UMAP_Projection_of_Populus_trichocarpa_Genotypes_Colored_by_DBSCAN_Cluster
Aninteractive UMAPplot derived from the top 8 PCs of LD-prunedPopulus trichocarpapangenome SNP data. Points are colored by DBSCAN clustering.
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UMAP_Projection_of_Populus_trichocarpa_Genotypes_Colored_by_ADMIXTURE_Ancestry
Aninteractive UMAP plotderived from the top 8 PCs of LD-prunedPopulus trichocarpapangenome SNP data. Points are colored by ADMIXTURE population structure (k=3) and geographic origins are searchable by genotype or river system using the search bar.
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Populus_trichocarpa_associated_microbiomes_Cascade_Mountains
Abiotic stresses like heat and drought pose a major threat to forest ecosystems and are anticipated to increase due to anthropogenic climate change. One mechanism by which plants cope with stress
PMI↗
Species‐Specific Epigenetic Signature Associates With Heat Stress Tolerance in the Perennial Tree Species Populus
Epigenetic regulation in annual plants is recognized as a key component of recurring stress acclimation and adaptation, but reports on perennial tree species are limited. In this study, two contrasting tree species, Populus trichocarpa and Populus deltoides, and an F1 hybrid cross between them showed species-specific epigenetic and physiological responses to heat stress (42°C) following priming (35°C). By analyzing whole-genome methylation, transcriptomics, proteomics, metabolomics, and photosynthesis parameters, we found that P. deltoides expresses specific epigenetic signatures in response to heat, resulting in improved photosynthetic efficiency compared to P. trichocarpa. Conversely, P. trichocarpa displayed stress signaling and defense mechanisms that could not sustain a net assimilation rate despite maintaining higher gas exchange. Heat stress following priming in hybrid plants increased transcript levels of thermotolerance-related transcription factors, such as SPL12. Selected regions in the promoter of SPL12 showed differential methylation between direct heat stress and priming followed by heat stress. As a result, upregulation of downstream genes and associated increases in protein and metabolite abundance for stress adaptation were exhibited. Consequently, hybrid plants showed enhanced photosynthesis and gas exchange rates, a trait lacking in P. trichocarpa. These results imply that priming may not be universally effective in enhancing plant performance under stress, particularly in perennial tree species. However, priming can acclimate the perennial tree species P. deltoides to withstand elevated temperature stress better. Our study has demonstrated that priming-based stress adaptation is species-specific but can be attained through crossbreeding, indicating its potential use in breeding programs.
DNA methylation↗
Luteolibacter sp. strain Populi
Luteolibacter sp. strain Populi is bacterium from the phylum Verrucomicrobiota, isolated from the rhizosphere of a black cottonwood tree, Populus trichocarpa, from the Cascade mountains in Washington. Its 6.6 Mb chromosome was completely sequenced using Oxford Nanopore long-reads and is predicted to encode 5301 proteins and 60 RNAs. The bacteria was isolated from the rhizosphere of a mature Populus trichocarpa from the Tieton riverwatershed of Washington state, USA (Lat: 46°42’9” N, Lon: 120°25 39’36” W). A rhizosphere sample (fine roots and adhering soil) was used to obtain a microbial fraction by centrifugation on Histodenz (12) and stained with 5µM Syto59 (Thermo Fisher Scientific Inc). A Cytopeia Influx cell sorter (BD, Franklin Lakes, NJ) was used to sort and array single cells (100 per plate) based on forward-side scatter and fluorescence intensity on asparagine-glucose nutrient agar (ATCC medium 184). The Luteolibacter sp. Populi genome sequence has been deposited in GenBank under the accession number CP161812. A draft genome annotated with Prokka and DRAM is available in this Narrative as Luteolibacter_sp_Prokka.240711.
59 BASIC BIOLOGICAL SCIENCES↗
Providing biological context for GWAS results using eQTL regulatory and co‐expression networks in Populus
Summary Our study utilized genome‐wide association studies (GWAS) to link nucleotide variants to traits in Populus trichocarpa , a species with rapid linkage disequilibrium decay. The aim was to overcome the challenge of interpreting statistical associations at individual loci without sufficient biological context, which often leads to reliance solely on gene annotations from unrelated model organisms. We employed an integrative approach that included GWAS targeting multiple traits using three individual techniques for lignocellulose phenotyping, expression quantitative trait loci (eQTL) analysis to construct transcriptional regulatory networks around each candidate locus and co‐expression analysis to provide biological context for these networks, using lignocellulose biosynthesis in Populus trichocarpa as a case study. The research identified three candidate genes potentially involved in lignocellulose formation, including one previously recognized gene (Potri.005G116800/VND1, a critical regulator of secondary cell wall formation) and two genes (Potri.012G130000/AtSAP9 and Potri.004G202900/BIC1) with newly identified putative roles in lignocellulose biosynthesis. Our integrative approach offers a framework for providing biological context to loci associated with trait variation, facilitating the discovery of new genes and regulatory networks.
59 BASIC BIOLOGICAL SCIENCES↗
Pyrolysis Molecular Beam Mass Spectrometry_Analysis_of_Natural_Variants_of_Poplulus_Trichocarpa_Leaves
Select leaves from natural variants of Poplar (Populus Trichocarpa) grown in a greenhouse at Oak Ridge National Laboratory were analyzed by Pyrolysis-Molecular Beam Mass Spectrometry (Py-MBMS). Leaves were harvested, cryomilled and kept frozen until analysis. Py-MBMS analysis was conducted using approximately 4 mg of biomass and each sample was analyzed in duplicate. A Frontier PY2020 unit pyrolyzed samples at 500°C for 30 s in 80 µL deactivated stainless steel cups. An Extrel Super-Sonic MBMS Model Max 1000 was used to collect mass spectral data fromm/z30 to 450 at 17 eV and processed using Merlin Automation software (V3). Spectral ion intensities were normalized to the total ion chromatogram signal for each sample for analysis of spectral variance. Lignin content (wt %) was estimated based on relative responses from standards of known Klason lignin content using mean-normalized ion intensities ofm/z120, 124 (G), 137 (G), 138 (G), 150 (G), 152, 154 (S), 164 (G), 167 (S), 168 (S), 178 (G), 180, 181, 182 (S), 194 (S), 208 (S) and 210 (S) where G indicates guaiacyl-derived ions, S indicates syringyl-derived ions, and other ions either derive from other lignin monomers or multiple sources. Ratios of S and G lignin monomer units (S/G) were obtained by dividing the sum of S-based ions by the sum of G-based ions using mean-normalized ion intensities.
CBI↗
Elucidating the drought-responsive changes in Poplar cuticular waxes: A GWAS analysis of genes involved in fatty acid biosynthesis
Drought and episodic drought events are major impending impacts of climate change, limiting the productivity of plants and especially trees due to their inherent high transpiration rates. One common mechanism used by plants to cope with drought stress is to change the composition of their leaf cuticular waxes. Cuticular waxes are essential for controlling non-stomatal water loss and are typically composed of a homologous series of very-long-chain fatty acid-derived compounds, as well as flavonoids, tocopherols, triterpenoids, and phytosterols. In this study, we compared the cuticular waxes of 339 natural accessions of Populus trichocarpa (black cottonwood) grown under control and drought conditions in a common garden. A Genome-Wide Association Study (GWAS) was then used to identify candidate genes associated with cuticular wax biosynthesis and/or its regulation. Although no major differences were observed in total wax load when subject to drought conditions, the amounts of the individual wax constituents were indeed responsive to drought. Specifically, changes in alkenes, alcohols, esters, and aldehydes were evident, and suggest that they contribute to the drought response/tolerance in poplar. GWAS uncovered several genes linked to fatty acid biosynthesis, including CER1, CER3, CER4, FATB, FAB1, FAR3, FAR4, KCS, and a homolog of SOH1, as well as other candidate genes that may be involved in coordinating the drought responses in poplar trees. Our findings provide new evidence that genotype-specific shifts in wax composition, rather than total wax accumulation, contribute to drought adaptation in poplar. Additionally, we show that genetic variation in key wax biosynthetic genes drives cuticular wax plasticity in P. trichocarpa under drought, identifying putative molecular targets for improving stress resilience in trees. This study expands our understanding of the adaptative mechanisms of the cuticle and their potential for enhancing drought tolerance in poplar species.
Alkanes↗
Intraspecific variability in plant and soil chemical properties in a common garden plantation of the energy crop Populus
Optimizing crops for synergistic soil carbon (C) sequestration can enhance CO 2 removal in food and bioenergy production systems. Yet, in bioenergy systems, we lack an understanding of how intraspecies variation in plant traits correlates with variation in soil biogeochemistry. This knowledge gap is exacerbated by both the heterogeneity and difficulty of measuring belowground traits. Here, we provide initial observations of C and nutrients in soil and root and stem tissues from a common garden field site of diverse, natural variant, Populus trichocarpa genotypes—established for aboveground biomass-to-biofuels research. Our goal was to explore the value of such field sites for evaluating genotype-specific effects on soil C, which ultimately informs the potential for optimizing bioenergy systems for both aboveground productivity and belowground C storage. To do this, we investigated variation in chemical traits at the scale of individual trees and genotypes and we explored correlations among stem, root, and soil samples. We observed substantial variation in soil chemical properties at the scale of individual trees and specific genotypes. While correlations among elements were observed both within and among sample types (soil, stem, root), above-belowground correlations were generally poor. We did not observe genotype-specific patterns in soil C in the top 10 cm, but we did observe genotype associations with soil acid-base chemistry (soil pH and base cations) and bulk density. Finally, a specific phenotype of interest (high vs low lignin) was unrelated to soil biogeochemistry. Our pilot study supports the usefulness of decade-old, genetically-variable, Populus bioenergy field test plots for understanding plant genotype effects on soil properties. Finally, this study contributes to the advancement of sampling methods and baseline data for Populus systems in the Pacific Northwest, USA. Further species- and region-specific efforts will enhance C predictability across scales in bioenergy systems and, ultimately, accelerate the identification of genotypes that optimize yield and carbon storage.
54 ENVIRONMENTAL SCIENCES↗
Dataset_for_Conserved_macromolecular_architecture_of_Poplar_secondary_cell_walls_revealed_by_ssNMR_and_atomistic_modeling
This dataset contains solid-state 13C NMR data and atomistic molecular dynamics simulation files supporting the study of nanoscale secondary cell wall architecture across 13 genetically diverse Populus trichocarpa genotypes grown under uniform greenhouse conditions in 13C-enriched CO2 atmospheres (~89% 13C enrichment).The dataset contains two collections of solid-state 13C NMR data. (1) 200 MHz data (Bruker Avance III HD, 4 mm HX probe, 10 kHz MAS): raw Bruker TopSpin experiment folders and DMFIT-exported ascii spectra for selective and non-selective 1D 13C-13C spin diffusion experiments (3000 ms mixing) used to quantify inter-polymer spatial proximities, and short-mixing (1 ms) reference spectra used for polymeric abundance quantification by spectral deconvolution. (2) 600 MHz data (Bruker Avance III, 1.6 mm PhoenixNMR HXY probe, 30 kHz MAS): raw Bruker TopSpin experiment folders containing 2D CORD, 2D CP-INADEQUATE, and 13C/1H relaxation (T1, T1rho) experiments for all 13 genotypes, with processed Excel workbooks per experiment type. Molecular dynamics simulation code, coordinate files, and analysis scripts (NAMD/CHARMM/Python) for six atomistic cell wall models are included. Summarized ssNMR data are compiled into a single excel file and subjected to statistical analysis. Multivariate analysis code (PCA, Pearson correlation) and summary data are provided as excel worksheets and Jupyter notebooks (Python 3).
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Leveraging hyperspectral imaging to identify drought tolerant Populus species and genotypes within species
The aim of this study was to identity variation in drought tolerance across genotypes of Populus deltoides, Populus trichocarpa, and hybrids of the two species. A panel of 102 Populus genotypes, comprising 37 genotypes of P. trichocarpa, 37 of P. deltoides and 28 unique hybrid genotypes (P. trichocarpa x P. deltoides and P. deltoides x P. trichocarpa) were evaluated in the greenhouse under two treatments, well-watered (WW) and drought (DS). Plant physiological data were collected throughout the experiment once the drought treatment began. Throughout the experiment, we tracked soil volumetric water content, pot weight, stomatal conductance, quantum yield of photosystem II, and electron transport rate. In addition to those measurements, upon completion of the experiment, we assessed above and belowground plant biomass, plant height and stem diameter, leaf number, specific leaf area, relative water content, total protein, and total chlorophyll. We obtained hyperspectral signatures of one leaf from each plant at the end of the experiment. Columns BC – LL are hyperspectral averages for one leaf from each plant at each wavelength as described in the column header.
Hyper-spectral imaging, Populus, plant stress tole↗
A multi-omic survey of black cottonwood tissues highlights coordinated transcriptomic and metabolomic mechanisms for plant adaptation to phosphorus deficiency
Phosphorus (P) deficiency in plants creates a variety of metabolic perturbations that decrease photosynthesis and growth. Phosphorus deficiency is especially challenging for the production of bioenergy feedstock plantation species, such as poplars (Populus spp.), where fertilization may not be practically or economically feasible. While the phenotypic effects of P deficiency are well known, the molecular mechanisms underlying whole-plant and tissue-specific responses to P deficiency, and in particular the responses of commercially valuable hardwoods, are less studied. We used a multi-tissue and multi-omics approach using transcriptomic, proteomic, and metabolomic analyses of the leaves and roots of black cottonwood (Populus trichocarpa) seedlings grown under P-deficient (5 µM P) and replete (100 µM P) conditions to assess this knowledge gap and to identify potential gene targets for selection for P efficiency. In comparison to seedlings grown at 100 µM P, P-deficient seedlings exhibited reduced dry biomass, altered chlorophyll fluorescence, and reduced tissue P concentrations. In line with these observations, growth, C metabolism, and photosynthesis pathways were downregulated in the transcriptome of the P-deficient plants. Additionally, we found evidence of strong lipid remodeling in the leaves. Metabolomic data showed that the roots of P-deficient plants had a greater relative abundance of phosphate ion, which may reflect extensive degradation of P-rich metabolites in plants exposed to long-term P-deficiency. With the notable exception of the KEGG pathway for Starch and Sucrose Metabolism (map00500), the responses of the transcriptome and the metabolome to P deficiency were consistent with one another. No significant changes in the proteome were detected in response to P deficiency. Collectively, our multi-omic and multi-tissue approach enabled the identification of important metabolic and regulatory pathways regulated across tissues at the molecular level that will be important avenues to further evaluate for P efficiency. These included stress-mediating systems associated with reactive oxygen species maintenance, lipid remodeling within tissues, and systems involved in P scavenging from the rhizosphere.
59 BASIC BIOLOGICAL SCIENCES↗
Expression quantitative trait loci mapping identified PtrXB38 as a key hub gene in adventitious root development in Populus
Summary Plant establishment requires the formation and development of an extensive root system with architecture modulated by complex genetic networks. Here, we report the identification of the PtrXB38 gene as an expression quantitative trait loci (eQTL) hotspot, mapped using 390 leaf and 444 xylem Populus trichocarpa transcriptomes. Among predicted targets of this trans ‐eQTL were genes involved in plant hormone responses and root development. Overexpression of PtrXB38 in Populus led to significant increases in callusing and formation of both stem‐born roots and base‐born adventitious roots. Omics studies revealed that genes and proteins controlling auxin transport and signaling were involved in PtrXB38‐mediated adventitious root formation. Protein–protein interaction assays indicated that PtrXB38 interacts with components of endosomal sorting complexes required for transport machinery, implying that PtrXB38‐regulated root development may be mediated by regulating endocytosis pathway. Taken together, this work identified a crucial root development regulator and sheds light on the discovery of other plant developmental regulators through combining eQTL mapping and omics approaches.
54 ENVIRONMENTAL SCIENCES↗
An orphan gene BOOSTER enhances photosynthetic efficiency and plant productivity
Organelle-to-nucleus DNA transfer is an ongoing process playing an important role in the evolution of eukaryotic life. Here, genome-wide association studies (GWAS) of non-photochemical quenching parameters in 743 Populus trichocarpa accessions identified a nuclear-encoded genomic region associated with variation in photosynthesis under fluctuating light. The identified gene, BOOSTER (BSTR), comprises three exons, two with apparent endophytic origin and the third containing a large fragment of plastid-encoded Rubisco large subunit. Higher expression of BSTR facilitated anterograde signaling between nucleus and plastid, which corresponded to enhanced expression of Rubisco, increased photosynthesis, and up to 35% greater plant height and 88% biomass in poplar accessions under field conditions. Overexpression of BSTR in Populus tremula × P. alba achieved up to a 200% in plant height. Similarly, Arabidopsis plants heterologously expressing BSTR gained up to 200% in biomass and up to 50% increase in seed.
60 APPLIED LIFE SCIENCES↗
Dynamic nitrogen fixation in an aerobic endophyte of Populus
Biological nitrogen fixation by microbial diazotrophs can contribute significantly to nitrogen availability in non-nodulating plant species. In this study of molecular mechanisms and gene expression relating to biological nitrogen fixation, the aerobic nitrogen-fixing endophyte Burkholderia vietnamiensis, strain WPB, isolated from Populus trichocarpa served as a model for endophyte–poplar interactions. Nitrogen-fixing activity was observed to be dynamic on nitrogen-free medium with a subset of colonies growing to form robust, raised globular like structures. Secondary ion mass spectrometry (NanoSIMS) confirmed that N-fixation was uneven within the population. A fluorescent transcriptional reporter (GFP) revealed that the nitrogenase subunit nifH is not uniformly expressed across genetically identical colonies of WPB and that only ~11% of the population was actively expressing the nifH gene. Higher nifH gene expression was observed in clustered cells through monitoring individual bacterial cells using single-molecule fluorescence in situ hybridization. Through 15 N 2 enrichment, we identified key nitrogenous metabolites and proteins synthesized by WPB and employed targeted metabolomics in active and inactive populations. We cocultivated WPB Pnif-GFP with poplar within a RhizoChip, a synthetic soil habitat, which enabled direct imaging of microbial nifH expression within root epidermal cells. We observed that nifH expression is localized to the root elongation zone where the strain forms a unique physical interaction with the root cells. This work employed comprehensive experimentation to identify novel mechanisms regulating both biological nitrogen fixation and beneficial plant–endophyte interactions.
15 N-tracking metabolomics↗
Functional diversification within the heme-binding split-barrel family
Due to neofunctionalization, a single fold can be identified in multiple proteins that have distinct molecular functions. Depending on the time that has passed since gene duplication and the number of mutations, the sequence similarity between functionally divergent proteins can be relatively high, eroding the value of sequence similarity as the sole tool for accurately annotating the function of uncharacterized homologs. Here, we combine bioinformatic approaches with targeted experimentation to reveal a large multifunctional family of putative enzymatic and nonenzymatic proteins involved in heme metabolism. This family (homolog of HugZ (HOZ)) is embedded in the “FMN-binding split barrel” superfamily and contains separate groups of proteins from prokaryotes, plants, and algae, which bind heme and either catalyze its degradation or function as nonenzymatic heme sensors. In prokaryotes these proteins are often involved in iron assimilation, whereas several plant and algal homologs are predicted to degrade heme in the plastid or regulate heme biosynthesis. In the plant Arabidopsis thaliana, which contains two HOZ subfamilies that can degrade heme in vitro (HOZ1 and HOZ2), disruption of AtHOZ1 (AT3G03890) or AtHOZ2A (AT1G51560) causes developmental delays, pointing to important biological roles in the plastid. In the tree Populus trichocarpa, a recent duplication event of a HOZ1 ancestor has resulted in localization of a paralog to the cytosol. Structural characterization of this cytosolic paralog and comparison to published homologous structures suggests conservation of heme-binding sites. This study unifies our understanding of the sequence-structure-function relationships within this multilineage family of heme-binding proteins and presents new molecular players in plant and bacterial heme metabolism.
59 BASIC BIOLOGICAL SCIENCES↗
Higher Wood Density Lowers Feedstock Cost and Has Minimal Impact on Biomass Conversion to Biofuels
Poplar and other woody feedstocks have the potential to provide up to 200 million tons of biomass per year that can be converted to liquid fuels. Most forestry strategies that aim to increase biomass productivity per hectare rely on short rotation plantations of fast-growing varieties. The improvement of the wood density as a key trait itself has largely been overlooked. We evaluated natural variation in wood density across a population of genetically diverse Populus trichocarpa trees grown in a common garden. Wood density varies greatly within this population but is heritable; higher wood density was not systematically associated with reduced growth, challenging assumptions of a trade-off between wood density and biomass accumulation. Furthermore, denser wood led to significant improvements throughout the supply chain including lowering biomass production and transportation costs. Higher density did not correlate with changes in biomass composition. Density did not impact bioconversion in the two feedstock-to-fuel pipelines tested (pretreatment by ionic liquids and fermentation to bisabolene or soaking in aqueous ammonia and fermentation to ethanol) on a representative subset of poplars. These findings highlight wood density as a promising breeding target for accelerating the development of high-yielding, conversion-efficient bioenergy crops and as an avenue for increasing landuse efficiency and reducing biomass transportation cost.
09 BIOMASS FUELS↗
Conserved Macromolecular Architecture of Poplar Secondary Cell Walls Revealed by ssNMR and Atomistic Modeling
The macromolecular architecture of plant secondary cell walls governs wood's mechanical and biochemical properties, yet its natural intra-species variability remains poorly characterized. Here, we combined 13C solid-state NMR (ssNMR), multivariate statistical analysis, and molecular modeling to profile nanoscale structure across 13 genetically diverse Populus trichocarpa genotypes grown in 13C-enriched atmospheres. SsNMR-derived phenotypes spanning composition, structure, mobility, and inter-polymer proximities reveal a conserved architecture, with a subtle yet coordinated variation organizing into dominant structural and secondary mobility axes. A representative atomistic model captures these features and reproduces experimental metrics. Molecular dynamics simulations support a weak but consistent positive correlation between cellulose abundance and crystalline-like order, with interior cellulose chains enriched in tg (trans-gauche) conformations without expanding crystalline cores. Together, experiment and simulation reveal a genetically buffered, broadly conserved nanoscale architecture across genotypes, where subtle fine-tuning of cellulose bundling and matrix packing balances mechanical performance with biological function.
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