Search

Key message: Fluorescence in situ hybridization with frozen sections of root tips showed difference of chromosome territories distribution between autosome and sex-chromosome homologous pairs in Populus trichocarpa. The spatial organization of chromatin within the interphase nucleus and the interactions between chromosome territories (CTs) are essential for various biologic processes. Three-dimensional fluorescence in situ hybridization (3D-FISH) is a powerful tool for analyzing CTs, but its application in plants is limited. In this study, we established a 3D-FISH technique using frozen sections of Populus trichocarpa root tips, which was an improvement over the use of paraffin sections and enabled us to acquire good FISH signals. Using chromosome-specific oligo probes, we were able to analyze CTs in interphase nuclei in three dimensions. The distribution of chromosome pairs 17 and 19 in the 3D-preserved nuclei of P. trichocarpa root tip cells were analyzed and showed that the autosome pair 17 associated more often than sex chromosome 19. This research lays a foundation for further study of the spatial position of chromosomes in the nucleus and the relationship between gene expression and spatial localization of chromosomes in poplar. [ABSTRACT FROM AUTHOR]

The Remorin (REM) gene family is a plant-specific, oligomeric, filamentous family protein located on the cell membrane, which is important for plant growth and stress responses. In this study, a total of 22 PtREMs were identified in the genome of Populus trichocarpa. Subcellular localization analysis showed that they were predictively distributed in the cell membrane and nucleus. Only five PtREMs members contain both Remorin_C- and Remorin_N-conserved domains, and most of them only contain the Remorin_C domain. A total of 20 gene duplication pairs were found, all of which belonged to fragment duplication. Molecular evolutionary analysis showed the PtREMs have undergone purified selection. Lots of cis-acting elements assigned into categories of plant growth and development, stress response, hormone response and light response were detected in the promoters of PtREMs. PtREMs showed distinct gene expression patterns in response to diverse stress conditions where the mRNA levels of PtREM4.1, PtREM4.2 and PtREM6.11 were induced in most cases. A co-expression network centered by PtREMs was constructed to uncover the possible functions of PtREMs in protein modification, microtube-based movement and hormone signaling. The obtained results shed new light on understanding the roles of PtREMs in coping with environmental stresses in poplar species. [ABSTRACT FROM AUTHOR]

The BTB (Broad-complex, tramtrack, and bric-a-brac) gene family, characterized by a highly conserved BTB domain, is implicated in a spectrum of biological processes, encompassing growth and development, as well as stress responses. Characterization and functional studies of BTB genes in poplar are still limited, especially regarding their response to hormones and biotic/abiotic stresses. In this study, we conducted an HMMER search in conjunction with BLASTp and identified 95 BTB gene models in Populus trichocarpa. Through domain motif and phylogenetic relationship analyses, these proteins were classified into eight families, NPH3, TAZ, Ankyrin, only BTB, BACK, Armadillo, TPR, and MATH. Collinearity analysis of poplar BTB genes with homologs in six other species elucidated evolutionary relationships and functional conservations. RNA-seq analysis of five tissues of poplar identified BTB genes as playing a pivotal role during developmental processes. Comprehensive RT-qPCR analysis of 11 BTB genes across leaves, roots, and xylem tissues revealed their responsive expression patterns under diverse hormonal and biotic/abiotic stress conditions, with varying degrees of regulation observed in the results. This study marks the first in-depth exploration of the BTB gene family in poplar, providing insights into the potential roles of BTB genes in hormonal regulation and response to stress. [ABSTRACT FROM AUTHOR]

Population demographic changes, alongside landscape, geographic and climate heterogeneity, can influence the timing, stability and extent of introgression where species hybridise. Thus, quantifying interactions across diverged lineages, and the relative contributions of interspecific genetic exchange and selection to divergence at the genome‐wide level is needed to better understand the drivers of hybrid zone formation and maintenance. We used seven latitudinally arrayed transects to quantify the contributions of climate, geography and landscape features to broad patterns of genetic structure across the hybrid zone of Populus trichocarpa and P. balsamifera and evaluated the demographic context of hybridisation over time. We found genetic structure differed among the seven transects. While ancestry was structured by climate, landscape features influenced gene flow dynamics. Demographic models indicated a secondary contact event may have influenced contemporary hybrid zone formation with the origin of a putative hybrid lineage that inhabits regions with higher aridity than either of the ancestral groups. Phylogenetic relationships based on chloroplast genomes support the origin of this hybrid lineage inferred from demographic models based on the nuclear data. Our results point towards the importance of climate and landscape patterns in structuring the contact zones between P. trichocarpa and P. balsamifera and emphasise the value whole genome sequencing can have to advancing our understanding of how neutral processes influence divergence across space and time. [ABSTRACT FROM AUTHOR]

Thaumatin-like proteins (TLPs) in plants are involved in diverse biotic and abiotic stresses, including antifungal activity, low temperature, drought, and high salinity. However, the roles of the TLP genes are rarely reported in early flowering. Here, the TLP gene family was identified in P. trichocarpa. The 49 PtTLP genes were classified into 10 clusters, and gene structures, conserved motifs, and expression patterns were analyzed in these PtTLP genes. Among 49 PtTLP genes, the PtTLP6 transcription level is preferentially high in stems, and GUS staining signals were mainly detected in the phloem tissues of the PtTLP6pro::GUS transgenic poplars. We generated transgenic Arabidopsis plants overexpressing the PtTLP6 gene, and its overexpression lines showed early flowering phenotypes. However, the expression levels of main flowering regulating genes were not significantly altered in these PtTLP6-overexpressing plants. Our data further showed that overexpression of the PtTLP6 gene led to a reactive oxygen species (ROS) burst in Arabidopsis, which might advance the development process of transgenic plants. In addition, subcellular localization of PtTLP6-fused green fluorescent protein (GFP) was in peroxisome, as suggested by tobacco leaf transient transformation. Overall, this work provides a comprehensive analysis of the TLP gene family in Populus and an insight into the role of TLPs in woody plants. [ABSTRACT FROM AUTHOR]

Summary: Community genetics seeks to understand the mechanisms by which natural genetic variation in heritable host phenotypes can encompass assemblages of organisms such as bacteria, fungi, and many animals including arthropods. Prior studies that focused on plant genotypes have been unable to identify genes controlling community composition, a necessary step to predict ecosystem structure and function as underlying genes shift within plant populations.We surveyed arthropods within an association population of Populus trichocarpa in three common gardens to discover plant genes that contributed to arthropod community composition. We analyzed our surveys with traditional single‐trait genome‐wide association analysis (GWAS), multitrait GWAS, and functional networks built from a diverse set of plant phenotypes.Plant genotype was influential in structuring arthropod community composition among several garden sites. Candidate genes important for higher level organization of arthropod communities had broadly applicable functions, such as terpenoid biosynthesis and production of dsRNA binding proteins and protein kinases, which may be capable of targeting multiple arthropod species.We have demonstrated the ability to detect, in an uncontrolled environment, individual genes that are associated with the community assemblage of arthropods on a host plant, further enhancing our understanding of genetic mechanisms that impact ecosystem structure. [ABSTRACT FROM AUTHOR]

The B-box proteins (BBXs) encode a family of zinc-finger transcription factors that regulate the plant circadian rhythm and early light morphogenesis. The double B-box (DBB) family is in the class of the B-box family, which contains two conserved B-box domains and lacks a CCT (CO, CO-like and TOC1) motif. In this study, the identity, classification, structures, conserved motifs, chromosomal location, cis elements, duplication events, and expression profiles of the PtrDBB genes were analyzed in the woody model plant Populus trichocarpa. Here, 12 PtrDBB genes (PtrDBB1–PtrDBB12) were identified and classified into four distinct groups, and all of them were homogeneously spread among eight out of seventeen poplar chromosomes. The collinearity analysis of the DBB family genes from P. trichocarpa and two other species (Z. mays and A. thaliana) indicated that segmental duplication gene pairs and high-level conservation were identified. The analysis of duplication events demonstrates an insight into the evolutionary patterns of DBB genes. The previously published transcriptome data showed that PtrDBB genes represented distinct expression patterns in various tissues at different stages. In addition, it was speculated that several PtrDBBs are involved in the responsive to drought stress, light/dark, and ABA and MeJA treatments, which implied that they might function in abiotic stress and phytohormone responses. In summary, our results contribute to the further understanding of the DBB family and provide a reference for potential functional studies of PtrDBB genes in P. trichocarpa. [ABSTRACT FROM AUTHOR]

Summary: Histone H3 lysine‐4 trimethylation (H3K4me3) activating drought‐responsive genes in plants for drought adaptation has long been established, but the underlying regulatory mechanisms are unknown.Here, using yeast two‐hybrid, bimolecular fluorescence complementation, biochemical analyses, transient and CRISPR‐mediated transgenesis in Populus trichocarpa, we unveiled in this adaptation a regulatory interplay between chromatin regulation and gene transactivation mediated by an epigenetic determinant, a PtrSDG2‐1–PtrCOMPASS (complex proteins associated with Set1)‐like H3K4me3 complex, PtrSDG2‐1–PtrWDR5a‐1–PtrRbBP5‐1–PtrAsh2‐2 (PtrSWRA).Under drought conditions, a transcription factor PtrAREB1‐2 interacts with PtrSWRA, forming a PtrSWRA–PtrAREB1‐2 pentamer, to recruit PtrSWRA to specific promoter elements of drought‐tolerant genes, such as PtrHox2, PtrHox46, and PtrHox52, for depositing H3K4me3 to promote and maintain activated state of such genes for tolerance. CRISPR‐edited defects in the pentamer impaired drought tolerance and elevated expression of PtrHox2, PtrHox46, or PtrHox52 improved the tolerance as well as growth in P. trichocarpa.Our findings revealed the identity of the underlying H3K4 trimethyltransferase and its interactive arrangement with the COMPASS for catalysis specificity and efficiency. Furthermore, our study uncovered how the H3K4 trimethyltransferase–COMPASS complex is recruited to the effector genes for elevating H3K4me3 marks for improved drought tolerance and growth/biomass production in plants. [ABSTRACT FROM AUTHOR]

Primary and secondary growth of trees are needed for increments in plant height and stem diameter, respectively, affecting the production of woody biomass for applications in timber, pulp/paper, and related biomaterials. These two types of growth are believed to be both regulated by distinct transcription factor (TF)-mediated regulatory pathways. Notably, we identified PtrLBD39, a highly stem phloem-specific TF in Populus trichocarpa and found that the ectopic expression of PtrLBD39 in P. trichocarpa markedly retarded both primary and secondary growth. In these overexpressing plants, the RNA-seq, ChIP-seq, and weighted gene co-expression network analysis (WGCNA) revealed that PtrLBD39 directly or indirectly regulates TFs governing vascular tissue development, wood formation, hormonal signaling pathways, and enzymes responsible for wood components. This regulation led to growth inhibition, decreased fibrocyte secondary cell wall thickness, and reduced wood production. Therefore, our study indicates that, following ectopic expression in P. trichocarpa, PtrLBD39 functions as a repressor influencing both primary and secondary growth. [ABSTRACT FROM AUTHOR]

Buds of poplar trees (Populus species) are often covered with sticky, usually polyphenol-rich, exudates. Moreover, accessible data showed that some Populus bud extracts may be excellent antibacterial agents, especially against Gram-positive bacteria. Due to the fragmentary nature of the data found, we conducted a systematic screening study. The antimicrobial activity of two extract types (semi-polar—ethanolic and polar—ethanolic-water (50/50; V/V)) from 27 bud samples of different poplar taxons were compared. Antimicrobial assays were performed against Gram-positive (five strains) and Gram-negative (six strains) bacteria as well as fungi (three strains) and covered the determination of minimal inhibitory, bactericidal, and fungicidal concentrations. The composition of extracts was later investigated by ultra-high-performance liquid chromatography coupled with ultraviolet detection (UHPLC-DAD) and with electrospray-quadrupole-time-of-flight tandem mass spectrometry (UHPLC-ESI-qTOF-MS). As a result, most of the extracts exhibited good (MIC ≤ 62.5 µg/mL) or moderate (62.5 < MIC ≤ 500 µg/mL) activity against Gram-positives and Helicobacter pylori, as well as fungi. The most active were ethanolic extracts from P. trichocarpa, P. trichocarpa clone 'Robusta', and P. tacamahaca × P. trichocarpa. The strongest activity was observed for P. tacamahaca × P. trichocarpa. Antibacterial activity was supposedly connected with the abundant presence of flavonoids (pinobanksin, pinobanksin 3-acetate, chrysin, pinocembrin, galangin, isosakuranetin dihydrochalcone, pinocembrin dihydrochalcone, and 2′,6′-dihydroxy-4′-methoxydihydrochalcone), hydroxycinnamic acids monoesters (p-methoxycinnamic acid cinnamyl ester, caffeic acid phenethylate and different isomers of prenyl esters), and some minor components (balsacones). [ABSTRACT FROM AUTHOR]

Populus trichocarpa (poplar) is a fast-growing model tree whose lignocellulosic biomass is a promising biofuel feedstock. Enhancing its viability and yield in non-arable drought-prone lands can reduce biomass costs and accelerate adoption as a biofuel crop. Data from extensive -omics and phenotypic studies were leveraged herein to reconstruct a multi-tissue (root, stem and leaf) genome-scale model (GSM) of poplar, i Potri3463, encompassing 14 360 reactions, 12 402 metabolites and 3463 genes. Two condition-specific GSMs were extracted from i Potri3463: i Potri3016C (control) and i Potri2999D (drought), supported by condition-specific transcript levels and reaction essentiality for growth. Physiological constraints consistent with experimental measurements of drought-stressed plants were imposed on growth, photorespiration and carbon assimilation rates. Calculated increased flux capacity through the violaxanthin cycle and GABA biosynthetic pathways agree with established key strategies for improving drought tolerance. Differential gene expression analysis was performed on existing transcriptomes of poplar under different watering regimes. Computational flux knockdown was applied to reactions with increased flux capacity under drought that were associated with at least one downregulated gene. Several such reactions were essential for maintaining observed biomass yield and their associated genes are candidates for overexpression to improve drought tolerance. Glutamine synthetase is one whose overexpression in poplar confirms in silico predictions. However, the two most promising candidates are genes encoding ferulate-5-hydroxylase, Potri.007G016400 and Potri.005G117500, as their overexpression in other plant species led to demonstrably improved drought tolerance while previous overexpression in poplar reduced biomass recalcitrance. i Potri3463 is the first poplar-specific whole-plant GSM and the second one available for a woody plant. [ABSTRACT FROM AUTHOR]

Background: The jasmonate ZIM domain (JAZ) protein is a key repressor of the jasmonate signal transduction pathway, which plays an important role in plant growth and development and defense responses. In this study, based on the published whole-genome data, we identified members of the JAZ gene family in Populus trichocarpa. Through a series of bioinformatic approaches, their expression patterns under various stress conditions have been analyzed to explore and excavate the endogenous resistance genes of poplar and provide a theoretical basis for breeding new varieties of poplar resistance. Results: A total of 13 PtJAZ genes have been identified in P. trichocarpa and designated as PtJAZ1–PtJAZ13. Those 13 PtJAZ genes were unevenly distributed on nine chromosomes, and they could be divided into four subfamilies. The gene structures and motif composition of the members derived from the same subfamily were similar. Collinearity analysis demonstrated that, compared with Arabidopsis thaliana and Oryza sativa, the most collinear pairs (13) were found in P. trichocarpa and Eucalyptus robusta. Cis-acting element analysis suggested that the promoter regions of PtJAZs contained a large number of hormones and stress response elements, of which abscisic acid (ABA) and methyl jasmonate (MeJA) hormone response elements were the most abundant. The PtJAZ genes not only had diverse expression patterns in different tissues, but they also responded to various abiotic and biotic stress conditions. The co-expression network and GO and KEGG analyses showed that JAZ genes were closely related to insect resistance. Conclusions: In this study, applying bioinformatic methods, 13 PtJAZ gene family members from P. trichocarpa were identified and comprehensively analyzed. By further studying the function of the poplar JAZ gene family, the aim is to select genes with better insect resistance and stress resistance so as to lay a solid foundation for the subsequent breeding of new poplar varieties. [ABSTRACT FROM AUTHOR]

Roots are the main organ for water uptake and the earliest part of a plant's response to drought, making them of great importance to our understanding of the root system's response to drought. However, little is known about the underlying molecular mechanisms that control root responses to drought stress. Here, we identified and functionally characterized the AP2/ERF family transcription factor (TF) PtrABR1 and the upstream target gene zinc-finger protein TF PtrYY1, which respond to drought stress by promoting the growth and development of lateral roots in Populus trichocarpa. A root-specific induction of PtrABR1 under drought stress was explored. The overexpression of PtrABR1 (PtrABR1-OE) promoted root growth and development, thereby increasing tolerance to drought stress. In addition, PtrYY1 is directly bound to the promoter of PtrABR1 under drought stress, and the overexpression of PtrYY1 (PtrYY1-OE) promoted lateral root growth and development and increased tolerance to drought stress. An RNA-seq analysis of PtrABR1-OE with wild-type (WT) poplar identified PtrGH3.6 and PtrPP2C44, which share the same pattern of expression changes as PtrABR1. A qRT-PCR and cis-element analysis further suggested that PtrGH3.6 and PtrPP2C44 may act as potential downstream targets of PtrABR1 genes in the root response pathway to drought stress. In conclusion, these results reveal a novel drought regulatory pathway in which PtrABR1 regulates the network through the upstream target gene PtrYY1 and the potential downstream target genes PtrGH3.6 and PtrPP2C44, thereby promoting root growth and development and improving tolerance to drought stress. [ABSTRACT FROM AUTHOR]

Polygalacturonases inhibiting proteins(PGIPs) are produced by plants to specifically recognize pathogen-secreted polygalacturonases, further inhibiting degradation of pectin, an important component of plant cell wall by pathogen, which enhances the resistance to plant pathogens. To explore the disease resistance mechanism of PGIP in a model tree species of Poplar, in this study, the members of PGIP(PtPGIP) family in Populus trichocarpa were identified, physical and chemical properties and structure of proteins, phylogeny, and gene expression patterns of PGIP(PtPGIP) family in Populus trichocarpa were analyzed. The results showed that four PtPGIP genes were identified in the Populus trichocarpa. PtPGIP1 and PtPGI2 were tandem duplications and located on Chr. 6, PtPGIP3 and PtPGIP4 were tandem duplications and located on Chr. 16, PtPGIP1 was a pseudogen, and PtPGIP2, PtPGIP3, and PtPGIP4 possessed a complete PGIP protein domain. All PtPGIPs were hydrophobic proteins, had good lipophilicity and 3 to 7 N-glycosylation sites. Subcellular localization prediction indicated that they were probably located outside the cell. PtPGIP2, PtPGIP3, and PtPGIP4 contained 10 LRR fragments except for the PtPGIP1. The conserved sequence xxLxLxx in LRR as well as protein molecular docking showed differences. PtPGIP genes revealed tissue-specific expression. The expression levels of PtPGIP1 were low in various tissues. While the expression levels of PtPGIP2 and PtPGIP4 in roots, seedlings, and inflorescences were higher than those in leaves, especially the expression level of PtPGIP2 was the highest in inflorescences, indicating that the PGIP gene family of Populus trichocarpa had undergone functional differentiation, which made it play an important role in coping with various adversity stresses. [ABSTRACT FROM AUTHOR]

The Golden2-like (GLK) transcription factors are plant-specific transcription factors (TFs) that perform extensive and significant roles in regulating chloroplast development. Here, genome-wide identification, classification, conserved motifs, cis-elements, chromosomal locations, evolution and expression patterns of the PtGLK genes in the woody model plant Populus trichocarpa were analyzed in detail. In total, 55 putative PtGLKs (PtGLK1-PtGLK55) were identified and divided into 11 distinct subfamilies according to the gene structure, motif composition and phylogenetic analysis. Synteny analysis showed that 22 orthologous pairs and highly conservation between regions of GLK genes across P. trichocarpa and Arabidopsis were identified. Furthermore, analysis of the duplication events and divergence times provided insight into the evolutionary patterns of GLK genes. The previously published transcriptome data indicated that PtGLK genes exhibited distinct expression patterns in various tissues and different stages. Additionally, several PtGLKs were significantly upregulated under the responses of cold stress, osmotic stress, and methyl jasmonate (MeJA) and gibberellic acid (GA) treatments, implying that they might take part in abiotic stress and phytohormone responses. Overall, our results provide comprehensive information on the PtGLK gene family and elucidate the potential functional characterization of PtGLK genes in P. trichocarpa. [ABSTRACT FROM AUTHOR]

The strictosidine synthase-like (SSL) gene family is a small plant immune-regulated gene family that plays a critical role in plant resistance to biotic/abiotic stresses. To date, very little has been reported on the SSL gene in plants. In this study, a total of thirteen SSLs genes were identified from poplar, and these were classified into four subgroups based on multiple sequence alignment and phylogenetic tree analysis, and members of the same subgroup were found to have similar gene structures and motifs. The results of the collinearity analysis showed that poplar SSLs had more collinear genes in the woody plants Salix purpurea and Eucalyptus grandis. The promoter analysis revealed that the promoter region of PtrSSLs contains a large number of biotic/abiotic stress response elements. Subsequently, we examined the expression patterns of PtrSSLs following drought, salt, and leaf blight stress, using RT-qPCR to validate the response of PtrSSLs to biotic/abiotic stresses. In addition, the prediction of transcription factor (TF) regulatory networks identified several TFs, such as ATMYB46, ATMYB15, AGL20, STOP1, ATWRKY65, and so on, that may be induced in the expression of PtrSSLs in response to adversity stress. In conclusion, this study provides a solid basis for a functional analysis of the SSL gene family in response to biotic/abiotic stresses in poplar. [ABSTRACT FROM AUTHOR]

Background: High-throughput metabolomics analytical methodology is needed for population-scale studies of bioenergy-relevant feedstocks such as poplar (Populus sp.). Here, the authors report the relative abundance of extractable aromatic metabolites in Populus trichocarpa leaves rapidly estimated using pyrolysis-molecular beam mass spectrometry (py-MBMS). Poplar leaves were analyzed in conjunction with and validated by GC/MS analysis of extracts to determine key spectral features used to build PLS models to predict the relative composition of extractable aromatic metabolites in whole poplar leaves. Results: The Pearson correlation coefficient for the relative abundance of extractable aromatic metabolites based on ranking between GC/MS analysis and py-MBMS analysis of the Boardman leaf set was 0.86 with R2 = 0.76 using a simplified prediction approach from select ions in MBMS spectra. Metabolites most influential to py-MBMS spectral features in the Clatskanie set included the following compounds: catechol, salicortin, salicyloyl-coumaroyl-glucoside conjugates, α-salicyloylsalicin, tremulacin, as well as other salicylates, trichocarpin, salicylic acid, and various tremuloidin conjugates. Ions in py-MBMS spectra with the highest correlation to the abundance of extractable aromatic metabolites as determined by GC/MS analysis of extracts, included m/z 68, 71, 77, 91, 94, 105, 107, 108, and 122, and were used to develop the simplified prediction approach without PLS models or a priori measurements. Conclusions: The simplified py-MBMS method is capable of rapidly screening leaf tissue for relative abundance of extractable aromatic secondary metabolites to enable prioritization of samples in large populations requiring comprehensive metabolomics that will ultimately inform plant systems biology models and advance the development of optimized biomass feedstocks for renewable fuels and chemicals. [ABSTRACT FROM AUTHOR]

Dissection of regulatory network that control wood structure is highly challenging in functional genomics. Nevertheless, due to the availability of genomic, transcriptomic and proteomic sequences, a large amount of information is available for use in achieving this goal. MicroRNAs, which compose a class of small non-coding RNA molecules that inhibit protein translation by targeting mRNA cleavage sites and thus regulate a wide variety of developmental and physiological processes in plants, are important parts of this regulatory network. These findings and the availability of sequence information have made it possible to carry out an in silico analysis to predict and annotate miRNAs and their target genes associated with an important factor affecting wood rigidity, microfibril angle (MFA), throughout the Populus trichocarpa Hook. genome. Our computational approach revealed miRNAs and their targets via ESTs, sequences putatively associated with microfibril angle. In total, 250 miRNAs were identified as RNA molecules with roles in the silencing and post-transcriptional regulation of the expression of nine genes. We found SHY2, IAA4 (ATAUX2–11), BZIP60, AP2, MYB15, ABI3, MYB17, LAF1 and MYB28 as important nodes in a network with possible role in MFA determination. Other co-expressed genes putatively involved in this regulatory system were also identified by construction of a co-expression network. The candidate genes from this study may help unravel the regulatory networks putatively linked to microfibril angle. [ABSTRACT FROM AUTHOR]

Carbamoyl phosphate synthetase (CPS) catalyses the synthesis of ammonia carbamoyl phosphate (CP), which plays a key role in the biosynthesis of arginine and pyrimidine nucleotides. There are two subunits of the CPS enzyme in Populus trichocarpa, CarA (small subunit) and CarB (large subunit). Only when they coexist can CPS catalyse synthesis of CP. However, it is not clear how CPS responds to nitrogen (N) to affect arginine and pyrimidine nucleotide biosynthesis.In this study, bioinformatics methods were used to analyse the expression patterns of genes encoding CarA and CarB, and qRT‐PCR and RNA‐seq were used to investigate their molecular responses under different N concentrations.Phylogenetic analysis revealed that the phylogenetic trees of CarA and CarB had similar topologies. qRT‐PCR showed that the PtCarA and PtCarB genes were regulated by N, while their N‐regulated patterns differed in different tissues. The expression patterns of PtCarA and PtCarB show a significant positive correlation according to qRT‐PCR and RNA‐seq. The analysis of promoter cis‐acting elements showed that the promoter regions of PtCarA1, PtCarA2 and PtCarB contained some identical cis‐acting elements. According to analysis of the phylogenetic tree, expression patterns and promoter elements, we speculate that there might be coevolution among PtCarA1, PtCarA2 and PtCarB.This study provides valuable information for further understanding the function of CPS in poplar, especially for N response, and provides new ideas for studying the evolution of gene families related to heteromultimers. [ABSTRACT FROM AUTHOR]

Auxin is a key regulator that virtually controls almost every aspect of plant growth and development throughout its life cycle. As the major components of auxin signaling, auxin response factors (ARFs) play crucial roles in various processes of plant growth and development. In this study, a total of 35 PtrARF genes were identified, and their phylogenetic relationships, chromosomal locations, synteny relationships, exon/intron structures, cis-elements, conserved motifs, and protein characteristics were systemically investigated. We also analyzed the expression patterns of these PtrARF genes and revealed that 16 of them, including PtrARF1, 3, 7, 11, 13–17, 21, 23, 26, 27, 29, 31, and 33, were preferentially expressed in primary stems, while 15 of them, including PtrARF2, 4, 6, 9, 10, 12, 18–20, 22, 24, 25, 28, 32, and 35, participated in different phases of wood formation. In addition, some PtrARF genes, with at least one cis-element related to indole-3-acetic acid (IAA) or abscisic acid (ABA) response, responded differently to exogenous IAA and ABA treatment, respectively. Three PtrARF proteins, namely PtrARF18, PtrARF23, and PtrARF29, selected from three classes, were characterized, and only PtrARF18 was a transcriptional self-activator localized in the nucleus. Moreover, Y2H and bimolecular fluorescence complementation (BiFC) assay demonstrated that PtrARF23 interacted with PtrIAA10 and PtrIAA28 in the nucleus, while PtrARF29 interacted with PtrIAA28 in the nucleus. Our results provided comprehensive information regarding the PtrARF gene family, which will lay some foundation for future research about PtrARF genes in tree development and growth, especially the wood formation, in response to cellular signaling and environmental cues. [ABSTRACT FROM AUTHOR]

Fasciclin-like arabinogalactan proteins (FLAs) play an important role in plant development and adaptation to the environment. However, the roles of FLAs in wood formation remain poorly understood. Here, we identified a total of 50 PtrFLA genes in poplar. They were classified into four groups: A to D, among which group A was the largest group with 28 members clustered into four branches. Most PtrFLAs of group A were dominantly expressed in developing xylem based on microarray and RT-qPCR data. The roles of PtrFLA40 and PtrFLA45 in group A were investigated via the Cas9/gRNA-induced mutation lines. Loss of PtrFLA40 and PtrFLA45 increased stem length and diameter in ptrfla40ptrfla45 double mutants, but not in ptrfla40 or ptrfla45 single mutants. Further, our findings indicated that the ptrfla40ptrfla45 mutants enlarged the cell size of xylem fibers and vessels, suggesting a negative modulation in stem xylem cell size. In addition, wood lignin content in the ptrfla40fla45 mutants was increased by nearly 9%, and the lignin biosynthesis-related genes were significantly up-regulated in the ptrfla40fla45 mutants, in agreement with the increase in wood lignin content. Overall, Cas9/gRNA-mediated mutations in PtrFLA40 and PtrFLA45 reveal redundant roles in modulating wood cell size and secondary cell wall (SCW) synthesis in poplar. [ABSTRACT FROM AUTHOR]

Aim: The biogeographic history affects adaptive evolution by altering the pattern of genetic variation and provides the background upon which other evolutionary processes are operating. Assessment of the consequence of such eco‐evolutionary interactions is crucial to population's genetic diversity maintenance. Yet, considering how biogeographic and multiple evolutionary processes interact in complex environments to alter genetic diversity in conservation planning remains unresolved. Location: The Pacific Northwest. Methods: Herein I document the impact of geographic landscapes and past climatic fluctuations on the evolutionary processes that drive population divergence of Populus trichocarpa by assembling genomic, climatic and species occurrence data. Results: Based on inferences of demographic history of the species and tests for niche divergence, results show that the British Columbia (BC)‐North and BC‐South populations are ancestral and subject to more recent bottlenecks than the Oregon population. Consistently, ecological niche modelling illustrates that compared to the Last Glacial Maximum (~21 Ka), the present niche suitability of the BC‐North and ‐South populations is overall marginally inferior to that of the Oregon population. However, genomic analysis demonstrates that the Oregon harbours the lowest genetic diversity, possibly due to an extended bottleneck experienced by the population. Model prediction indicates that in the 2050s, the impact of climate change is minor compared to the niche suitability of the species as a whole, but climate change will likely result in considerable genetic shifts over two generations especially in the Oregon and BC‐South populations, which will attenuate along a northward gradient. Main conclusions: This study underpins the long‐lasting impact of demographic histories and natural selection on genetic diversity, rather than highlighting the consequence of recent demographic events (e.g. bottleneck in the Holocene epoch; ~12 Ka) potentially cascading to eroding genetic diversity and yielding conservation urgency. [ABSTRACT FROM AUTHOR]

Programmed cell death (PCD) is essential for wood development in trees. However, the determination of crucial factors involved in xylem PCD of wood development is still lacking. Here, two Populus trichocarpa typical aspartic protease (AP) genes, AP17 and AP45 , modulate xylem maturation, especially fibre PCD, during wood formation. AP17 and AP45 were dominantly expressed in the fibres of secondary xylem, as suggested by GUS expression in APpro::GUS transgenic plants. Cas9/gRNA-induced AP17 or AP45 mutants delayed secondary xylem fibre PCD, and ap17ap45 double mutants showed more serious defects. Conversely, AP17 overexpression caused premature PCD in secondary xylem fibres, indicating a positive modulation in wood fibre PCD. Loss of AP17 and AP45 did not alter wood fibre wall thickness, whereas the ap17ap45 mutants showed a low lignin content in wood. However, AP17 overexpression led to a significant decrease in wood fibre wall thickness and lignin content, revealing the involvement in secondary cell wall synthesis during wood formation. In addition, the ap17ap45 mutant and AP17 overexpression plants resulted in a significant increase in saccharification yield in wood. Overall, AP17 and AP45 are crucial modulators in xylem maturation during wood development, providing potential candidate genes for engineering lignocellulosic wood for biofuel utilization. [ABSTRACT FROM AUTHOR]

Populus trichocarpa (Black cottonwood) is a dominant timber-yielding tree that has become a notable model plant for genome-level insights in forest trees. The efficient transport and solubility of various glycoside-associated compounds is linked to Family-1 UDP-glycosyltransferase (EC 2.4.1.x; UGTs) enzymes. These glycosyltransferase enzymes play a vital role in diverse plant functions, such as regulation of hormonal homeostasis, growth and development (seed, flower, fiber, root, etc.), xenobiotic detoxification, stress response (salt, drought, and oxidative), and biosynthesis of secondary metabolites. Here, we report a genome-wide analysis of the P. trichocarpa genome that identified 191 putative UGTs distributed across all chromosomes (with the exception of chromosome 20) based on 44 conserved plant secondary product glycosyltransferase (PSPG) motif amino acid sequences. Phylogenetic analysis of the 191 Populus UGTs together with 22 referenced UGTs from Arabidopsis and maize clustered the putative UGTs into 16 major groups (A–P). Whole-genome duplication events were the dominant pattern of duplication among UGTs in Populus. A well-conserved intron insertion was detected in most intron-containing UGTs across eight examined eudicots, including Populus. Most of the UGT genes were found preferentially expressed in leaf and root tissues in general. The regulation of putative UGT expression in response to drought, salt and heat stress was observed based on microarray and available RNA sequencing datasets. Up- and down-regulated UGT expression models were designed, based on transcripts per kilobase million values, confirmed their maximally varied expression under drought, salt and heat stresses. Co-expression networking of putative UGTs indicated their maximum co-expression with cytochrome P450 genes involved in triterpenoid biosynthesis. Our results provide an important resource for the identification of functional UGT genes to manipulate abiotic stress responsive glycosylation in Populus. [ABSTRACT FROM AUTHOR]

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. • Poplar NF-YA family member PtrNF-YA6 has an important role in secondary cell wall formation. • PtrNF-YA6 protein is localized in the nucleus and specifically recognizes and binds SNBE and SMRE elements. • PtrNF-YA6 overexpression resulted in reduced xylem area and thinning of secondary cell walls. • PtrNF-YA6 can directly inhibit the expression of hemicellulose synthesis-related genes. [ABSTRACT FROM AUTHOR]

SUMMARY: Long non‐coding RNAs (lncRNAs) are emerging as versatile regulators in diverse biological processes. However, little is known about their cis‐ and trans‐regulatory contributions in gene expression under salt stress. Using 27 RNA‐seq data sets from Populus trichocarpa leaves, stems and roots, we identified 2988 high‐confidence lncRNAs, including 1183 salt‐induced differentially expressed lncRNAs. Among them, 301 lncRNAs have potential for positively affecting their neighboring genes, predominantly in a cis‐regulatory manner rather than by co‐transcription. Additionally, a co‐expression network identified six striking salt‐associated modules with a total of 5639 genes, including 426 lncRNAs, and in these lncRNA sequences, the DNA/RNA binding motifs are enriched. This suggests that lncRNAs might contribute to distant gene expression of the salt‐associated modules in a trans‐regulatory manner. Moreover, we found 30 lncRNAs that have potential to simultaneously cis‐ and trans‐regulate salt‐responsive homologous genes, and Ptlinc‐NAC72, significantly induced under long‐term salt stress, was selected for validating its regulation of the expression and functional roles of the homologs PtNAC72.A and PtNAC72.B (PtNAC72.A/B). The transient transformation of Ptlinc‐NAC72 and a dual‐luciferase assay of Ptlinc‐NAC72 and PtNAC72.A/B promoters confirmed that Ptlinc‐NAC72 can directly upregulate PtNAC72.A/B expression, and a presence/absence assay was further conducted to show that the regulation is probably mediated by Ptlinc‐NAC72 recognizing the tandem elements (GAAAAA) in the PtNAC72.A/B 5′ untranslated region (5′‐UTR). Finally, the overexpression of Ptlinc‐NAC72 produces a hypersensitive phenotype under salt stress. Altogether, our results shed light on the cis‐ and trans‐regulation of gene expression by lncRNAs in Populus and provides an example of long‐term salt‐induced Ptlinc‐NAC72 that could be used to mitigate growth costs by conferring plant resilience to salt stress. Significance Statement: Evidence of the cis‐ and trans‐regulation of gene expression by lncRNAs under salt stress is lacking. Here, we demonstrate the limits of cis‐regulation versus the limitless trans‐regulation of lncRNAs in potentially regulating gene expression under salt stress, and also demonstrate that Ptlinc‐NAC72 functions in delayed negative feedback under long‐term salt stress that could be exploited to maximize salt tolerance, thereby mitigating growth costs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

SUMMARY: Enzymes are essential components of all biological systems. The key characteristics of proteins functioning as enzymes are their substrate specificities and catalytic efficiencies. In plants, most genes encoding enzymes are members of large gene families. Within such families, the contributions of active site motifs to the functional divergence of duplicate genes have not been well elucidated. In this study, we identified 41 glutaredoxin (GRX) genes in the Populus trichocarpa genome. GRXs are ubiquitous enzymes in plants that play important roles in developmental and stress tolerance processes. In poplar, GRX genes were divided into four classes based on clear differences in gene structure and expression pattern, subcellular localization, enzymatic activity, and substrate specificity of the encoded proteins. Using site‐directed mutagenesis, this study revealed that the divergence of the active site motif among different classes of GRX proteins resulted in substrate switches and thus provided new insights into the molecular evolution of these important plant enzymes. [ABSTRACT FROM AUTHOR]

Lignin is essential for the characteristics and quality of timber. Nitrogen has significant effects on lignin contents in plants. Nitrogen has been found to affect wood quality in plantations and lignin content in plants. Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) is an important methyltransferase in lignin biosynthesis. However, the classification of woody plant CCoAOMT gene family members and the regulation mechanism of nitrogen are not clear. Bioinformatics methods were used to predict the members, classification, and transcriptional distribution of the CCoAOMT gene family in Populus trichocarpa. The results showed that there were five PtCCoAOMTs identified, and they could be divided into three sub-groups according to their structural and phylogenetic features. The results of tissue expression specificity analysis showed that: PtCCoAOMT1 was highly expressed in roots and internodes; PtCCoAOMT2 was highly expressed in roots, nodes, and internodes, PtCCoAOMT3 was highly expressed in stems; PtCCoAOMT4 was highly expressed in young leaves, and, PtCCoAOMT5 was highly expressed in roots. Different forms and concentrations of nitrogen had varying effects on the expression patterns of genes in different plant tissue types. The results of real-time PCR showed that the expression levels of PtCCoAOMT1 and PtCCoAOMT2 in stems increased significantly under different forms of nitrogen. PtCCoAOMT3 and PtCCoAOMT4 were induced by nitrate nitrogen in upper stems and lower leaves, respectively. PtCCoAOMT4 and PtCCoAOMT5 were induced by different concentrations of nitrate nitrogen in lower stems and roots, respectively. These results could provide valuable information for revealing the differences between functions and expression patterns of the various CCoAOMT gene family members under different forms and concentrations of exogenous nitrogen in poplar. [ABSTRACT FROM AUTHOR]

Aims: This study explored the specific molecular mechanism of Laccaria bicolor to help Populus trichocarpa resist infection by Botryosphaeria dothidea. Methods and Results: Transcriptome technology was used to sequence P. trichocarpa under disease stress, and a total of 6379 differentially expressed genes (DEGs) were identified. A total of 536 new DEGs were induced by L. bicolor during the infection of B. dothidea. L. bicolor helps to prevent and alleviate the infection of B. dothidea by regulating related genes in the cell wall pathway, signal transduction pathway, disease‐resistant protein synthesis pathway and antioxidant enzyme synthesis pathway of P. trichocarpa. Conclusion: The inoculation of L. bicolor can regulate the expression of genes in the cell wall pathway and enhance the physical defense capabilities of plants. Under disease stress conditions, L. bicolor can regulate signal transduction pathways, disease‐resistant related pathways and reactive oxygen species (ROS) clearance pathways to help P. trichocarpa alleviate the disease. Significance and Impact of the Study: The research reveals the mechanism of L. bicolor inducing resistance to canker of P. trichocarpa from the molecular level and provides a theoretical basis for the practical application of mycorrhizal fungi to improve plant disease resistance. [ABSTRACT FROM AUTHOR]

Xylem is a complex tissue that forms the bulk of tree bodies and has several functions, including to conduct water, store water and nutrients, and biomechanically support the plant body. We examined how xylem functional traits varied at different positions within 9-year-old Populus balsamifera subsp. trichocarpa. Whole trees were excavated, and xylem samples were collected at 1-m increments along the main root-to-shoot axis of six trees, from root tip to shoot tip. We examined biomechanical and water-storage traits of the xylem, including using a non-invasive imaging technique to examine water content within long, intact branches (high-resolution computed tomography; microCT). Xylem density, strength, and stiffness were greater in shoots than roots. Along the main root-to-shoot axis, xylem strength and stiffness were greatest at shoot tips, and the tissue became linearly weaker and less stiff down the plant and through the root. Roots had greater water storage with lower biomechanical support, and shoots had biomechanically stronger and stiffer xylem with lower water storage. These findings support trade-offs among xylem functions between roots and shoots. Understanding how xylem functions differ throughout tree bodies is important in understanding whole-tree functioning and how terrestrial plants endure numerous environmental challenges over decades of growth. [ABSTRACT FROM AUTHOR]

Summary: Secondary cell wall biosynthesis has been shown to be regulated by a suite of transcription factors. Here, we identified a new xylem vessel‐specific NAC domain transcription factor, secondary wall‐associated NAC domain protein5 (SND5), in Arabidopsis thaliana and studied its role in regulating secondary wall biosynthesis.We showed that the expression of SND5 and its close homolog, SND4/ANAC075, was specifically associated with secondary wall‐containing cells and dominant repression of their functions severely reduced secondary wall thickening in these cells. Overexpression of SND4/5 as well as their homologs SND2/3 fused with the activation domain of the viral protein VP16 led to ectopic secondary wall deposition in cells that are normally parenchymatous. SND2/3/4/5 regulated the expression of the same downstream target genes as do the secondary wall NAC master switches (SWNs) by binding to and activating the secondary wall NAC binding elements (SNBEs).Furthermore, we demonstrated that the poplar (Populus trichocarpa) orthologs of SND2/3/4/5 also activated SNBEs and regulated secondary wall biosynthesis during wood formation.Together, these findings indicate that SND2/3/4/5 and their poplar orthologs regulate the expression of secondary wall‐associated genes through activating SNBEs and they are positioned at an upper level in the SWN‐mediated transcriptional network. [ABSTRACT FROM AUTHOR]

Plant Pectin acetylesterase (PAE) belongs to family CE13 of carbohydrate esterases in the CAZy database. The ability of PAE to regulate the degree of acetylation of pectin, an important polysaccharide in the cell wall, affects the structure of plant cell wall. In this study, ten PtPAE genes were identified and characterized in Populus trichocarpa genome using bioinformatics methods, and the physiochemical properties such as molecular weight, isoelectric points, and hydrophilicity, as well as the secondary and tertiary structure of the protein were predicted. According to phylogenetic analysis, ten PtPAEs can be divided into three evolutionary clades, each of which had similar gene structure and motifs. Tissue-specific expression profiles indicated that the PtPAEs had different expression patterns. Real-time quantitative PCR (RT-qPCR) analysis showed that transcription level of PtPAEs was regulated by different CO2 and nitrogen concentrations. These results provide important information for the study of the phylogenetic relationship and function of PtPAEs in Populus trichocarpa. [ABSTRACT FROM AUTHOR]

We applied miRNA expression profiling method to Populus trichocarpa stems of the three developmental stages, primary stem (PS), transitional stem (TS), and secondary stem (SS), to investigate miRNA species and their regulation on lignocellulosic synthesis and related processes. We obtained 892, 872, and 882 known miRNAs and 1727, 1723, and 1597 novel miRNAs, from PS, TS, and SS, respectively. Comparisons of these miRNA species among different developmental stages led to the identification of 114, 306, and 152 differentially expressed miRNAs (DE-miRNAs), which had 921, 2639, and 2042 candidate target genes (CTGs) in the three respective stages of the same order. Correlation analysis revealed 47, 439, and 71 DE-miRNA-CTG pairs of high negative correlation in PS, TS, and SS, respectively. Through biological process analysis, we finally identified 34, 6, and 76 miRNA-CTG pairs from PS, TS, and SS, respectively, and the miRNA target genes in these pairs regulate or participate lignocellulosic biosynthesis-related biological processes: cell division and differentiation, cell wall modification, secondary cell wall biosynthesis, lignification, and programmed cell death processes. This is the first report on an integrated analysis of genome-wide mRNA and miRNA profilings during multiple phases of poplar stem development. Our analysis results imply that individual miRNAs modulate secondary growth and lignocellulosic biosynthesis through regulating transcription factors and lignocellulosic biosynthetic pathway genes, resulting in more dynamic promotion, suppression, or regulatory circuits. This study advanced our understanding of many individual miRNAs and their essential, diversified roles in the dynamic regulation of secondary growth in woody tree species. [ABSTRACT FROM AUTHOR]

Plant Pectin acetylesterase (PAE) belongs to family CE13 of carbohydrate esterases in the CAZy database. The ability of PAE to regulate the degree of acetylation of pectin, an important polysaccharide in the cell wall, affects the structure of plant cell wall. In this study, ten PtPAE genes were identified and characterized in Populus trichocarpa genome using bioinformatics methods, and the physiochemical properties such as molecular weight, isoelectric points, and hydrophilicity, as well as the secondary and tertiary structure of the protein were predicted. According to phylogenetic analysis, ten PtPAEs can be divided into three evolutionary clades, each of which had similar gene structure and motifs. Tissue-specific expression profiles indicated that the PtPAEs had different expression patterns. Real-time quantitative PCR (RT-qPCR) analysis showed that transcription level of PtPAEs was regulated by different CO2 and nitrogen concentrations. These results provide important information for the study of the phylogenetic relationship and function of PtPAEs in Populus trichocarpa. [ABSTRACT FROM AUTHOR]

Background: Entering and exiting winter dormancy present important trade-offs between growth and survival at northern latitudes. Many forest trees display local adaptation across latitude in traits associated with these phenology transitions. Transfers of a species outside its native range introduce the species to novel combinations of environmental conditions potentially requiring different combinations of alleles to optimize growth and survival. In this study, we performed genome wide association analyses and a selection scan in a P. trichocarpa mapping population derived from crossings between clones collected across the native range and introduced into Sweden. GWAS analyses were performed using phenotypic data collected across two field seasons and in a controlled phytotron experiment. Results: We uncovered 584 putative candidate genes associated with spring and autumn phenology traits as well as with growth. Many regions harboring variation significantly associated with the initiation of leaf shed and leaf autumn coloring appeared to have been evolving under positive selection in the native environments of P. trichocarpa. A comparison between the candidate genes identified with results from earlier GWAS analyses performed in the native environment found a smaller overlap for spring phenology traits than for autumn phenology traits, aligning well with earlier observations that spring phenology transitions have a more complex genetic basis than autumn phenology transitions. Conclusions: In a small and structured introduced population of P. trichocarpa, we find complex genetic architectures underlying all phenology and growth traits, and identify multiple putative candidate genes despite the limitations of the study population. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) are a recently discovered type of non‐coding RNA derived from pre‐mRNAs. R‐loops consist of a DNA:RNA hybrid and the associated single‐stranded DNA. In Arabidopsis thaliana, circRNA:DNA R‐loops regulate alternative splicing (AS) of SEPALLATA3 (SEP3). However, the occurrence and functions of circRNAs and R‐loops in Populus trichocarpa are largely unexplored. Here, we performed circRNA‐enriched sequencing in the stem‐differentiating xylem (SDX) of P. trichocarpa and identified 2,742 distinct circRNAs, including circ‐CESA4, circ‐IRX7, and circ‐GUX1, which are generated from genes involved in cellulose, and hemicellulose biosynthesis, respectively. To investigate the roles of circRNAs in modulating alternative splicing (AS), we detected 7,836 AS events using PacBio Iso‐Seq and identified 634 circRNAs that overlapped with 699 AS events. Furthermore, using DNA:RNA hybrid immunoprecipitation followed by sequencing (DRIP‐seq), we identified 8,932 R‐loop peaks that overlapped with 181 circRNAs and 672 AS events. Notably, several SDX‐related circRNAs overlapped with R‐loop peaks, pointing to their possible roles in modulating AS in SDX. Indeed, overexpressing circ‐IRX7 increased the levels of R‐loop structures and decreased the frequency of intron retention in linear IRX7 transcripts. This study provides a valuable R‐loop atlas resource and uncovers the interplay between circRNAs and AS in SDX of P. trichocarpa. [ABSTRACT FROM AUTHOR]

Poplar plantations harbor large potential as a renewable source of biomass for bioenergy and other industrial applications. The overall aim of this study is to analyze growth, phenology, stem form, and branching characteristics of 32 poplar clones grown in a trial in southern Sweden for their suitability to be grown as industrial feedstock. In a linear mixed model, performed for diameter at breast height and stem volume, the precision was improved by the use of two competition indices. The significance of phenology and quality characteristics for growth performance and ranking of poplar clones was evaluated through genotypic correlations, and multivariate hierarchical cluster analysis used to group the material. All traits showed moderate to high broad sense heritability. In general, higher stem volume was positively correlated with later leaf senescence, and uncorrelated with spring phenology. Selection efficiency for stem diameter and height was greatly improved between age 3 and 6 years allowing a better precision in selecting a subset of clones to be further tested in production plots and pilot plantations. Two commercial Populus maximowiczii Henry × trichocarpa Torr. & Gray cultivars performed best, while some intraspecific hybrids of P. trichocarpa are considered useful to genetically diversify commercial plantations in Southern Sweden (Belgian clones) or establish plantations in north-central parts of Sweden (Swedish clones). The cluster analysis emphasized growth traits and the grouping of the clones corresponded to their origin (or parentage). The results will facilitate decisions on the use of studied material in breeding, further testing and commercial deployment of poplar plantations in Sweden. [ABSTRACT FROM AUTHOR]

Glycoside hydrolase family 1 (GH1) β-glucosidases (BGLUs) are encoded by a large number of genes and are involved in many developmental processes and stress responses in plants. Due to their importance in plant growth and development, genome-wide analyses have been conducted in the model plant species Arabidopsis thaliana , rice and maize but not in woody plant species, which have important economic and ecological value. In this study, we systematically analyzed Populus BGLU s (PtBGLU s) and demonstrated the involvement of several genes under stress conditions. Forty-four PtBGLU s were identified in Populus databases; these genes were located on 11 chromosomes, and the proteins of several PtBGLU genes were highly similar. More than 90% of PtBGLUs contain three conserved motifs. Collinearity results showed that 44 PtBGLU genes resulted from 12 tandem and 5 segmental duplication events. Phylogenetic analysis revealed that 128 BGLU genes from Populus trichocarpa , A. thaliana and Oryza sativa could be classified into 4 subgroups and subgroup Ⅱ and Ⅳ were differently having PtBGLU s and AtBGLU s. We further investigated whether several PtBGLU s responded to drought stress and ABA treatment, and the results showed that most of the selected BGLU genes were expressed in response to stress, which is consistent with previous studies involving rice and Arabidopsis homologous genes. Large numbers of stress-, hormone-, and development-related elements in the PtBGLU promoters suggest that BGLU genes may be involved in complex networks. Taken together, our results provide valuable information for an improved understanding of β-glucosidase function in woody plants. • Systematically analyzed forty-four Populus trichocarpa BGLU s located on 11 chromosomes. • Tandem and segmental replication are important for the expansion and evolution of PtBGLU s. • Most of the selected BGLU genes were involved in response to drought stress or ABA treatments. [ABSTRACT FROM AUTHOR]

Plant UDP-glucose 6-dehydrogenase (UGDH) is an important enzyme for the formation of hemicellulose and pectin. Previous studies on UGDH have primarily focused on the biosynthesis of cell wall polysaccharides, while few studies have focused on their regulation by exogenous nitrogen. In the present study, four genes encoding PtUGDH proteins were analyzed by bioinformatics methods. And, the expression profiles of PtUGDH genes under different nitrogen treatments were evaluated with qRT-PCR. The results showed that PtUGDHs have conserved NAD coenzyme binding motif GAGYVGG and the catalytic motif GFGGSCFQKDIL. According to the phylogenetic analysis, PtUGDHs were divided into two subgroups. PtUGDH3 and PtUGDH4 were closely related to AtUGDH1 (important for normal development of Arabidopsis cell wall structure). Chromosomal distribution and genome synteny analysis revealed four segmental-duplicated gene pairs on chr4, 8, 10 and 17. Tissue-specific data from PlantGenIE showed that PtUGDH3 and PtUGDH4 were highly expressed in stems. The qRT-PCR detection showed that the expression of PtUGDH3 in the lower stem and PtUGDH2 of upper leaves were significantly increased induced by low ammonium or nitrate condition. This comprehensive analysis of the UGDH family in poplar provides new insights into their regulation by nitrogen, and would increase our understanding of the roles of UGDHs in hemicellulose and pectin biosynthesis in the cell wall and during poplar development. [ABSTRACT FROM AUTHOR]

Regulation of gene expression related to chromatin modification at the transcriptional silencing and RNA interference (RNAi) at the post-transcriptional level. RNA-dependent RNA polymerase (RDR) and Argonaute (AGO), along with Dicer-like (DCL) from the core components of RNAi, play integral roles in these processes. Here, 14 PtAGOs , 5 PtDCLs , and 9 PtRDRs were identified in P. trichocarpa and compared them with those of another Salicaceae willow (Salix suchowensis Cheng). Maximum-likelihood trees revealed that each AGO , DCL , and RDR family members were divided into four subfamilies. Forty-three orthologous pairs were identified between the P. trichocarpa and S. suchowensis RNAi-toolbox genes. Sixteen collinear gene pairs were detected in highly microsynteny regions with containing more than ten pairs of conserved flanking-genes, indicated that they were considered to have evolved from the large-scale duplication events. Many of the RNAi-toolbox genes were up-regulated, suggesting P. trichocarpa should have evolved specialized regulatory mechanisms in response to cold, salt, drought and heat stresses. Some RNAi-toolbox genes were most highly expressed in stem, suggesting these genes may function in the regulation of small RNAs during P. trichocarpa stem development. Our results provided the integrative analysis and highlighted the function and duplication of the RNAi-toolbox genes in P. trichocarpa. • Fourteen PtAGOs , five PtDCLs , and nine PtRDRs were found in poplar genome. • Differential evolution of the RNAi members reveals conservative and divergent patterns in poplar. • Poplar duplicated gene pairs were all derived from the large-scale duplications. • Expression patterns of RNAi genes seem to be characteristic of stem development and stress responses. [ABSTRACT FROM AUTHOR]

Background: As an important epigenetic mark, 5-methylcytosine (5mC) methylation is involved in many DNA-dependent biological processes and plays a role during development and differentiation of multicellular organisms. However, there is still a lack of knowledge about the dynamic aspects and the roles of global 5mC methylation in wood formation in tree trunks. In this study, we not only scrutinized single-base resolution methylomes of primary stems (PS), transitional stems (TS), and secondary stems (SS) of Populus trichocarpa using a high-throughput bisulfite sequencing technique, but also analyzed the effects of 5mC methylation on the expression of genes involved in wood formation. Results: The overall average percentages of CG, CHG, and CHH methylation in poplar stems were ~ 53.6%, ~ 37.7%, and ~ 8.5%, respectively, and the differences of 5mC in genome-wide CG/CHG/CHH contexts among PS, TS, and SS were statistically significant (p < 0.05). The evident differences in CG, CHG, and CHH methylation contexts among 2 kb proximal promoters, gene bodies, and 2 kb downstream regions were observed among PS, TS, and SS. Further analysis revealed a perceptible global correlation between 5mC methylation levels of gene bodies and transcript levels but failed to reveal a correlation between 5mC methylation levels of proximal promoter regions and transcript levels. We identified 653 and 858 DMGs and 4978 and 4780 DEGs in PS vs TS and TS vs SS comparisons, respectively. Only 113 genes of 653 DMGs and 4978 DEGs, and 114 genes of 858 DMGs and 4780 DEG were common. Counterparts of some of these common genes in other species, including Arabidopsis thaliana, are known to be involved in secondary cell wall biosynthesis and hormone signaling. This indicates that methylation may directly modulate wood formation genes and indirectly attune hormone signaling genes, which in turn impact wood formation. Conclusions: DNA methylation only marginally affects pathway genes or regulators involved in wood formation, suggesting that further studies of wood formation should lean towards the indirect effects of methylation. The information and data we provide here will be instrumental for understanding the roles of methylation in wood formation in tree species. [ABSTRACT FROM AUTHOR]

Background and Aims Soil salinization and aridification are swiftly engulfing the limited land resources on which humans depend, restricting agricultural production. Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is important in the biosynthesis of terpenoids, which are involved in plant growth, development and responses to environmental stresses. This study aimed to provide guidance for producing salt- and drought-resistant poplar. Methods A protein expression system was used to obtain PtHMGR protein, and high-performance liquid chromatography was used to detect the activity of PtHMGR protein in vitro. In addition, a simplified version of the leaf infection method was used for transformation of 'Nanlin895' poplar (Populus × euramericana). qRT–PCR was used to identify expression levels of genes. Key Results PtHMGR catalysed a reaction involving HMG-CoA and NADPH to form mevalonate. Overexpression of PtHMGR in Populus × euramericana 'Nanlin895' improved drought and salinity tolerance. In the presence of NaCl and PEG6000, the rates of rooting and survival of PtHMGR -overexpressing poplars were higher than those of wild-type poplars. The transgenic lines also exhibited higher proline content and peroxidase and superoxide dismutase activities, and a lower malondialdehyde level under osmotic stress. In addition, the expression of genes related to reactive oxygen species (ROS) scavenging and formation was altered by osmotic stress. Moreover, the effect of osmotic stress on transcript levels of stress-related genes differed between the transgenic and wild-type poplars. Conclusion PtHMGR catalysed a reaction involving HMG-CoA and NADPH to form mevalonate in vitro. Overexpression of PtHMGR promoted root development, increased the expression of ROS scavenging-related genes, decreased the expression of ROS formation-related genes, and increased the activity of antioxidant enzymes in transgenic poplars, enhancing their tolerance of osmotic stress. In addition, overexpression of PtHMGR increased expression of the stress-related genes KIN1 , COR15 and AAO3 and decreased that of ABI , MYB , MYC2 and RD22 , enhancing the stress resistance of poplar. [ABSTRACT FROM AUTHOR]

β-d-xylosidase (BXL) hydrolyzes xylobiose and xylo-oligosaccharides into xylose monomers, and is a rate-limiting enzyme in the degradation of hemicellulose in the cell wall. In this study, ten genes encoding putative BXL proteins were identified in the Populus trichocarpa genome by bioinformatics methods. In the phylogenetic analysis, the PtBXLs formed two subfamilies. PtBXL8 and PtBXL9 were closely related to AtBXL1, an important enzyme in the normal development of the Arabidopsis cell wall structure. Chromosomal distribution and genome synteny analyses revealed two tandem-duplicated gene pairs PtBXL3/4 and PtBXL6/7 on chromosomes II and V, respectively, and six segmental-duplicated gene pairs on chromosomes II, V, VIII, X, and XIV among the PtBXL gene family. Tissue-specific expression data from PlantGenIE indicated that PtBXL2, 4, 5, and 10 were highly expressed in stems. Quantitative real-time RT-PCR analyses revealed that PtBXL4, 5, and 9 were up-regulated in the upper stem in response to the low and high ammonium and nitrate treatments. The influence of nitrogen on the expression of PtBXL4, 5, and 9 genes may affect the formation of the plant secondary cell wall. This comprehensive analysis of the BXL family in poplar provides new insights into their regulation by nitrogen and increases our understanding of the roles of BXLs in hemicellulose metabolism in the secondary cell wall and during plant development. [ABSTRACT FROM AUTHOR]

Cellulose synthase A genes (CesA s) are responsible for cellulose biosynthesis in plant cell walls. In this study, functions of secondary wall cellulose synthases PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B were characterized during wood formation in Populus trichocarpa (Torr. & Gray). CesA RNAi knockdown transgenic plants exhibited stunted growth, narrow leaves, early necrosis, reduced stature, collapsed vessels, thinner fiber cell walls and extended fiber lumen diameters. In the RNAi knockdown transgenics, stems exhibited reduced mechanical strength, with reduced modulus of rupture (MOR) and modulus of elasticity (MOE). The reduced mechanical strength may be due to thinner fiber cell walls. Vessels in the xylem of the transgenics were collapsed, indicating that water transport in xylem may be affected and thus causing early necrosis in leaves. A dramatic decrease in cellulose content was observed in the RNAi knockdown transgenics. Compared with wildtype, the cellulose content was significantly decreased in the PtrCesA4 , PtrCesA7 and PtrCesA8 RNAi knockdown transgenics. As a result, lignin and xylem contents were proportionally increased. The wood composition changes were confirmed by solid-state NMR, two-dimensional solution-state NMR and sum-frequency-generation vibration (SFG) analyses. Both solid-state nuclear magnetic resonance (NMR) and SFG analyses demonstrated that knockdown of PtrCesA s did not affect cellulose crystallinity index. Our results provided the evidence for the involvement of PtrCesA4 , PtrCesA7-A/B and PtrCesA8-A/B in secondary cell wall formation in wood and demonstrated the pleiotropic effects of their perturbations on wood formation. [ABSTRACT FROM AUTHOR]

Interferon gamma-induced lysosomal thiol reductase (GILT) is abundantly expressed in antigen-presenting cells and participates in the treatment and presentation of antigens by major histocompatibility complex II. Also, GILT catalyzes the reduction of disulfide bonds, which plays an important role in cellular immunity. (1) Background: At present, the studies of GILT have mainly focused on animals. In plants, GILT homologous gene (Arabidopsis thaliana OSH1: AtOSH1) was discovered in the forward screen of mutants with compromised responses to sulphur nutrition. However, the complete properties and functions of poplar OSH1 are unclear. In addition, CdCl2 stress is swiftly engulfing the limited land resources on which humans depend, restricting agricultural production. (2) Methods: A prokaryotic expression system was used to produce recombinant PtOSH1 protein, andWestern blotting was performed to identify its activity. In addition, a simplified version of the floral-dip method was used to transform A. thaliana. (3) Results: Here, we describe the identification and characterization of OSH1 from Populus trichocarpa. The deduced PtOSH1 sequence contained CQHGX2ECX2NX4C and CXXC motifs. The transcript level of PtOSH1 was increased by cadmium (Cd) treatment. In addition, recombinant PtOSH1 reduced disulfide bonds. A stress assay showed that PtOSH1-overexpressing (OE) A. thaliana lines had greater resistance to Cd than wild-type (WT) plants. Also, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in PtOSH1-OE plants were significantly higher than those in WT A. thaliana. These results indicate that PtOSH1 likely plays an important role in the response to Cd by regulating the reactive oxygen species (ROS)-scavenging system. (4) Conclusions: PtOSH1 catalyzes the reduction of disulfide bonds and behaves as a sulfhydryl reductase under acidic conditions. The overexpression of PtOSH1 in A. thaliana promoted root development, fresh weight, and dry weight; upregulated the expression levels of ROS scavenging-related genes; and improved the activity of antioxidant enzymes, enhancing plant tolerance to cadmium (Cd) stress. This study aimed to provide guidance that will facilitate future studies of the function of PtOSH1 in the response of plants to Cd stress. [ABSTRACT FROM AUTHOR]

Background: Natural selection on fitness-related traits can be temporally heterogeneous among populations. As climate changes, understanding population-level responses is of scientific and practical importance. We examined 18 phenotypic traits associated with phenology, biomass, and ecophysiology in 403 individuals of natural Populus trichocarpa populations, growing in a common garden. Results: Compared with tree origin settings, propagules likely underwent drought exposures in the common garden due to significantly low rainfall during the years of measurement. All study traits showed population differentiation reflecting adaptive responses due to local genetic adaptation. Phenology and biomass traits were strongly under selection and showed plastic responses between years, co-varying with latitude. While phenological events (e.g., bud set and growth period) and biomass were under positive directional selection, post-bud set period, particularly from final bud set to the onset of leaf drop, was selected against. With one exception to water-use efficiency, ecophysiology traits were under negative directional selection. Moreover, extended phenological events jointly evolved with source niches under increased temperature and decreased rainfall exposures. High biomass coevolved with climatic niches of high temperature; low rainfall promoted high photosynthetic rates evolution. Conclusions: This work underpins that P. trichocarpa is likely to experience increased fitness (height gain) by evolving toward extended bud set and growth period, abbreviated post-bud set period, and increased drought resistance, potentially constituting a powerful mechanism for long-lived tree species in surviving unpredictably environmental extremes (e.g., drought). [ABSTRACT FROM AUTHOR]

Terpenoids are one of the main types of secondary metabolites in plants and are crucial for plant growth, development and defense. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a rate-limiting enzyme for terpene biosynthesis in the MVA pathways. In this study, six PtHMGR genes (PtHMGR1-PtHMGR6) were identified in Populus trichocarpa and distributed on six chromosomes. Collinear analysis of PtHMGR genes suggested that six replication gene pairs were acquired by segmental duplication events in the Populus genome. In addition, the promoters of PtHMGRs contained abundant light-response elements, hormone-response elements and abiotic stress-response elements. Expression profile analysis demonstrated that the PtHMGR genes were induced by various abiotic stresses and hormone treatments. Furthermore, transgenic expression of PtHMGR5 in Populus significantly reduced membrane damage and increased drought (mannitol) tolerance. In conclusion, our findings provide valuable information regarding PtHMGR genes, which is of great significance for the cultivation of improved plants in response to drought (mannitol) stress. • Genome-wide analysis identified 6 putative HMGR genes in Populus. • PtHMGR genes responded to various abiotic stresses. • PtHMGR genes were expressed under hormonal treatments. • Overexpression of PtHMGR5 enhanced drought tolerance in Populus. [ABSTRACT FROM AUTHOR]

Pretreatment is commonly used to reduce recalcitrance of the lignin-carbohydrate matrix. In this study, leading pretreatment technologies, including dilute sulfuric acid, liquid hot water, alkaline, and organosolv pretreatments, were applied to the selected Populus trichocarpa genotype with relatively low lignin content to elucidate cellulose physicochemical property changes and digestibility-related factors. Pretreated Populus trichocarpa (BESC 131) exhibited higher accessibility and glucose yield than the untreated biomass. Chemical composition and Fourier transform infrared (FTIR) analysis results revealed that hemicellulose and lignin were removed to a varying extent depending on the pretreatment techniques applied. The degree of polymerization of the cellulose was decreased to the largest extent after dilute acid pretreatment, followed by organosolv, alkaline, and liquid hot water pretreatments. Cellulose crystallinity index was slightly changed after the pretreatments; however, its differences were not remarkable between those pretreatment techniques. Among four different pretreatments, organosolv was the most effective pretreatment technology in terms of sugar release, which was three times higher than that of the untreated native biomass. Among all of the tested cell wall traits, the lignin content of Populus trichocarpa was the most remarkable feature associated with glucose release, though Populus trichocarpa recalcitrance was not solely dependent on any single factor. [ABSTRACT FROM AUTHOR]

Summary: Occurrence of stomata on both leaf surfaces (amphistomaty) promotes higher stomatal conductance and photosynthesis while simultaneously increasing exposure to potential disease agents in black cottonwood (Populus trichocarpa).A genome‐wide association study (GWAS) with 2.2M single nucleotide polymorphisms generated through whole‐genome sequencing found 280 loci associated with variation in adaxial stomatal traits, implicating genes regulating stomatal development and behavior. Strikingly, numerous loci regulating plant growth and response to biotic and abiotic stresses were also identified.The most significant locus was a poplar homologue of SPEECHLESS (PtSPCH1). Individuals possessing PtSPCH1 alleles associated with greater adaxial stomatal density originated primarily from environments with shorter growing seasons (e.g. northern latitudes, high elevations) or with less precipitation. PtSPCH1 was expressed in developing leaves but not developing stem xylem. In developing leaves, RNA sequencing showed patterns of coordinated expression between PtSPCH1 and other GWAS‐identified genes.The breadth of our GWAS results suggests that the evolution of amphistomaty is part of a larger, complex response in plants. Suites of genes underpin this response, retrieved through genetic association to adaxial stomata, and show coordinated expression during development. We propose that the occurrence of amphistomaty in P. trichocarpa involves PtSPCH1 and reflects selection for supporting rapid growth over investment in immunity. [ABSTRACT FROM AUTHOR]