Wood Formation Research Articles

Integrated transcriptome and proteome analysis reveals brassinosteroid-mediated regulation of cambium initiation and patterning in woody stem.

Wood formation involves sequential developmental events requiring the coordination of multiple hormones. Brassinosteroids (BRs) play a key role in wood development, but little is known about the cellular and molecular processes that underlie wood formation in tree species. Here, we generated transgenic poplar lines with edited PdBRI1 genes, which are orthologs of Arabidopsis vascular-enriched BR receptors, and showed how inhibition of BR signaling influences wood development at the mRNA and/or proteome level. Six Populus PdBRI1 genes formed three gene pairs, each of which was highly expressed in basal stems. Simultaneous mutation of PdBRI1-1, -2, -3 and- 6, which are orthologs of the Arabidopsis vascular-enriched BR receptors BRI1, BRL1 and BRL3, resulted in severe growth defects. In particular, the stems of these mutant lines displayed a discontinuous cambial ring and patterning defects in derived secondary vascular tissues. Abnormal cambial formation within the cortical parenchyma was also observed in the stems of pdbri1-1;2;3;6. Transgenic poplar plants expressing edited versions of PdBRI1-1 or PdBRI1-1;2;6 exhibited phenotypic alterations in stem development at 4.5months of growth, indicating that there is functional redundancy among these PdBRI1 genes. Integrated analysis of the transcriptome and proteome of pdbri1-1;2;3;6 stems revealed differential expression of a number of genes/proteins associated with wood development and hormones. Concordant (16%) and discordant (84%) regulation of mRNA and protein expression, including wood-associated mRNA/protein expression, was found in pdbri1-1;2;3;6 stems. This study found a dual role of BRs in procambial cell division and xylem differentiation and provides insights into the multiple layers of gene regulation that contribute to wood formation in Populus.

Molecular understanding of wood formation in trees

Trees convert and store the majority of their photosynthetic products in wood which is an essential renewable resource much in demand by human society. Formation of wood follows a process of consecutive cell developmental stages, from vascular cambium proliferation, cell expansion and differentiation, secondary cell wall deposition to programmed cell death, which is controlled by the functionality of complex molecular networks. What are the molecular networks involved in wood formation? How do the molecular networks act in a way to generate wood tissue during tree growth? What are the regulatory modules that lead to the formation of various wood characteristics? The answers to these questions are fundamental to understanding how trees grow, as well as how we can genetically engineer trees with desired properties of wood for human needs. In recent years, a great deal of interest has been invested in the elucidation of wood formation at the molecular level. This review summarizes the current state of understanding of the molecular process that guides wood formation in trees.

Lectin affinity-based glycoproteome analysis of the developing xylem in poplar

Glycosylation is a significant post-translational modification of the proteins, and some glycoproteins serve as the players in plant cell wall synthesis and modification. Wood is a highly developed cell wall organization, and protein glycosylation as a regulatory way might be involved in wood formation. Here, a lectin affinity-based glycoproteome was performed in stem developing xylem of poplar. After enrichment, trypsin digestion, LC-MS/MS analysis and peptide identification, we identified 154 glycoproteins from poplar developing xylem, which were classified into 9 functional groups mainly including protein acting on carbohydrates, oxido-reductase, proteases, and protein kinases. Further, N- and/or O-glycosylation sites of the identified proteins were analyzed by the bioinformatic tools, and deglycosylation experiments in the selected PtSOD and PtHAD proteins verified the reliability of the identified glycoproteins. Analysis of protein subcellular localization showed that total 63 percent of the identified glycoproteins were extracellular proteins or located in the plasma membrane. Poplar eFP and RT-qPCR data showed that a number of the genes encoding these glycoproteins such as laccase, peroxidase and cysteine protease, have highly preferential expression profiles in the developing xylem. Together with the literatures, most identified glycoproteins could be involved in wood cell wall synthesis and modification in poplar. Thus, our study provides some potential wood formation-related glycoproteins to be determined during tree stem development.

Transcription factor PagLBD21 functions as a repressor of secondary xylem development in <i>Populus</i>

During secondary growth in trees, xylem cells differentiated from cambium cells begin to synthesize secondary cell walls that are primarily composed of cellulose, hemicellulose and lignin, and are deposited between primary cell walls and plasma membranes, leading to in wood formation. Identification of regulatory genes functioning during this developmental process is valuable for increasing wood production. In this study, we functionally characterized an LBD (LATERAL ORGAN BOUNDARIES DOMAIN) transcription factor PagLBD21 as a repressor of secondary xylem development in <italic>Populus</italic>. Compared to wild type plants, transgenic plants overexpressing <italic>PagLBD21</italic> (<italic>PagLBD21OE</italic>) exhibited decreased xylem widths in cross-sections. Consistent with the functional analysis, RNA sequencing (RNA-seq) analysis revealed that genes functioning in xylem development and secondary cell wall biosynthesis pathways were significantly down-regulated in <italic>PagLBD21OE</italic> plants. We also performed DNA affinity purification followed by sequencing (DAP-seq) to identify genome-wide target genes of PagLBD21. Furthermore, we compared the RNA-seq and DAP-seq datasets of PagLBD21 and PagLBD3, and the results showed that there was a significant overlap between their target genes, suggesting these two LBD transcription factors are functionally redundant during secondary growth.

Reflections by a member of the Editorial Board

I thank Co-Editors Ass. Prof. Dr. Jože Kropivšek and Prof. Dr. Katarina Čufar for the opportunity to serve as an Editorial Board member of Les/Wood, an international journal firmly grounded in the broad sweep of Slovene wood science and technology. The Editorial Board works to extend the relevance and impact of Les/Wood within and beyond Slovenia to the international community of wood science and technology in the broadest sense. We see the journal as a tool to support research and development of specific technologies, as well as to develop common terminology.&#x0D; I would like to use this opportunity to sketch out how I came to be involved with wood science in Slovenia. In retrospect that involvement seems inevitable, but the path was not always clear at the time! After completing my postgraduate education in plant pathology and mycology in the USA in 1982, I accepted my current position as supervisory plant physiologist for the USDA Forest Service, a US federal land management agency with a strong commitment to research &amp; development and to international forestry.&#x0D; Throughout my career, my personal research has involved tree growth and wood decay in response to injury, infection, and environmental change. These tree responses affect forest health, the economic value of wood for art and industry, the tree-ring record of environmental conditions, and the performance of trees in urban and community environments.&#x0D; My USDA FS mentor and colleague Dr. Walter Shortle met Prof. Dr. Niko Torelli and Prof. Dr. Katarina Čufar at the IAWA conference in Hamburg (1983) and at the IUFRO World Conference 1996 in Ljubljana, where the two of them were local organizers. Afterwards they obtained a joint Yugoslav American Project, “Possible alterations of wood in air polluted trees”, which defined my earlier collaborations with Professor Torelli and Katarina Čufar on silver fir decline in Slovenia and Europe. Later, Katarina Čufar, Dr. Tom Levanič, and I identified changes in the climatic responses of silver fir in Slovenia and of red spruce in the northeastern US. I also worked with Dr. Primož Oven on special considerations for the health and safety of city trees. Later, Katarina Čufar and I both served on the Executive Council of the Tree Ring Society, an international association for dendrochronological research. We co-taught a tree biology section at the Dendro Fieldweek as part of the World Dendro conference 2010 in Finland. We found that our combination of research experience both intensified and broadened our understanding and presentation of wood structure and function. Since then, we have worked to provide Slovene students and scientists a platform to share techniques and experiences that both extend the science and community of scientists in the international research community.&#x0D; As an active researcher, I see Les/Wood as noteworthy because of the potential breadth of articles reporting on topics from wood mechanical properties to technologies and industrial processing to underlying anatomy and the biological processes that result in wood formation and preservation. In addition to the high level of scholarship and technical expertise within Les/Wood, I find collegiality of spirit, openness of approach, and pride of history and context. These qualities lift up and support ongoing and future research for Slovenia, its geographic surroundings, and the broader world research community. I’m excited at the prospect of Les/Wood extending its traditional strengths to meet the critical need for wood science to optimize wood utilization, ecological understanding, and cultural patrimony.

The chromosome-scale genomes of Dipterocarpus turbinatus and Hopea hainanensis (Dipterocarpaceae) provide insights into fragrant oleoresin biosynthesis and hardwood formation.

SummaryDipterocarpaceae are typical tropical plants (dipterocarp forests) that are famous for their high economic value because of their production of fragrant oleoresins, top‐quality timber and usage in traditional Chinese medicine. Currently, the lack of Dipterocarpaceae genomes has been a limiting factor to decipher the fragrant oleoresin biosynthesis and gain evolutionary insights into high‐quality wood formation in Dipterocarpaceae. We generated chromosome‐level genome assemblies for two representative Dipterocarpaceae species viz. Dipterocarpus turbinatus Gaertn. f. and Hopea hainanensis Merr. et Chun. Our whole‐genome duplication (WGD) analysis revealed that Dipterocarpaceae underwent a shared WGD event, which showed significant impacts on increased copy numbers of genes related to the biosynthesis of terpene, BAHD acyltransferases, fatty acid and benzenoid/phenylpropanoid, which probably confer to the formation of their characteristic fragrant oleoresin. Additionally, compared with common soft wood plants, the expansion of gene families was also found to be associated with wood formation, such as in CESA (cellulose synthase), CSLE (cellulose synthase‐like protein E), laccase and peroxidase in Dipterocarpaceae genomes, which might also contribute to the formation of harder, stronger and high‐density timbers. Finally, an integrative analysis on a combination of genomic, transcriptomic and metabolic data from different tissues provided further insights into the molecular basis of fragrant oleoresins biosynthesis and high‐quality wood formation of Dipterocarpaceae. Our study contributes the first two representative genomes for Dipterocarpaceae, which are valuable genetic resources for further researches on the fragrant oleoresins and superior‐quality timber, genome‐assisted breeding and improvement, and conservation biology of this family.

Cutting tree rings into time slices: how intra-annual dynamics of wood formation help decipher the space-for-time conversion.

Tree-ring anatomy, microdensity and isotope records provide valuable intra-annual information. However, extracting signals at that scale is challenged by the complexity of xylogenesis, where two major processes - cell enlargement and wall thickening - occur at different times and rates. We characterized the space-for-time association in the tree rings of three conifer species by examining the duration, overlapping, inter-tree synchronicity and interannual stability during cell enlargement and wall thickening across regular tree-ring sectors (portions of equal tangential width). The number of cells and cell differentiation rates determined the duration of sector formation, which augmented more rapidly throughout the ring for wall thickening than for enlargement. Increasing the number of sectors above c. 15 had a limited effect on improving time resolution because consecutive sector formation overlapped greatly in time, especially in narrow rings and during wall thickening. Increasing the number of sectors also resulted in lower synchronicity and stability of intermediate-sector enlargement, whereas all sectors showed high synchronicity and stability during wall thickening. Increasing the number of sectors had a stronger effect on enhancing time-series resolution for enlargement- than for wall-thickening-related traits, which would nevertheless produce more reliable intra-annual chronologies as a result of the more similar calendars across trees and years in wall thickening.

Cell-wall fluorescence highlights the phases of xylogenesis

Summary The monitoring of xylogenesis makes it possible to follow tree growth responses to stress factors in real-time, by observing the course of wood cell division and differentiation. Proper microscopy techniques are of key importance to exactly identify the xylem cells during the different phases of differentiation. We aimed to apply epifluorescence microscopy to follow the lignification process during the different phases of xylogenesis in Mediterranean softwood and hardwood. Microcores from trees of Pinus halepensis Mill. and Arbutus unedo L. were collected at a site in southern Italy, during the period June-December. Fluorescence imaging of sections stained with a water solution of safranin and Astra blue clearly highlighted the contrast between lignified and un-lignified tissue. The proposed methodology is useful to quickly and unambiguously detect the different stages of cell differentiation, as well as the progress in the lignification process. Moreover, it proved to be easily applied to demanding wood materials, such as Mediterranean woods and can be helpful to better track stress responses and the development of anomalies during wood formation, such as intra-annual density fluctuations.

In-depth temperature and smoke production of charring wood under a constant external heat flux

The combustion characteristics of charring wood have been studied experimentally in a well-ventilated environment of a smoke chamber. A numerical simulation has also been performed for a limited case, with the Fire Dynamics Simulator, to estimate the burning environment. A horizontally placed specimen (ponderosa pine) with a moisture content of 0% or 20% is exposed to a radiant flux (25 kW/m2), with or without flaming ignition. Simultaneous measurements of the specimen’s in-depth temperature and the mass loss determine the charring front (rate) at 300 °C and the gasification rate, respectively. These condensed-phase conditions relate directly to real-time variations of gas-phase quantities: the specific optical density of smoke and the concentrations of toxic gases measured by a Fourier transform infrared gas analyzer. In-depth temperature trends are similar whether the flame exists, whereas the smoke and toxicants’ concentrations are substantially different. After the charring front moves through the specimen, the oxidative pyrolysis continues under the irradiation at high temperatures (up to ∼550 °C). Carbon monoxide and acrolein are produced continuously throughout the test, and the results indicate strong correlations. Although char formation of wood is favorable for fire safety, consequent incomplete combustion produces smoke and toxicants.

Full-length transcriptomic identification of R2R3-MYB family genes related to secondary cell wall development in Cunninghamia lanceolata (Chinese fir)

BackgroundR2R3-MYB is a class of transcription factor crucial in regulating secondary cell wall development during wood formation. The regulation of wood formation in gymnosperm has been understudied due to its large genome size. Using Single-Molecule Real-Time sequencing, we obtained full-length transcriptomic libraries from the developmental stem of Cunninghamia lanceolata, a perennial conifer known as Chinese fir. The R2R3-MYB of C. lanceolata (hereafter named as ClMYB) associated with secondary wall development were identified based on phylogenetic analysis, expression studies and functional study on transgenic line.ResultsThe evolutionary relationship of 52 ClMYBs with those from Arabidopsis thaliana, Eucalyptus grandis, Populus trichocarpa, Oryza sativa, two gymnosperm species, Pinus taeda, and Picea glauca were established by neighbour-joining phylogenetic analysis. A large number of ClMYBs resided in the woody-expanded subgroups that predominated with the members from woody dicots. In contrast, the woody-preferential subgroup strictly carrying the members of woody dicots contained only one candidate. The results suggest that the woody-expanded subgroup emerges before the gymnosperm/angiosperm split, while most of the woody-preferential subgroups are likely lineage-specific to woody dicots. Nine candidates shared the same subgroups with the A. thaliana orthologs, with known function in regulating secondary wall development. Gene expression analysis inferred that ClMYB1/2/3/4/5/26/27/49/51 might participate in secondary wall development, among which ClMYB1/2/5/26/27/49 were significantly upregulated in the highly lignified compression wood region, reinforcing their regulatory role associated with secondary wall development. ClMYB1 was experimentally proven a transcriptional activator that localised in the nucleus. The overexpression of ClMYB1 in Nicotiana benthamiana resulted in an increased lignin deposition in the stems. The members of subgroup S4, ClMYB3/4/5 shared the ERF-associated amphiphilic repression motif with AtMYB4, which is known to repress the metabolism of phenylpropanoid derived compounds. They also carried a core motif specific to gymnosperm lineage, suggesting divergence of the regulatory process compared to the angiosperms.ConclusionsThis work will enrich the collection of full-length gymnosperm-specific R2R3-MYBs related to stem development and contribute to understanding their evolutionary relationship with angiosperm species.

QtlXplorer: an online systems genetics browser in the Eucalyptus Genome Integrative Explorer (EucGenIE)

BackgroundAffordable high-throughput DNA and RNA sequencing technologies are allowing genomic analysis of plant and animal populations and as a result empowering new systems genetics approaches to study complex traits. The availability of intuitive tools to browse and analyze the resulting large-scale genetic and genomic datasets remain a significant challenge. Furthermore, these integrative genomics approaches require innovative methods to dissect the flow and interconnectedness of biological information underlying complex trait variation. The Plant Genome Integrative Explorer (PlantGenIE.org) is a multi-species database and domain that houses online tools for model and woody plant species including Eucalyptus. Since the Eucalyptus Genome Integrative Explorer (EucGenIE) is integrated within PlantGenIE, it shares genome and expression analysis tools previously implemented within the various subdomains (ConGenIE, PopGenIE and AtGenIE). Despite the success in setting up integrative genomics databases, online tools for systems genetics modelling and high-resolution dissection of complex trait variation in plant populations have been lacking.ResultsWe have developed qtlXplorer (https://eucgenie.org/QTLXplorer) for visualizing and exploring systems genetics data from genome-wide association studies including quantitative trait loci (QTLs) and expression-based QTL (eQTL) associations. This module allows users to, for example, find co-located QTLs and eQTLs using an interactive version of Circos, or explore underlying genes using JBrowse. It provides users with a means to build systems genetics models and generate hypotheses from large-scale population genomics data. We also substantially upgraded the EucGenIE resource and show how it enables users to combine genomics and systems genetics approaches to discover candidate genes involved in biotic stress responses and wood formation by focusing on two multigene families, laccases and peroxidases.ConclusionsqtlXplorer adds a new dimension, population genomics, to the EucGenIE and PlantGenIE environment. The resource will be of interest to researchers and molecular breeders working in Eucalyptus and other woody plant species. It provides an example of how systems genetics data can be integrated with functional genetics data to provide biological insight and formulate hypotheses. Importantly, integration within PlantGenIE enables novel comparative genomics analyses to be performed from population-scale data.

Tree growth forces and wood properties

Living wood in the tree performs a muscular action by generating forces at sapwood periphery and residual strains in dead sapwood fibres. Dissymmetric force generation around tree trunk is the motor system allowing movement, posture control and tree reshaping after accidents. Rather young trees are able to restore the verticality of their trunk after accidental rotation of the soil-root system due to wind or landslide, leading to typically curved stems shape. The very high dissymmetry of forces for the motor action is associated with the occurrence of reaction wood on one side of the inclined stem during many successive years. A method to reconstitute this biomechanical history from observations after tree felling, on either green or dry wood, is discussed. A selection of 17 such trees coming from 15 different species (13 different families), tropical or temperate, hardwoods or softwoods, were selected and peripheral residual strains were measured in situ before felling, on 8 position for each stem. Matched wooden rods were sawn and measured for their mechanical and physical properties at green and dry states, allowing the estimation of tree growth stress, i.e., the force created by the living wood. It was possible to build easy-to-use conversion coefficients between growth stress indicator (GSI), measured in situ by the single hole method, and growth strain and growth stress with the knowledge of basic density and green longitudinal elastic modulus. Maturation strain, specific modulus (as a proxy of micro-fibril angle) and longitudinal shrinkage are properties independent from basic density, whose variation among species was very large. On the whole range of compression wood, normal wood and tension wood strong relationships between these 3 properties were observed, but altogether no single model based on cell-wall microfibril angle only could be used. Growth forces are the product of 4 parameters: ring width, basic density, basic specific modulus and maturation strain, all of them being the result of wood formation. Thanks to the wide range of wood types and species, simple and highly significant formulas were obtained for the relationship between basic and dry density, green and dry longitudinal modulus of elasticity, basic and dry specific modulus. To estimate ring width in green state from values in dry state, radial shrinkage needs to be measured afterwards on dry specimens. Maturation strains is less accurately linked to late measurements on dry wood, but longitudinal shrinkage offers a rather good solution for an estimation provided that the wood type (softwood, hardwood with-G layer, hardwood without G-Layer) is known.

Biologia Futura: progress and future perspectives of long non-coding RNAs in forest trees.

Forest trees are affected by climate change, anthropogenic pressure, as well as abiotic and biotic stresses. Conventional tree breeding has so far been limited to enhance overall productivity, and our understanding of the genetic basis of quantitative traits is still inadequate. Quantum leaps in next-generation sequencing technologies and bioinformatics have permitted the exploration and identification of various non-coding regions of the genome other than protein coding genes. These genomic regions produce various types of non-coding RNAs and regulate myriads of biological functions at epigenetic, transcriptional and translational levels. Recently, long non-coding RNAs (lncRNAs) which act as molecular switch have been identified to be pivotal molecules in forest trees. This review focuses on progress made in regulatory mechanisms in various developmental phases like wood formation, adventitious rooting and flowering and stress responses. It was predicted that complex regulatory interactions among lncRNA, miRNA and gene exist. LncRNAs can function as a sponge for miRNAs, reducing the suppressive effect of miRNAs on target mRNAs and perhaps adding a new layer of regulatory interactions among non-coding RNA classes in trees. Furthermore, network analysis revealed the interactions of lncRNA and genes during the expression of several important genes. The insights generated about lncRNAs in forest trees would enable improvement of economically important traits including the devastating abiotic and biotic stresses. In addition, solid understanding on the wide range of regulatory functions of lncRNAs on traits influencing biomass productivity and adaptation would aid the applications of biotechnology in genetic improvement of forest trees.

Protoplast isolation and transcriptome analysis of developing xylem in Pinus massoniana (Pinaceae).

With active physiological and biochemical activities, tissue-specific protoplasts from cambial derivatives, could serve as a specific source for information on xylogenesis for softwood species resistant to stable genetic transformation and lacking available mutants. In this study, protoplasts were isolated from developing xylem of the Chinese red pine, Pinus massoniana, by enzymolysis. High-quality RNAs were extracted from developing xylem and their protoplasts for constructing transcriptome libraries. Using Illumina HiSeq 2500 PE150 platform, a total of 362,328,426 clean paired-end reads (54.35G) were generated from multiple cDNA libraries and assembled into 146,422 unigenes. The transcriptome data were further analysed to identify 1567 differentially expressed genes (DEGs) between the isolated protoplasts and developing xylem of P. massoniana (Masson pine), 1126 DEGs were upregulated in protoplasts relative to developing xylem cells and 441 were downregulated. Most of the differentially expressed genes in biological process terms are related to plant response, which may be due to the response to cell wall removal. Further, the expression pattern of 71 unigenes involved in lignin biosynthesis was verified by RNA-seq. This study is the first to report the transcriptome profiles of the developing xylem and its protoplasts of coniferous trees, which provide a new perspective and valuable resource for tracking transcriptional regulatory events in wood formation of Masson pine.

Wood transcriptome analysis and expression variation of lignin biosynthetic pathway transcripts in Ailanthus excelsa Roxb., a multi-purpose tropical tree species

Safety matches are an essential consumer commodity which faces considerable shortage of raw material due to specific requirements like color, wax stability, consistent burning and splinting ability in wood. Ailanthus excelsa Roxb. is a multi-purpose tropical tree species which is predominantly used for production of match splints. It is a data deficient species with no information on molecular regulation governing the wood phenotypes. Hence, with the aim to identify the lignin biosynthetic pathway transcripts, transcriptome sequencing of pooled developing secondary wood tissues of a 22-month-old tree was conducted. The processed reads were de novo assembled and 48,493 unigenes were annotated. Quantitative real time PCR was conducted in six even-aged trees to document the natural expression profiles of nine major transcripts from the monolignol pathway. Significant differential expression of Phenylalanine ammonia-lyase (AePAL), Cinnamate 4-hydroxylase (AeC4H), Cinnamyl-alcohol dehydrogenase (AeCAD), Laccase (AeLACI) and Caffeic Acid 3-O-Methyl Transferase (AeCOMT) was documented across all genotypes studied. Transcriptome-wise microsatellites were also mined and is the first co-dominant marker resource in this species. The genomic resource generated in A. excelsa will facilitate understanding the molecular mechanisms of wood formation and accelerate trait-based breeding program through diversity assessment, population structure analysis, clonal discrimination and marker assisted selection.

A Talk between Flavonoids and Hormones to Reorient the Growth of Gymnosperms.

Plants reorient the growth of affected organs in response to the loss of gravity vector. In trees, this phenomenon has received special attention due to its importance for the forestry industry of conifer species. Sustainable management is a key factor in improving wood quality. It is of paramount importance to understand the molecular and genetic mechanisms underlying wood formation, together with the hormonal and environmental factors that affect wood formation and quality. Hormones are related to the modulation of vertical growth rectification. Many studies have resulted in a model that proposes differential growth in the stem due to unequal auxin and jasmonate allocation. Furthermore, many studies have suggested that in auxin distribution, flavonoids act as molecular controllers. It is well known that flavonoids affect auxin flux, and this is a new area of study to understand the intracellular concentrations and how these compounds can control the gravitropic response. In this review, we focused on different molecular aspects related to the hormonal role in flavonoid homeostasis and what has been done in conifer trees to identify molecular players that could take part during the gravitropic response and reduce low-quality wood formation.

PmMYB4, a Transcriptional Activator from Pinus massoniana, Regulates Secondary Cell Wall Formation and Lignin Biosynthesis

Wood formation originates in the biosynthesis of lignin and further leads to secondary cell wall (SCW) biosynthesis in woody plants. Masson pine (Pinus massoniana Lamb) is an economically important industrial timber tree, and its wood yield affects the stable development of the paper industry. However, the regulatory mechanisms of SCW formation in Masson pine are still unclear. In this study, we characterized PmMYB4, which is a Pinus massoniana MYB gene involved in SCW biosynthesis. The open reading frame (ORF) of PmMYB4 was 1473 bp, which encoded a 490 aa protein and contained two distinctive R2 and R3 MYB domains. It was shown to be a transcription factor, with the highest expression in semi-lignified stems. We overexpressed PmMYB4 in tobacco. The results indicated that PmMYB4 overexpression increased lignin deposition, SCW thickness, and the expression of genes involved in SCW formation. Further analysis indicated that PmMYB4 bound to AC-box motifs and might directly activate the promoters of genes (PmPAL and PmCCoAOMT) involved in SCW biosynthesis. In addition, PmMYB4-OE(over expression) transgenic lines had higher lignin and cellulose contents and gene expression than control plants, indicating that PmMYB4 regulates SCW mainly by targeting lignin biosynthetic genes. In summary, this study illustrated the MYB-induced SCW mechanism in Masson pine and will facilitate enhanced lignin and cellulose synthesis in genetically engineered trees.

Origin of Intra-annual Density Fluctuations in a Semi-arid Area of Northwestern China.

Intra-annual density fluctuation (IADF) is a structural modification of the tree ring in response to fluctuations in the weather. The expected changes in monsoon flow would lead to heterogeneous moisture conditions during the growing season and increase the occurrence of IADF in trees of the arid ecosystems of continental Asia. To reveal the timings and physiological mechanisms behind IADF formation, we monitored cambial activity and wood formation in Chinese pine (Pinus tabuliformis) during 2017–2019 at three sites in semi-arid China. We compared the dynamics of xylem formation under a drought event, testing the hypothesis that drought affects the process of cell enlargement and thus induces the production of IADF. Wood microcores collected weekly from April to October were used for anatomical analyses to estimate the timings of cambial activity, and the phases of enlargement, wall thickening, and lignification of the xylem. The first cells started enlargement from late April to early May. The last latewood cells completed differentiation in mid-September. Trees produced IADF in 2018. During that year, a drought in June limited cell production in the cambium, only 36% of the xylem cells being formed in IADF trees, compared to 68% in normal tree rings. IADF cells enlarged under drought in early July and started wall thickening during the rainfall events of late July. The drought restricted cell enlargement and affected wall thickening, resulting in narrow cells with wide walls. Cambium and cell enlargement recovered from the abundant rainfall, producing a new layer with large earlywood tracheids. IADF is a specific adaptation of trees to cope with water deficit events occurring during xylem formation. Our findings confirmed the hypothesis that the June-July drought induces latewood-like IADFs by limiting the process of cell enlargement in the xylem. Our finding suggests a higher occurrence of IADF in trees of arid and semi-arid climates of continental Asia if the changes to monsoon flows result in more frequent drought events during the earlywood formation in June.

Transcriptome and microRNA Sequencing Identified miRNAs and Target Genes in Different Developmental Stages of the Vascular Cambium in Cryptomeria fortunei Hooibrenk.

Cryptomeria fortunei Hooibrenk is an important fast-growing coniferous timber species that is widely used in landscaping. Recently, research on timber quality has gained substantial attention in the field of tree breeding. Wood is the secondary xylem formed by the continuous inward division and differentiation of the vascular cambium; therefore, the development of the vascular cambium is particularly important for wood quality. In this study, we analyzed the transcriptomes of the cambial zone in C. fortunei during different developmental stages using Illumina HiSeq sequencing, focusing on general transcriptome and microRNA (miRNA) data. We performed functional annotation of the differentially expressed genes (DEGs) in the different stages identified by transcriptome sequencing and generated 15 miRNA libraries yielding 4.73 Gb of clean reads. The most common length of the filtered miRNAs was 21nt, accounting for 33.1% of the total filtered reads. We annotated a total of 32 known miRNA families. Some miRNAs played roles in hormone signal transduction (miR159, miR160, and miR166), growth and development (miR166 and miR396), and the coercion response (miR394 and miR395), and degradome sequencing showed potential cleavage sites between miRNAs and target genes. Differential expression of miRNAs and target genes and functional validation of the obtained transcriptome and miRNA data provide a theoretical basis for further elucidating the molecular mechanisms of cellular growth and differentiation, as well as wood formation in the vascular cambium, which will help improve the wood quality of C. fortunei.

Extraction and high-throughput sequencing of oak heartwood DNA: Assessing the feasibility of genome-wide DNA methylation profiling.

Tree ring features are affected by environmental factors and therefore are the basis for dendrochronological studies to reconstruct past environmental conditions. Oak wood often provides the data for these studies because of the durability of oak heartwood and hence the availability of samples spanning long time periods of the distant past. Wood formation is regulated in part by epigenetic mechanisms such as DNA methylation. Studies of the methylation state of DNA preserved in oak heartwood thus could identify epigenetic tree ring features informing on past environmental conditions. In this study, we aimed to establish protocols for the extraction of DNA, the high-throughput sequencing of whole-genome DNA libraries (WGS) and the profiling of DNA methylation by whole-genome bisulfite sequencing (WGBS) for oak (Quercus robur) heartwood drill cores taken from the trunks of living standing trees spanning the AD 1776-2014 time period. Heartwood contains little DNA, and large amounts of phenolic compounds known to hinder the preparation of high-throughput sequencing libraries. Whole-genome and DNA methylome library preparation and sequencing consistently failed for oak heartwood samples more than 100 and 50 years of age, respectively. DNA fragmentation increased with sample age and was exacerbated by the additional bisulfite treatment step during methylome library preparation. Relative coverage of the non-repetitive portion of the oak genome was sparse. These results suggest that quantitative methylome studies of oak hardwood will likely be limited to relatively recent samples and will require a high sequencing depth to achieve sufficient genome coverage.