Genome-wide characterization of post-transcriptional processes related to wood formation in Dalbergia odorifera.

BACKGROUND: Alternative polyadenylation (APA), alternative splicing (AS), and long non-coding RNAs (lncRNAs) play regulatory roles in post-transcriptional processes in plants. However, little is known about their involvement in xylem development in Dalbergia odorifera, a valuable rosewood species with medicinal and commercial significance. We addressed this by conducting Isoform Sequencing (Iso-Seq) using PacBio's SMRT technology and combined it with RNA-seq analysis (RNA sequencing on Illumina platform) after collecting xylem samples from the transition zone and the sapwood of D. odorifera. RESULTS: We identified 14,938 full-length transcripts, including 9,830 novel isoforms, which has updated the D. odorifera genome annotation. Our analysis has revealed that 4,164 genes undergo APA, whereas 3,084 genes encounter AS. We have also annotated 118 lncRNAs. Furthermore, RNA-seq analysis identified 170 differential alternative splicing (DAS) events, 344 genes with differential APA site usage (DE-APA), and 6 differentially expressed lncRNAs in the transition zone when compared to the sapwood. AS, APA, and lncRNAs are differentially regulated during xylem development. Differentially expressed APA genes were enriched for terpenoid and flavonoid metabolism, indicating their role in the heartwood formation. Additionally, DE-APA genes were associated with cell wall biosynthesis and terpenoid metabolism, implying an APA's role in wood formation. A DAS gene (involved in chalcone accumulation) with a significantly greater inclusion of the last exon in the transition zone than in the sapwood was identified. We also found that differentially expressed lncRNAs targeted the genes related to terpene synthesis. CONCLUSIONS: This study enhances our understanding of the molecular regulatory mechanisms underlying wood formation in D. odorifera, and provides valuable genetic resources and insights for its molecular-assisted breeding.

Comparative physiological, biochemical, metabolomic, and transcriptomic analyses reveal the formation mechanism of heartwood for Acacia melanoxylon.

Acacia melanoxylon is well known as a valuable commercial tree species owing to its high-quality heartwood (HW) products. However, the metabolism and regulatory mechanism of heartwood during wood development remain largely unclear. In this study, both microscopic observation and content determination proved that total amount of starches decreased and phenolics and flavonoids increased gradually from sapwood (SW) to HW. We also obtained the metabolite profiles of 10 metabolites related to phenolics and flavonoids during HW formation by metabolomics. Additionally, we collected a comprehensive overview of genes associated with the biosynthesis of sugars, terpenoids, phenolics, and flavonoids using RNA-seq. A total of ninety-one genes related to HW formation were identified. The transcripts related to plant hormones, programmed cell death (PCD), and dehydration were increased in transition zone (TZ) than in SW. The results of RT-PCR showed that the relative expression level of genes and transcription factors was also high in the TZ, regardless of the horizontal or vertical direction of the trunk. Therefore, the HW formation took place in the TZ for A. melanoxylon from molecular level, and potentially connected to plant hormones, PCD, and cell dehydration. Besides, the increased expression of sugar and terpenoid biosynthesis-related genes in TZ further confirmed the close connection between terpenoid biosynthesis and carbohydrate metabolites of A. melanoxylon. Furthermore, the integrated analysis of metabolism data and RNA-seq data showed the key transcription factors (TFs) regulating flavonoids and phenolics accumulation in HW, including negative correlation TFs (WRKY, MYB) and positive correlation TFs (AP2, bZIP, CBF, PB1, and TCP). And, the genes and metabolites from phenylpropanoid and flavonoid metabolism and biosynthesis were up-regulated and largely accumulated in TZ and HW, respectively. The findings of this research provide a basis for comprehending the buildup of metabolites and the molecular regulatory processes of HW formation in A. melanoxylon.

The Integration of Transcriptome and Metabolome Analyses Provides Insights into the Determinants of the Wood Properties in Toona ciliata.

Toona ciliata, also known as Chinese mahogany, is a high-quality and fast-growing wood species with a high economic value. The wood properties of T. ciliata of different provenances vary significantly. In this study, we conducted comprehensive transcriptome and metabolome analyses of red and non-red T. ciliata wood cores of different provenances to compare their wood properties and explore the differential metabolites and genes that govern the variation in their wood properties. Through combined analyses, three differential genes and two metabolites were identified that are possibly related to lignin synthesis. The lignin content in wood cores from T. ciliata of different provenances shows significant variation following systematic measurement and comparisons. The gene Tci09G002190, one of the three differential genes, was identified as a member of the CAD (Cinnamyl alcohol dehydrogenase) gene family of T. ciliata, which is associated with lignin synthesis. Our data provide insights into the determinants of the wood properties in T. ciliata, providing a solid foundation for research into the subsequent mechanisms of the formation of T. ciliata wood.

PagEXPA1 combines with PagCDKB2;1 to regulate plant growth and the elongation of fibers in Populus alba × Populus glandulosa.

Expansins are important plant cell wall proteins. They can loosen and soften the cell walls and lead to wall extension and cell expansion. To investigate their role in wood formation and fiber elongation, the PagEXPA1 that highly expressed in cell differentiation and expansion tissues was cloned from 84K poplar (Populus alba × P. glandulosa). The subcellular localization showed that PagEXPA1 located in the cell wall and it was highly expressed in primary stems and young leaves. Compared with non-transgenic 84K poplar, overexpression of PagEXPA1 can promote plant-growth, lignification, and fiber cell elongation, while PagEXPA1 Cas9-editing mutant lines exhibited the opposite phenotype. Transcriptome analysis revealed that DEGs were mainly enriched in some important processes, which are associated with cell wall formation and cellulose synthesis. The protein interaction prediction and expression analysis showed that PagCDKB2:1 and PagEXPA1 might have an interaction relationship. The luciferase complementary assay and bimolecular fluorescence complementary assay validated that PagEXPA1 can combined with PagCDKB2;1. So they promoted the expansion of xylem vascular tissues and the development of poplar though participating in the regulation of cell division and differentiation by programming the cell-cycle. It provides good foundation for molecular breeding of fast-growing and high-quality poplar varieties.

Overexpression of the PtrNF-YA6 gene inhibits secondary cell wall thickening in poplar.

The NF-Y gene family in plants plays a crucial role in numerous biological processes, encompassing hormone response, stress response, as well as growth and development. In this study, we first used bioinformatics techniques to identify members of the NF-YA family that may function in wood formation. We then used molecular biology techniques to investigate the role and molecular mechanism of PtrNF-YA6 in secondary cell wall (SCW) formation in Populus trichocarpa. We found that PtrNF-YA6 protein was localized in the nucleus and had no transcriptional activating activity. Overexpression of PtrNF-YA6 had an inhibitory effect on plant growth and development and significantly suppressed hemicellulose synthesis and SCW thickening in transgenic plants. Yeast one-hybrid and ChIP-PCR assays revealed that PtrNF-YA6 directly regulated the expression of hemicellulose synthesis genes (PtrGT47A-1, PtrGT8C, PtrGT8F, PtrGT43B, PtrGT47C, PtrGT8A and PtrGT8B). In conclusion, PtrNF-YA6 can inhibit plant hemicellulose synthesis and SCW thickening by regulating the expression of downstream SCW formation-related target genes.

Dissection of figured wood trait in curly birch (Betula pendula Roth var. carelica (Mercklin) Hämet-Ahti) using high-throughput genotyping.

Curly (Karelian) birch is a special variety of Betula pendula Roth distributed in the northwestern part of Europe. Karelian birch is well-known for its valuable figured curly wood also known as "wooden marble". The genetic basis underlying curly wood formation has been debated since last century, however, there was no data about loci responsible for the curly wood trait. In the present study, we analyzed two full-sibs populations derived from experimental crosses of curly birches and segregating for the trait. RADseq genotyping was applied to reveal how many loci are involved in 'curliness' formation and to search for genetic variants associated with this trait. One single interval on chromosome 10 was detected containing possible candidate genes. InDel marker BpCW1 was suggested for the first time for marker-assisted selection of trees with curly wood at their earliest stages of development.

Analyses of high spatial resolution datasets identify genes associated with multi-layered secondary cell wall thickening in Pinus bungeana.

BACKGROUND AND AIMS: Secondary cell wall (SCW) thickening is a major cellular developmental stage determining wood structure and properties. Although the molecular regulation of cell wall deposition during tracheary element differentiation has been well established in primary growth systems, less is known about the gene regulatory processes involved in the multi-layered SCW thickening of mature trees. METHODS: Using third-generation [long-read single-molecule real-time (SMRT)] and second-generation [short-read sequencing by synthesis (SBS)] sequencing methods, we established a Pinus bungeana transcriptome resource with comprehensive functional and structural annotation for the first time. Using these approaches, we generated high spatial resolution datasets for the vascular cambium, xylem expansion regions, early SCW thickening, late SCW thickening and mature xylem tissues of 71-year-old Pinus bungeana trees. KEY RESULTS: A total of 79 390 non-redundant transcripts, 31 808 long non-coding RNAs and 5147 transcription factors were annotated and quantified in different xylem tissues at all growth and differentiation stages. Furthermore, using this high spatial resolution dataset, we established a comprehensive transcriptomic profile and found that members of the NAC, WRKY, SUS, CESA and LAC gene families are major players in early SCW formation in tracheids, whereas members of the MYB and LBD transcription factor families are highly expressed during late SCW thickening. CONCLUSIONS: Our results provide new molecular insights into the regulation of multi-layered SCW thickening in conifers. The high spatial resolution datasets provided can serve as important gene resources for improving softwoods.

Transcription factor PagMYB31 positively regulates cambium activity and negatively regulates xylem development in poplar.

Wood formation involves consecutive developmental steps, including cell division of vascular cambium, xylem cell expansion, secondary cell wall (SCW) deposition, and programmed cell death. In this study, we identified PagMYB31 as a coordinator regulating these processes in Populus alba × Populus glandulosa and built a PagMYB31-mediated transcriptional regulatory network. PagMYB31 mutation caused fewer layers of cambial cells, larger fusiform initials, ray initials, vessels, fiber and ray cells, and enhanced xylem cell SCW thickening, showing that PagMYB31 positively regulates cambial cell proliferation and negatively regulates xylem cell expansion and SCW biosynthesis. PagMYB31 repressed xylem cell expansion and SCW thickening through directly inhibiting wall-modifying enzyme genes and the transcription factor genes that activate the whole SCW biosynthetic program, respectively. In cambium, PagMYB31 could promote cambial activity through TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF)/PHLOEM INTERCALATED WITH XYLEM (PXY) signaling by directly regulating CLAVATA3/ESR-RELATED (CLE) genes, and it could also directly activate WUSCHEL HOMEOBOX RELATED4 (PagWOX4), forming a feedforward regulation. We also observed that PagMYB31 could either promote cell proliferation through the MYB31-MYB72-WOX4 module or inhibit cambial activity through the MYB31-MYB72-VASCULAR CAMBIUM-RELATED MADS2 (VCM2)/PIN-FORMED5 (PIN5) modules, suggesting its role in maintaining the homeostasis of vascular cambium. PagMYB31 could be a potential target to manipulate different developmental stages of wood formation.

Genotyping SNPs in lignin biosynthesis gene (CAD1) and transcription factors (MYB1 and MYB2) exhibits association with wood density in teak (Tectona grandis L.f.).

BACKGROUND: Teak (Tectona grandis L.f.), an important source of tropical timber with immense economic value, is a highly outcrossing forest tree species. 150 unrelated accessions of teak (Tectona grandis L.f.) plus trees assembled as clones at National Teak Germplasm Bank, Chandrapur, Maharashtra, India was investigated for association mapping of candidate lignin biosynthesis gene (CAD1) and transcription factors (MYB1 and MYB2). METHODS AND RESULTS: The CAD1, MYB1 and MYB2 were amplified using specifically designed primers. The amplified sequences were then sequenced and genotyped for 112 SNPs/11 indels. We evaluated the association between SNPs and wood density in teak accessions using GLM and MLM statistical models, with Bonferroni correction applied. The teak accessions recorded an average wood density of 416.69 kg.m-3 (CV 4.97%) and comprised of three loosely structured admixed sub-populations (K = 3), containing 72.05% genetic variation within sub-populations with low intragenic LD (0-21% SNP pairs) at P < 0.05 and high LD decay (33-934 bp) at R2 = 0.1. GLM and MLM models discounting systematic biases (Q and K matrices) to avoid false discovery revealed five loci at rare variants (MAF 0.003) and three loci at common variants (MAF 0.05) to be significantly (P < 0.05) associated with the wood density. However, the stringent Bonferroni correction (4.06-7.04 × 10-4) yielded only a single associated locus (B1485C/A) from exon of MYB1 transcription factor, contributing to about 10.35% phenotypic variation in wood density trait. CONCLUSION: Scored SNP locus (B1485C/A) can be developed as a molecular probe for selection of improved planting stock with proven wood density trait for a large-scale teak plantation.

Genome editing of wood for sustainable pulping.

Wood is an abundant and renewable feedstock for pulping and biorefining, but the aromatic polymer lignin greatly limits its efficient use. Sulis et al. recently reported a multiplex CRISPR editing strategy targeting multiple lignin biosynthetic genes to achieve combined lignin modifications, improve wood properties, and make pulping more sustainable.

Wood of trees: Cellular structure, molecular formation, and genetic engineering.

Wood is an invaluable asset to human society due to its renewable nature, making it suitable for both sustainable energy production and material manufacturing. Additionally, wood derived from forest trees plays a crucial role in sequestering a significant portion of the carbon dioxide fixed during photosynthesis by terrestrial plants. Nevertheless, with the expansion of the global population and ongoing industrialization, forest coverage has been substantially decreased, resulting in significant challenges for wood production and supply. Wood production practices have changed away from natural forests toward plantation forests. Thus, understanding the underlying genetic mechanisms of wood formation is the foundation for developing high-quality, fast-growing plantation trees. Breeding ideal forest trees for wood production using genetic technologies has attracted the interest of many. Tremendous studies have been carried out in recent years on the molecular, genetic, and cell-biological mechanisms of wood formation, and considerable progress and findings have been achieved. These studies and findings indicate enormous possibilities and prospects for tree improvement. This review will outline and assess the cellular and molecular mechanisms of wood formation, as well as studies on genetically improving forest trees, and address future development prospects.

Allelic variations of WAK106-E2Fa-DPb1-UGT74E2 module regulate fibre properties in Populus tomentosa.

Wood formation, intricately linked to the carbohydrate metabolism pathway, underpins the capacity of trees to produce renewable resources and offer vital ecosystem services. Despite their importance, the genetic regulatory mechanisms governing wood fibre properties in woody plants remain enigmatic. In this study, we identified a pivotal module comprising 158 high-priority core genes implicated in wood formation, drawing upon tissue-specific gene expression profiles from 22 Populus samples. Initially, we conducted a module-based association study in a natural population of 435 Populus tomentosa, pinpointing PtoDPb1 as the key gene contributing to wood formation through the carbohydrate metabolic pathway. Overexpressing PtoDPb1 led to a 52.91% surge in cellulose content, a reduction of 14.34% in fibre length, and an increment of 38.21% in fibre width in transgenic poplar. Moreover, by integrating co-expression patterns, RNA-sequencing analysis, and expression quantitative trait nucleotide (eQTN) mapping, we identified a PtoDPb1-mediated genetic module of PtoWAK106-PtoDPb1-PtoE2Fa-PtoUGT74E2 responsible for fibre properties in Populus. Additionally, we discovered the two PtoDPb1 haplotypes that influenced protein interaction efficiency between PtoE2Fa-PtoDPb1 and PtoDPb1-PtoWAK106, respectively. The transcriptional activation activity of the PtoE2Fa-PtoDPb1 haplotype-1 complex on the promoter of PtoUGT74E2 surpassed that of the PtoE2Fa-PtoDPb1 haplotype-2 complex. Taken together, our findings provide novel insights into the regulatory mechanisms of fibre properties in Populus, orchestrated by PtoDPb1, and offer a practical module for expediting genetic breeding in woody plants via molecular design.

Woody plant cell walls: Fundamentals and utilization.

Cell walls in plants, particularly forest trees, are the major carbon sink of the terrestrial ecosystem. Chemical and biosynthetic features of plant cell walls were revealed early on, focusing mostly on herbaceous model species. Recent developments in genomics, transcriptomics, epigenomics, transgenesis, and associated analytical techniques are enabling novel insights into formation of woody cell walls. Here, we review multilevel regulation of cell wall biosynthesis in forest tree species. We highlight current approaches to engineering cell walls as potential feedstock for materials and energy and survey reported field tests of such engineered transgenic trees. We outline opportunities and challenges in future research to better understand cell type biogenesis for more efficient wood cell wall modification and utilization for biomaterials or for enhanced carbon capture and storage.

Transcriptome and miRNAs Profiles Reveal Regulatory Network and Key Regulators of Secondary Xylem Formation in "84K" Poplar.

Secondary xylem produced by stem secondary growth is the main source of tree biomass and possesses great economic and ecological value in papermaking, construction, biofuels, and the global carbon cycle. The secondary xylem formation is a complex developmental process, and the underlying regulatory networks and potential mechanisms are still under exploration. In this study, using hybrid poplar (Populus alba × Populus glandulosa clone 84K) as a model system, we first ascertained three representative stages of stem secondary growth and then investigated the regulatory network of secondary xylem formation by joint analysis of transcriptome and miRNAs. Notably, 7507 differentially expressed genes (DEGs) and 55 differentially expressed miRNAs (DEMs) were identified from stage 1 without initiating secondary growth to stage 2 with just initiating secondary growth, which was much more than those identified from stage 2 to stage 3 with obvious secondary growth. DEGs encoding transcription factors and lignin biosynthetic enzymes and those associated with plant hormones were found to participate in the secondary xylem formation. MiRNA-target analysis revealed that a total of 85 DEMs were predicted to have 2948 putative targets. Among them, PagmiR396d-PagGRFs, PagmiR395c-PagGA2ox1/PagLHW/PagSULTR2/PagPolyubiquitin 1, PagmiR482d-PagLAC4, PagmiR167e-PagbHLH62, and PagmiR167f/g/h-PagbHLH110 modules were involved in the regulating cambial activity and its differentiation into secondary xylem, cell expansion, secondary cell wall deposition, and programmed cell death. Our results give new insights into the regulatory network and mechanism of secondary xylem formation.

Transcriptomic monitoring of Douglas-fir heartwood formation.

OBJECTIVES: Molecular cues linked to heartwood formation open new (complementary) perspectives to genetic breeding programs of Douglas-fir, a tree species largely cultivated in Europe for the natural durability and civil engineering properties of its wood. DATA DESCRIPTION: RNAs from a single genotype of Douglas-fir, extracted from three distinct wood zones (outer sapwood, inner sapwood and transition zone) at four vegetative seasons to generate an extensive RNA-seq dataset used to apprehend the in-wood dynamic and seasonality of heartwood formation in this hardwood model species. Previously published data collected on somatic embryos of the same genotype could be merged with the present dataset to upgrade grade the Douglas-fir reference transcriptome.

Genes expression profiles in vascular cambium of Eucalyptus urophylla × Eucalyptus grandis at different ages.

BACKGROUND: Wood is a secondary xylem generated by vascular cambium. Vascular cambium activities mainly include cambium proliferation and vascular tissue formation through secondary growth, thereby producing new secondary phloem inward and secondary xylem outward and leading to continuous tree thickening and wood formation. Wood formation is a complex biological process, which is strictly regulated by multiple genes. Therefore, molecular level research on the vascular cambium of different tree ages can lead to the identification of both key and related genes involved in wood formation and further explain the molecular regulation mechanism of wood formation. RESULTS: In the present study, RNA-Seq and Pac-Bio Iso-Seq were used for profiling gene expression changes in Eucalyptus urophylla × Eucalyptus grandis (E. urograndis) vascular cambium at four different ages. A total of 59,770 non-redundant transcripts and 1892 differentially expressed genes (DEGs) were identified. The expression trends of the DEGs related to cell division and differentiation, cell wall biosynthesis, phytohormone, and transcription factors were analyzed. The DEGs encoding expansin, kinesin, cycline, PAL, GRP9, KNOX, C2C2-dof, REV, etc., were highly expressed in E. urograndis at three years old, leading to positive effects on growth and development. Moreover, some gene family members, such as NAC, MYB, HD-ZIP III, RPK, and RAP, play different regulatory roles in wood formation because of their sophisticated transcriptional network and function redundantly. CONCLUSIONS: These candidate genes are a potential resource to further study wood formation, especially in fast-growing and adaptable eucalyptus. The results may also serve as a basis for further research to unravel the molecular mechanism underlying wood formation.

Seasonal Developing Xylem Transcriptome Analysis of Pinus densiflora Unveils Novel Insights for Compression Wood Formation.

Wood is the most important renewable resource not only for numerous practical utilizations but also for mitigating the global climate crisis by sequestering atmospheric carbon dioxide. The compressed wood (CW) of gymnosperms, such as conifers, plays a pivotal role in determining the structure of the tree through the reorientation of stems displaced by environmental forces and is characterized by a high content of lignin. Despite extensive studies on many genes involved in wood formation, the molecular mechanisms underlying seasonal and, particularly, CW formation remain unclear. This study examined the seasonal dynamics of two wood tissue types in Pinus densiflora: CW and opposite wood (OW). RNA sequencing of developing xylem for two consecutive years revealed comprehensive transcriptome changes and unique differences in CW and OW across seasons. During growth periods, such as spring and summer, we identified 2255 transcripts with differential expression in CW, with an upregulation in lignin biosynthesis genes and significant downregulation in stress response genes. Notably, among the laccases critical for monolignol polymerization, PdeLAC17 was found to be specifically expressed in CW, suggesting its vital role in CW formation. PdeERF4, an ERF transcription factor preferentially expressed in CW, seems to regulate PdeLAC17 activity. This research provides an initial insight into the transcriptional regulation of seasonal CW development in P. densiflora, forming a foundation for future studies to enhance our comprehension of wood formation in gymnosperms.

The first multi-tissue genome-scale metabolic model of a woody plant highlights suberin biosynthesis pathways in Quercus suber.

Over the last decade, genome-scale metabolic models have been increasingly used to study plant metabolic behaviour at the tissue and multi-tissue level under different environmental conditions. Quercus suber, also known as the cork oak tree, is one of the most important forest communities of the Mediterranean/Iberian region. In this work, we present the genome-scale metabolic model of the Q. suber (iEC7871). The metabolic model comprises 7871 genes, 6231 reactions, and 6481 metabolites across eight compartments. Transcriptomics data was integrated into the model to obtain tissue-specific models for the leaf, inner bark, and phellogen, with specific biomass compositions. The tissue-specific models were merged into a diel multi-tissue metabolic model to predict interactions among the three tissues at the light and dark phases. The metabolic models were also used to analyse the pathways associated with the synthesis of suberin monomers, namely the acyl-lipids, phenylpropanoids, isoprenoids, and flavonoids production. The models developed in this work provide a systematic overview of the metabolism of Q. suber, including its secondary metabolism pathways and cork formation.

Association Mapping and Expression Analysis of the Genes Involved in the Wood Formation of Poplar.

Xylogenesis is a complex and sequential biosynthetic process controlled by polygenes. Deciphering the genetic architecture of this complex quantitative trait could provide valuable information for increasing wood biomass and improving its properties. Here, we performed genomic resequencing of 64 24-year-old trees (64 hybrids of section Aigeiros and their parents) grown in the same field and conducted full-sib family-based association analyses of two growth and six woody traits using GEMMA as a choice of association model selection. We identified 1342 significantly associated single nucleotide polymorphisms (SNPs), 673 located in the region upstream and downstream of 565 protein-encoding genes. The transcriptional regulation network of secondary cell wall (SCW) biosynthesis was further constructed based on the published data of poplar miRNA, transcriptome, and degradome. These provided a certain scientific basis for the in-depth understanding of the mechanism of poplar timber formation and the molecular-assisted breeding in the future.