAs) regulate target gene expression by pairing to target mRNAs, leading to mRNA degradation or translation inhibition. Out of several miRNAs in Arabidopsis, miR397b and miR857 regulate secondary growth by modulating lignin polymerization and deposition in secondary xylem cells by targeting laccases. Interestingly, the phytohormone ethylene is also suggested to have a role in lignin biosynthesis in tension wood formation. Despite this information, it is not known whether ethylene has any role in controlling secondary growth via miRNAs-mediated pathways. In this study, we elucidate that ethylene acts upstream to the miR397b/miR857-laccases module and negatively regulates lignin biosynthesis by directly activating the expression of both the miRNAs. The binding of EIN3 to the promoter of miR397b is further validated by yeast one-hybrid assay. In addition to its role in lignification, ethylene also regulates leaf serration by directly regulating the expression of NAC transcription factors, like CUP-SHAPED COTYLEDON2 (CUC2) and CUC3. Together, our study suggests a novel mechanism involving ethylene and miRNAs in lignin biosynthesis and leaf serration in Arabidopsis thaliana.

The 2018 hot drought pushed conifer wood formation to the limit of its plasticity: Consequences for woody biomass production and tree ring structure.

Hot droughts are expected to increase in Europe and disturb forest ecosystem functioning. Wood formation of trees has the potential to adapt to those events by compensatory mechanisms between the rates and durations of tracheid differentiation to form the typical pattern of vital wood anatomical structures. We monitored xylogenesis and measured wood anatomy of mature silver fir (Abies alba Mill.) and Scots pine (Pinus sylvestris L.) trees along an elevational gradient in the Black Forest during the hot drought year of 2018. We assessed the kinetics of tracheid differentiation and the final tracheid dimensions and quantified the relationship between rates and durations of cell differentiation over the growing season. Cell differentiation kinetics were decoupled, and temperature and water availability signals were imprinted in the tree ring structure. The sudden decline in woody biomass production provided evidence for a disruption in carbon sequestration processes due to heat and drought stress. Growth processes of Scots pine (pioneer species) were mainly affected by the spring drought, whereas silver fir (climax species) growth processes were more disturbed by the summer drought. Our study provides novel insights on the plasticity of wood formation and carbon allocation in temperate conifer tree species in response to extreme climatic events.

CHARACTERISTICS OF THE REDOX STATE OF THE LIGNIN-CARBOHYDRATE WOOD MATRIX AT THE INITIAL STAGES OF DEVELOPMENT ON THE EXAMPLE OF SCOTS PINE

The studies of wood formation at the initial stages allowed confirming the determining role of the redox transformations of monolignols in the lignification of plant matrix. The change in the composition of the phenolic fraction (conjugated and non-conjugated phenolic structures) of the phenol-quinone redox system of the cell wall was studied using the derivative and differential UV spectroscopy. It was shown that the accumulation of low-molecular phenolic compounds in the initial period of ontogenesis occurs mainly in the "needles" of plants. It was confirmed that the seedlings being removed to the hardening site adapt to the existing temperature regime. It is characterized by lower content of low-molecular phenolic compounds preventing the development of oxidative processes. The plant is exposed to oxidative stress at low temperatures, when the enzymatic (peroxidase activity) system of plant protection is activated. The change in the coniferyl alcohol content as a marker of the lignification is shown. The correlation of changes in peroxidase activity with the total content of phenolic compounds and coniferyl alcohol was shown. The IR spectroscopy method showed a change in the ratio of carbohydrate and lignin components at the initial stages of the development of seedlings of scots pine.

Dimerization of PtrMYB074 and PtrWRKY19 mediates transcriptional activation of PtrbHLH186 for secondary xylem development in Populus trichocarpa.

Summary Wood formation is controlled by transcriptional regulatory networks (TRNs) involving regulatory homeostasis determined by combinations of transcription factor (TF)–DNA and TF–TF interactions. Functions of TF–TF interactions in wood formation are still in the early stages of identification.PtrMYB074 is a woody dicot‐specific TF in a TRN for wood formation in Populus trichocarpa. Here, using yeast two‐hybrid and bimolecular fluorescence complementation, we conducted a genome‐wide screening for PtrMYB074 interactors and identified 54 PtrMYB074‐TF pairs. Of these pairs, 53 are novel.We focused on the PtrMYB074‐PtrWRKY19 pair, the most highly expressed and xylem‐specific interactor, and its direct transregulatory target, PtrbHLH186, the xylem‐specific one of the pair's only two direct TF target genes. Using transient and CRISPR‐mediated transgenesis in P. trichocarpa coupled with chromatin immunoprecipitation and electrophoretic mobility shift assays, we demonstrated that PtrMYB074 is recruited by PtrWRKY19 and that the PtrMYB074‐PtrWRKY19 dimers are required to transactive PtrbHLH186. Overexpressing PtrbHLH186 in P. trichocarpa resulted in retarded plant growth, increased guaiacyl lignin, a higher proportion of smaller stem vessels and strong drought‐tolerant phenotypes.Knowledge of the PtrMYB074‐PtrWRKY19‐PtrbHLH186 regulation may help design genetic controls of optimal growth and wood formation to maximize beneficial wood properties while minimizing negative effects on growth.

An earlier start of the thermal growing season enhances tree growth in cold humid areas but not in dry areas.

Climatic warming alters the onset, duration and cessation of the vegetative season. While previous studies have shown a tight link between thermal conditions and leaf phenology, less is known about the impacts of phenological changes on tree growth. Here, we assessed the relationships between the start of the thermal growing season and tree growth across the extratropical Northern Hemisphere using 3,451 tree-ring chronologies and daily climatic data for 1948-2014. An earlier start of the thermal growing season promoted growth in regions with high ratios of precipitation to temperature but limited growth in cold-dry regions. Path analyses indicated that an earlier start of the thermal growing season enhanced growth primarily by alleviating thermal limitations on wood formation in boreal forests and by lengthening the period of growth in temperate and Mediterranean forests. Semi-arid and dry subalpine forests, however, did not benefit from an earlier onset of growth and a longer growing season, presumably due to associated water loss and/or more frequent early spring frosts. These emergent patterns of how climatic impacts on wood phenology affect tree growth at regional to hemispheric scales hint at how future phenological changes may affect the carbon sequestration capacity of extratropical forest ecosystems.

Landslide-induced changes in tree-ring anatomy: A new dendrogeomorphic avenue?

The dendrogeomorphic dating of past landslides can be limited by the speed or magnitude of movements, as they are sometimes too small to induce growth responses in trees standing on unstable slopes. As a result, movements of low magnitude are generally assumed to remain undetectable in standard macroscopic analysis of growth disturbances in tree-ring series. To close this gap and to enlarge the suite of approaches available for the dendrogeomorphic detection of landslide activity in growth-ring records of trees, this study focuses on the detection of wood-anatomical growth responses in trees following low-magnitude landslides.Anatomical growth responses of five individuals of common spruce (Picea abies (L.) Karst.) growing on a monitored landslide have been analyzed in this study on a total of 200 tree rings from 40 increment cores extracted at two vertical levels of tree stems. Analysis included seven anatomical parameters commonly used in quantitative wood anatomy, with a focus on the year 2017 year during which the landslide body moved by 8 mm (prior to the start of the growing season) according to extensometer records. We report that mean tracheid lumen area (TLA) decreased significantly due to landsliding, whereas the mean number of traumatic resin ducts (TRD) and rays increased significantly after the occurrence of the landslide. By contrast, the intensity of stem tilting was visibly too small to induce the formation of compression wood but strong enough to induce larger number of rays, which are known to enhance stability as well. We also realize that the low intensity of the landslide movement also resulted in rather localized anatomical anomalies along the stem, with detected responses decreasing in intensity with increasing distance from the ground. We conclude that anatomical responses of P. abies trees can be used to detect past activity in low-magnitude landsliding and that this new proxy can thus extend the applicability of tree-ring approaches to landslide research and to the detection of events that have hitherto remained unidentified in reconstructions.

Differences in wood anatomy and chemistry of a Eucalyptus urophylla clone explained by site climate conditions

Environmental conditions can change both the quantity and quality of wood formation. This study aimed to evaluate anatomical and chemical changes in the wood of a Eucalyptus urophylla S.T. Blake clone cultivated in four sites of wide climatic conditions in Brazil. Radial samples were used to evaluate xylem anatomy along the growth cycles. Samples with a quarter of a disk were used to perform chemical analyses of extractives, total lignin (LG), syringyl/guaiacyl (S/G), holocellulose, and elemental analysis of wood. The elements Na, K, Ca, Mg, P, Mn, Fe, Zn, Ni, Cu, Cr, Cd, F, and Cl were also quantified. Correlations using the mean values of the variables per site were higher than those using values per tree growth cycle (years). Mean annual air temperature showed the highest correlations with wood density (r = −0.89) and the anatomical characteristics (vessel area: r = −0.68; fiber wall thickness: r = −0.70; vessel frequency: r = 0.74; and fiber lumen diameter: r = 0.90). Only LG and S/G showed significant correlations with the meteorological variables, with drier sites presenting a higher S/G. The anatomical characteristics change with regionwide climate features, while wood chemical characteristics showed weaker relations with climatic variations.

Genome Wide Association Study Identifies Candidate Genes Related to the Earlywood Tracheid Properties in Picea crassifolia Kom.

Picea crassifolia Kom. is one of the timber and ecological conifers in China and its wood tracheid traits directly affect wood formation and adaptability under harsh environment. Molecular studies on P. crassifolia remain inadequate because relatively few genes have been associated with these traits. To identify markers and candidate genes that can potentially be used for genetic improvement of wood tracheid traits, we examined 106 clones of P. crassifolia, and investigated phenotypic data for 14 wood tracheid traits before specific-locus amplified fragment sequencing (SLAF-seq) was employed to perform a genome wide association study (GWAS). Subsequently, the results were used to screen single nucleotide polymorphism (SNP) loci and candidate genes that exhibited a significant correlation with the studied traits. We developed 4,058,883 SLAF-tags and 12,275,765 SNP loci, and our analyses identified a total of 96 SNP loci that showed significant correlations with three earlywood tracheid traits using a mixed linear model (MLM). Next, candidate genes were screened in the 100 kb zone (50 kb upstream, 50 kb downstream) of each of the SNP loci, whereby 67 candidate genes were obtained in earlywood tracheid traits, including 34 genes of known function and 33 genes of unknown function. We provide the most significant SNP for each trait-locus combination and candidate genes occurring within the GWAS hits. These resources provide a foundation for the development of markers that could be used in wood traits improvement and candidate genes for the development of earlywood tracheid in P. crassifolia.

Transcriptome and miRNAome analysis reveals components regulating tissue differentiation of bamboo shoots.

Primary thickening determines bamboo yield and wood property. However, little is known about the regulatory networks involved in this process. This study identified a total of 58,652 genes and 150 miRNAs via transcriptome and small RNA sequencing using the underground thickening shoot samples of wild-type (WT) Moso bamboo (Phyllostachys edulis) and a thick wall (TW) variant (P. edulis "Pachyloen") at five developmental stages (WTS1/TWS1-WTS5/TWS5). A total of 14,029 (65.17%) differentially expressed genes and 68 (45.33%) differentially expressed miRNAs were identified from the WT, TW, and WTTW groups. The first two groups were composed of four pairwise combinations, each between two successive stages (WTS2/TWS2_versus_WTS1/TWS1, WTS3/TWS3_versus_WTS2/TWS2, WTS4/TWS4_versus_WTS3/TWS3, and WTS5/TWS5_versus_WTS4/TWS4), and the WTTW group was composed of five combinations, each between two relative stages (TWS1-5_versus_WTS1-5). Additionally, among the phytohormones, zeatin showed more remarkable changes in concentrations than indole-3-acetic acid, gibberellic acid, and abscisic acid throughout the five stages in the WT and the TW groups. Moreover, 125 cleavage sites were identified for 387 miRNA-mRNA pairs via degradome sequencing (P < 0.05). The dual-luciferase reporter assay confirmed that 13 miRNAs bound to 12 targets. Fluorescence in situ hybridization localized miR166 and miR160 in the shoot apical meristem and the procambium of Moso bamboo shoots at the S1 stage. Thus, primary thickening is a complex process regulated by miRNA-gene-phytohormone networks, and the miRNAome and transcriptome dynamics regulate phenotypic plasticity. These findings provide insights into the molecular mechanisms underlying wood formation and properties and propose targets for bamboo breeding.

Stem xylem traits and wood formation affect sex-specific responses to drought and rewatering in Populus cathayana.

The increased frequency and intensity of drought pose great threats to the survival of trees, especially in dioecious tree species with sexual differences in mortality and biased sex ratios. The sex-specific mechanisms underlying stem xylem anatomy and function and carbon metabolism in drought resistance and recovery were investigated in dioecious Populus cathayana Rehder. The sex-specific drought resistance and subsequent recovery were linked to the xylem anatomy and carbon metabolism. Females had a greater xylem vessel area per vessel, biomass and theoretically hydraulic efficiency under well-watered conditions. Conversely, males had a lower xylem lumen area, but greater vessel numbers, and a higher cell wall thickness, suggesting a theoretically conservative water-use strategy and drought resistance. The recovery of photosynthetic ability after drought in males was largely dependent on the recovery of xylem function and the regulation of the xylem carbohydrate metabolism. Additionally, the number of upregulated genes related to xylem cell wall biogenesis was greater in males relative to females under drought stress and subsequent rewatering, which facilitated drought resistance and xylem function restoration in males. These results suggested that sex-specific drought resistance and restoration were related to xylem anatomy and function, carbohydrate metabolism and cell turgor maintenance.