Providing biological context for GWAS results using eQTL regulatory and co-expression networks in Populus.

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.

Expression quantitative trait loci mapping identified PtrXB38 as a key hub gene in adventitious root development in Populus.

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.

Machine Learning-Based Classification of Lignocellulosic Biomass from Pyrolysis-Molecular Beam Mass Spectrometry Data.

High-throughput analysis of biomass is necessary to ensure consistent and uniform feedstocks for agricultural and bioenergy applications and is needed to inform genomics and systems biology models. Pyrolysis followed by mass spectrometry such as molecular beam mass spectrometry (py-MBMS) analyses are becoming increasingly popular for the rapid analysis of biomass cell wall composition and typically require the use of different data analysis tools depending on the need and application. Here, the authors report the py-MBMS analysis of several types of lignocellulosic biomass to gain an understanding of spectral patterns and variation with associated biomass composition and use machine learning approaches to classify, differentiate, and predict biomass types on the basis of py-MBMS spectra. Py-MBMS spectra were also corrected for instrumental variance using generalized linear modeling (GLM) based on the use of select ions relative abundances as spike-in controls. Machine learning classification algorithms e.g., random forest, k-nearest neighbor, decision tree, Gaussian Naïve Bayes, gradient boosting, and multilayer perceptron classifiers were used. The k-nearest neighbors (k-NN) classifier generally performed the best for classifications using raw spectral data, and the decision tree classifier performed the worst. After normalization of spectra to account for instrumental variance, all the classifiers had comparable and generally acceptable performance for predicting the biomass types, although the k-NN and decision tree classifiers were not as accurate for prediction of specific sample types. Gaussian Naïve Bayes (GNB) and extreme gradient boosting (XGB) classifiers performed better than the k-NN and the decision tree classifiers for the prediction of biomass mixtures. The data analysis workflow reported here could be applied and extended for comparison of biomass samples of varying types, species, phenotypes, and/or genotypes or subjected to different treatments, environments, etc. to further elucidate the sources of spectral variance, patterns, and to infer compositional information based on spectral analysis, particularly for analysis of data without a priori knowledge of the feedstock composition or identity.

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 CO2 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.

Heterologous expression of Arabidopsis laccase2, laccase4 and peroxidase52 driven under developing xylem specific promoter DX15 improves saccharification in populus.

BACKGROUND: Secondary cell wall holds considerable potential as it has gained immense momentum to replace the lignocellulosic feedstock into fuels. Lignin one of the components of secondary cell wall tightly holds the polysaccharides thereby enhancing the recalcitrance and complexity in the biomass. Laccases (LAC) and peroxidases (PRX) are the major phenyl-oxidases playing key functions during the polymerization of monolignols into lignin. Yet, the functions of laccase and peroxidases gene families remained largely unknown. Hence, the objective of this conducted study is to understand the role of specific LAC and PRX in Populus wood formation and to further investigate how the altered Lac and Prx expression affects biomass recalcitrance and plant growth. This study of heterologous expression of Arabidopsis Lac and Prx genes was conducted in poplar to avoid any otherwise occurring co-suppression mechanism during the homologous overexpression of highly expressed native genes. In the pursuit of optimizing lignocellulosic biomass for biofuel production, the present study focuses on harnessing the enzymatic potential of Arabidopsis thaliana Laccase2, Laccase4, and Peroxidase52 through heterologous expression. RESULTS: We overexpressed selected Arabidopsis laccase2 (AtLac2), laccase4 (AtLac4), and peroxidase52 (AtPrx52) genes, based on their high transcript expression respective to the differentiating xylem tissues in the stem, in hybrid poplar (cv. 717) expressed under the developing xylem tissue-specific promoter, DX15 characterized the transgenic populus for the investigation of growth phenotypes and recalcitrance efficiency. Bioinformatics analyses conducted on AtLac2 and AtLac4 and AtPrx52, revealed the evolutionary relationship between the laccase gene and peroxidase gene homologs, respectively. Transgenic poplar plant lines overexpressing the AtLac2 gene (AtLac2-OE) showed an increase in plant height without a change in biomass yield as compared to the controls; whereas, AtLac4-OE and AtPrx52-OE transgenic lines did not show any such observable growth phenotypes compared to their respective controls. The changes in the levels of lignin content and S/G ratios in the transgenic poplar resulted in a significant increase in the saccharification efficiency as compared to the control plants. CONCLUSIONS: Overall, saccharification efficiency was increased by 35-50%, 21-42%, and 8-39% in AtLac2-OE, AtLac4-OE, and AtPrx52-OE transgenic poplar lines, respectively, as compared to their controls. Moreover, the bioengineered plants maintained normal growth and development, underscoring the feasibility of this approach for biomass improvement without compromising overall plant fitness. This study also sheds light on the potential of exploiting regulatory elements of DX15 to drive targeted expression of lignin-modifying enzymes, thereby providing a promising avenue for tailoring biomass for improved biofuel production. These findings contribute to the growing body of knowledge in synthetic biology and plant biotechnology, offering a sustainable solution to address the challenges associated with lignocellulosic biomass recalcitrance.

Atomistic, macromolecular model of the Populus secondary cell wall informed by solid-state NMR.

Plant secondary cell walls (SCWs) are composed of a heterogeneous interplay of three major biopolymers: cellulose, hemicelluloses, and lignin. Details regarding specific intermolecular interactions and higher-order architecture of the SCW superstructure remain ambiguous. Here, we use solid-state nuclear magnetic resonance (ssNMR) measurements to infer refined details about the structural configuration, intermolecular interactions, and relative proximity of all three major biopolymers within air-dried Populus wood. To enhance the utility of these findings and enable evaluation of hypotheses in a physics-based environment in silico, the NMR observables are articulated into an atomistic, macromolecular model for biopolymer assemblies within the plant SCW. Through molecular dynamics simulation, we quantitatively evaluate several variations of atomistic models to determine structural details that are corroborated by ssNMR measurements.

Design and Validation of a High-Throughput Reductive Catalytic Fractionation Method.

Reductive catalytic fractionation (RCF) is a promising method to extract and depolymerize lignin from biomass, and bench-scale studies have enabled considerable progress in the past decade. RCF experiments are typically conducted in pressurized batch reactors with volumes ranging between 50 and 1000 mL, limiting the throughput of these experiments to one to six reactions per day for an individual researcher. Here, we report a high-throughput RCF (HTP-RCF) method in which batch RCF reactions are conducted in 1 mL wells machined directly into Hastelloy reactor plates. The plate reactors can seal high pressures produced by organic solvents by vertically stacking multiple reactor plates, leading to a compact and modular system capable of performing 240 reactions per experiment. Using this setup, we screened solvent mixtures and catalyst loadings for hydrogen-free RCF using 50 mg poplar and 0.5 mL reaction solvent. The system of 1:1 isopropanol/methanol showed optimal monomer yields and selectivity to 4-propyl substituted monomers, and validation reactions using 75 mL batch reactors produced identical monomer yields. To accommodate the low material loadings, we then developed a workup procedure for parallel filtration, washing, and drying of samples and a 1H nuclear magnetic resonance spectroscopy method to measure the RCF oil yield without performing liquid-liquid extraction. As a demonstration of this experimental pipeline, 50 unique switchgrass samples were screened in RCF reactions in the HTP-RCF system, revealing a wide range of monomer yields (21-36%), S/G ratios (0.41-0.93), and oil yields (40-75%). These results were successfully validated by repeating RCF reactions in 75 mL batch reactors for a subset of samples. We anticipate that this approach can be used to rapidly screen substrates, catalysts, and reaction conditions in high-pressure batch reactions with higher throughput than standard batch reactors.

Economic and Sustainability Impacts of Yield and Composition Variation in Bioenergy Crops: Switchgrass (Panicum virgatum L.).

Economically viable production of biobased products and fuels requires high-yielding, high-quality, sustainable process-advantaged crops, developed using bioengineering or advanced breeding approaches. Understanding which crop phenotypic traits have the largest impact on biofuel economics and sustainability outcomes is important for the targeted feedstock crop development. Here, we evaluated biomass yield and cell-wall composition traits across a large natural variant population of switchgrass (Panicum virgatum L.) grown across three common garden sites. Samples from 331 switchgrass genotypes were collected and analyzed for carbohydrate and lignin components. Considering plant survival and biomass after multiple years of growth, we found that 84 of the genotypes analyzed may be suited for commercial production in the southeastern U.S. These genotypes show a range of growth and compositional traits across the population that are apparently independent of each other. We used these data to conduct techno-economic analyses and life cycle assessments evaluating the performance of each switchgrass genotype under a standard cellulosic ethanol process model with pretreatment, added enzymes, and fermentation. We find that switchgrass yield per area is the largest economic driver of the minimum fuel selling price (MSFP), ethanol yield per hectare, global warming potential (GWP), and cumulative energy demand (CED). At any yield, the carbohydrate content is significant but of secondary importance. Water use follows similar trends but has more variability due to an increased dependence on the biorefinery model. Analyses presented here highlight the primary importance of plant yield and the secondary importance of carbohydrate content when selecting a feedstock that is both economical and sustainable.

Rapid screening of secondary aromatic metabolites in Populus trichocarpa leaves.

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.

Abundance of Major Cell Wall Components in Natural Variants and Pedigrees of Populus trichocarpa.

The rapid analysis of biopolymers including lignin and sugars in lignocellulosic biomass cell walls is essential for the analysis of the large sample populations needed for identifying heritable genetic variation in biomass feedstocks for biofuels and bioproducts. In this study, we reported the analysis of cell wall lignin content, syringyl/guaiacyl (S/G) ratio, as well as glucose and xylose content by high-throughput pyrolysis-molecular beam mass spectrometry (py-MBMS) for >3,600 samples derived from hundreds of accessions of Populus trichocarpa from natural populations, as well as pedigrees constructed from 14 parents (7 × 7). Partial Least Squares (PLS) regression models were built from the samples of known sugar composition previously determined by hydrolysis followed by nuclear magnetic resonance (NMR) analysis. Key spectral features positively correlated with glucose content consisted of m/z 126, 98, and 69, among others, deriving from pyrolyzates such as hydroxymethylfurfural, maltol, and other sugar-derived species. Xylose content positively correlated primarily with many lignin-derived ions and to a lesser degree with m/z 114, deriving from a lactone produced from xylose pyrolysis. Models were capable of predicting glucose and xylose contents with an average error of less than 4%, and accuracy was significantly improved over previously used methods. The differences in the models constructed from the two sample sets varied in training sample number, but the genetic and compositional uniformity of the pedigree set could be a potential driver in the slightly better performance of that model in comparison with the natural variants. Broad-sense heritability of glucose and xylose composition using these data was 0.32 and 0.34, respectively. In summary, we have demonstrated the use of a single high-throughput method to predict sugar and lignin composition in thousands of poplar samples to estimate the heritability and phenotypic plasticity of traits necessary to develop optimized feedstocks for bioenergy applications.

Importance of suberin biopolymer in plant function, contributions to soil organic carbon and in the production of bio-derived energy and materials.

Suberin is a hydrophobic biopolymer of significance in the production of biomass-derived materials and in biogeochemical cycling in terrestrial ecosystems. Here, we describe suberin structure and biosynthesis, and its importance in biological (i.e., plant bark and roots), ecological (soil organic carbon) and economic (biomass conversion to bioproducts) contexts. Furthermore, we highlight the genomics and analytical approaches currently available and explore opportunities for future technologies to study suberin in quantitative and/or high-throughput platforms in bioenergy crops. A greater understanding of suberin structure and production in lignocellulosic biomass can be leveraged to improve representation in life cycle analysis and techno-economic analysis models and enable performance improvements in plant biosystems as well as informed crop system management to achieve economic and environmental co-benefits.

Comparison of methodologies used to determine aromatic lignin unit ratios in lignocellulosic biomass.

BACKGROUND: Multiple analytical methods have been developed to determine the ratios of aromatic lignin units, particularly the syringyl/guaiacyl (S/G) ratio, of lignin biopolymers in plant cell walls. Chemical degradation methods such as thioacidolysis produce aromatic lignin units that are released from certain linkages and may induce chemical changes rendering it difficult to distinguish and determine the source of specific aromatic lignin units released, as is the case with nitrobenzene oxidation methodology. NMR methods provide powerful tools used to analyze cell walls for lignin composition and linkage information. Pyrolysis-mass spectrometry methods are also widely used, particularly as high-throughput methodologies. However, the different techniques used to analyze aromatic lignin unit ratios frequently yield different results within and across particular studies, making it difficult to interpret and compare results. This also makes it difficult to obtain meaningful insights relating these measurements to other characteristics of plant cell walls that may impact biomass sustainability and conversion metrics for the production of bio-derived fuels and chemicals. RESULTS: The authors compared the S/G lignin unit ratios obtained from thioacidolysis, pyrolysis-molecular beam mass spectrometry (py-MBMS), HSQC liquid-state NMR and solid-state (ss) NMR methodologies of pine, several genotypes of poplar, and corn stover biomass. An underutilized approach to deconvolute ssNMR spectra was implemented to derive S/G ratios. The S/G ratios obtained for the samples did not agree across the different methods, but trends were similar with the most agreement among the py-MBMS, HSQC NMR and deconvoluted ssNMR methods. The relationship between S/G, thioacidolysis yields, and linkage analysis determined by HSQC is also addressed. CONCLUSIONS: This work demonstrates that different methods using chemical, thermal, and non-destructive NMR techniques to determine native lignin S/G ratios in plant cell walls may yield different results depending on species and linkage abundances. Spectral deconvolution can be applied to many hardwoods with lignin dominated by S and G units, but the results may not be reliable for some woody and grassy species of more diverse lignin composition. HSQC may be a better method for analyzing lignin in those species given the wealth of information provided on additional aromatic moieties and bond linkages. Additionally, trends or correlations in lignin characteristics such as S/G ratios and lignin linkages within the same species such as poplar may not necessarily exhibit the same trends or correlations made across different biomass types. Careful consideration is required when choosing a method to measure S/G ratios and the benefits and shortcomings of each method discussed here are summarized.

Genetic Modification of KNAT7 Transcription Factor Expression Enhances Saccharification and Reduces Recalcitrance of Woody Biomass in Poplars.

The precise role of KNAT7 transcription factors (TFs) in regulating secondary cell wall (SCW) biosynthesis in poplars has remained unknown, while our understanding of KNAT7 functions in other plants is continuously evolving. To study the impact of genetic modifications of homologous and heterologous KNAT7 gene expression on SCW formation in transgenic poplars, we prepared poplar KNAT7 (PtKNAT7) overexpression (PtKNAT7-OE) and antisense suppression (PtKNAT7-AS) vector constructs for the generation of transgenic poplar lines via Agrobacterium-mediated transformation. Since the overexpression of homologous genes can sometimes result in co-suppression, we also overexpressed Arabidopsis KNAT7 (AtKNAT7-OE) in transgenic poplars. In all these constructs, the expression of KNAT7 transgenes was driven by developing xylem (DX)-specific promoter, DX15. Compared to wild-type (WT) controls, many SCW biosynthesis genes downstream of KNAT7 were highly expressed in poplar PtKNAT7-OE and AtKNAT7-OE lines. Yet, no significant increase in lignin content of woody biomass of these transgenic lines was observed. PtKNAT7-AS lines, however, showed reduced expression of many SCW biosynthesis genes downstream of KNAT7 accompanied by a reduction in lignin content of wood compared to WT controls. Syringyl to Guaiacyl lignin (S/G) ratios were significantly increased in all three KNAT7 knockdown and overexpression transgenic lines than WT controls. These transgenic lines were essentially indistinguishable from WT controls in terms of their growth phenotype. Saccharification efficiency of woody biomass was significantly increased in all transgenic lines than WT controls. Overall, our results demonstrated that developing xylem-specific alteration of KNAT7 expression affects the expression of SCW biosynthesis genes, impacting at least the lignification process and improving saccharification efficiency, hence providing one of the powerful tools for improving bioethanol production from woody biomass of bioenergy crops and trees.

Accurate determination of genotypic variance of cell wall characteristics of a Populus trichocarpa pedigree using high-throughput pyrolysis-molecular beam mass spectrometry.

BACKGROUND: Pyrolysis-molecular beam mass spectrometry (py-MBMS) analysis of a pedigree of Populus trichocarpa was performed to study the phenotypic plasticity and heritability of lignin content and lignin monomer composition. Instrumental and microspatial environmental variability were observed in the spectral features and corrected to reveal underlying genetic variance of biomass composition. RESULTS: Lignin-derived ions (including m/z 124, 154, 168, 194, 210 and others) were highly impacted by microspatial environmental variation which demonstrates phenotypic plasticity of lignin composition in Populus trichocarpa biomass. Broad-sense heritability of lignin composition after correcting for microspatial and instrumental variation was determined to be H2 = 0.56 based on py-MBMS ions known to derive from lignin. Heritability of lignin monomeric syringyl/guaiacyl ratio (S/G) was H2 = 0.81. Broad-sense heritability was also high (up to H2 = 0.79) for ions derived from other components of the biomass including phenolics (e.g., salicylates) and C5 sugars (e.g., xylose). Lignin and phenolic ion abundances were primarily driven by maternal effects, and paternal effects were either similar or stronger for the most heritable carbohydrate-derived ions. CONCLUSIONS: We have shown that many biopolymer-derived ions from py-MBMS show substantial phenotypic plasticity in response to microenvironmental variation in plantations. Nevertheless, broad-sense heritability for biomass composition can be quite high after correcting for spatial environmental variation. This work outlines the importance in accounting for instrumental and microspatial environmental variation in biomass composition data for applications in heritability measurements and genomic selection for breeding poplar for renewable fuels and materials.

Molecular weight distribution of raw and catalytic fast pyrolysis oils: comparison of analytical methodologies.

Comprehensive analysis of the molecular weight distribution of raw and catalytic fast pyrolysis oils derived from biomass remains a key technical hurdle to understanding oil quality as it relates to downstream use and multiple methods may be necessary to accurately represent all components present. Here, we report the molecular weight distribution metrics of fast pyrolysis (FP) and catalytic fast pyrolysis (CFP) oils as determined by gel permeation chromatography (GPC) combined with UV-diode array (UV), differential refractive index (RI), and multi-angle laser light scattering (MALS) detection. The measured molar mass distributions revealed that FP oil consisted of a higher proportion of larger products relative to the low molecular weight products contained in the CFP oil. GPC/RI and UV methods showed FP oil to have higher weight-average molecular weight (M w) and number-average molecular weight (M n) than CFP oil based on elution time. However, GPC/MALS, determined the two oils to have similar overall molecular weight distribution metrics (M w and M n) and yielded values significantly higher than those determined by RI and UV detectors relative to external standards. Overall, the use of a multiple detection GPC method could enable a more accurate comparison and determination of true molecular weight metrics of bio-oils.

Selective One-Dimensional 13C-13C Spin-Diffusion Solid-State Nuclear Magnetic Resonance Methods to Probe Spatial Arrangements in Biopolymers Including Plant Cell Walls, Peptides, and Spider Silk.

Two-dimensional (2D) and 3D through-space 13C-13C homonuclear spin-diffusion techniques are powerful solid-state nuclear magnetic resonance (NMR) tools for extracting structural information from 13C-enriched biomolecules, but necessarily long acquisition times restrict their applications. In this work, we explore the broad utility and underutilized power of a chemical shift-selective one-dimensional (1D) version of a 2D 13C-13C spin-diffusion solid-state NMR technique. The method, which is called 1D dipolar-assisted rotational resonance (DARR) difference, is applied to a variety of biomaterials including lignocellulosic plant cell walls, microcrystalline peptide fMLF, and black widow dragline spider silk. 1D 13C-13C spin-diffusion methods described here apply in select cases in which the 1D 13C solid-state NMR spectrum displays chemical shift-resolved moieties. This is analogous to the selective 1D nuclear Overhauser effect spectroscopy (NOESY) experiment utilized in liquid-state NMR as a faster (1D instead of 2D) and often less ambiguous (direct sampling of the time domain data, coupled with increased signal averaging) alternative to 2D NOESY. Selective 1D 13C-13C spin-diffusion methods are more time-efficient than their 2D counterparts such as proton-driven spin diffusion (PDSD) and dipolar-assisted rotational resonance. The additional time gained enables measurements of 13C-13C spin-diffusion buildup curves and extraction of spin-diffusion time constants TSD, yielding detailed structural information. Specifically, selective 1D DARR difference buildup curves applied to 13C-enriched hybrid poplar woody stems confirm strong spatial interaction between lignin and acetylated xylan polymers within poplar plant secondary cell walls, and an interpolymer distance of ∼0.45-0.5 nm was estimated. Additionally, Tyr/Gly long-range correlations were observed on isotopically enriched black widow spider dragline silks.

Genome-Wide Association Study of Wood Anatomical and Morphological Traits in Populus trichocarpa.

To understand the genetic mechanisms underlying wood anatomical and morphological traits in Populus trichocarpa, we used 869 unrelated genotypes from a common garden in Clatskanie, Oregon that were previously collected from across the distribution range in western North America. Using GEMMA mixed model analysis, we tested for the association of 25 phenotypic traits and nine multitrait combinations with 6.741 million SNPs covering the entire genome. Broad-sense trait heritabilities ranged from 0.117 to 0.477. Most traits were significantly correlated with geoclimatic variables suggesting a role of climate and geography in shaping the variation of this species. Fifty-seven SNPs from single trait GWAS and 11 SNPs from multitrait GWAS passed an FDR threshold of 0.05, leading to the identification of eight and seven nearby candidate genes, respectively. The percentage of phenotypic variance explained (PVE) by the significant SNPs for both single and multitrait GWAS ranged from 0.01% to 6.18%. To further evaluate the potential roles of candidate genes, we used a multi-omic network containing five additional data sets, including leaf and wood metabolite GWAS layers and coexpression and comethylation networks. We also performed a functional enrichment analysis on coexpression nearest neighbors for each gene model identified by the wood anatomical and morphological trait GWAS analyses. Genes affecting cell wall composition and transport related genes were enriched in wood anatomy and stomatal density trait networks. Signaling and metabolism related genes were also common in networks for stomatal density. For leaf morphology traits (leaf dry and wet weight) the networks were significantly enriched for GO terms related to photosynthetic processes as well as cellular homeostasis. The identified genes provide further insights into the genetic control of these traits, which are important determinants of the suitability and sustainability of improved genotypes for lignocellulosic biofuel production.

Electrocatalytic CO2 Reduction over Cu3P Nanoparticles Generated via a Molecular Precursor Route.

The design of nanoparticles (NPs) with tailored morphologies and finely tuned electronic and physical properties has become a key strategy for controlling selectivity and improving conversion efficiency in a variety of important electrocatalytic transformations. Transition metal phosphide NPs, in particular, have emerged as a versatile class of catalytic materials due to their multifunctional active sites and composition- and phase-dependent properties. Access to targeted transition metal phosphide NPs with controlled features is necessary to tune the catalytic activity. To this end, we have established a solution-synthesis route utilizing a molecular precursor containing M-P bonds to generate solid metal phosphide NPs with controlled stoichiometry and morphology. We expand here the application of molecular precursors in metal phosphide NP synthesis to include the preparation of phase-pure Cu3P NPs from the thermal decomposition of [Cu(H)(PPh3)]6. The mechanism of [Cu(H)(PPh3)]6 decomposition and subsequent formation of Cu3P was investigated through modification of the reaction parameters. Identification and optimization of the critical reaction parameters (i.e., time, temperature, and oleylamine concentration) enabled the synthesis of phase-pure 9-11 nm Cu3P NPs. To probe the multifunctionality of this materials system, Cu3P NPs were investigated as an electrocatalyst for CO2 reduction. At low overpotential (-0.30 V versus RHE) in 0.1 M KHCO3 electrolyte, Cu3P-modified carbon paper electrodes produced formate (HCOO-) at a maximum Faradaic efficiency of 8%.

Genetic variation of biomass recalcitrance in a natural Salix viminalis (L.) population.

BACKGROUND: Salix spp. are high-productivity crops potentially used for lignocellulosic biofuels such as bioethanol. In general, pretreatment is needed to facilitate the enzymatic depolymerization process. Biomass resistance to degradation, i.e., biomass recalcitrance, is a trait which can be assessed by measuring the sugar released after combined pretreatment and enzymatic hydrolysis. We have examined genetic parameters of enzymatic sugar release and other traits related to biorefinery use in a population of 286 natural Salix viminalis clones. Furthermore, we have evaluated phenotypic and genetic correlations between these traits and performed a genomewide association mapping analysis using a set of 19,411 markers. RESULTS: Sugar release (glucose and xylose) after pretreatment and enzymatic saccharification proved highly variable with large genetic and phenotypic variations, and chip heritability estimates (h 2) of 0.23-0.29. Lignin syringyl/guaiacyl (S/G) ratio and wood density were the most heritable traits (h 2 = 0.42 and 0.59, respectively). Sugar release traits were positively correlated, phenotypically and genetically, with biomass yield and lignin S/G ratio. Association mapping revealed seven marker-trait associations below a suggestive significance threshold, including one marker associated with glucose release. CONCLUSIONS: We identified lignin S/G ratio and shoot diameter as heritable traits that could be relatively easily evaluated by breeders, making them suitable proxy traits for developing low-recalcitrance varieties. One marker below the suggestive threshold for marker associations was identified for sugar release, meriting further investigation while also highlighting the difficulties in employing genomewide association mapping for complex traits.

The effect of coumaryl alcohol incorporation on the structure and composition of lignin dehydrogenation polymers.

BACKGROUND: Lignin dehydrogenation polymers (DHPs) are polymers generated from phenolic precursors for the purpose of studying lignin structure and polymerization processes. METHODS: Here, DHPs were synthesized using a Zutropfverfahren method with horseradish peroxidase and three lignin monomers, sinapyl (S), coumaryl (H), and coniferyl (G) alcohols, in the presence of hydrogen peroxide. The H monomer was reacted with G and a 1:1 molar mixture of S:G monomers at H molar compositions of 0, 5, 10, and 20 mol% to study how the presence of the H monomer affected the structure and composition of the recovered polymers. RESULTS: At low H concentrations, solid-state NMR spectra suggest that the H and G monomers interact to form G:H polymers that have a lower average molecular weight than the solely G-based polymer or the G:H polymer produced at higher H concentrations. Solid-state NMR and pyrolysis-MBMS analyses suggest that at higher H concentrations, the H monomer primarily self-polymerizes to produce clusters of H-based polymer that are segregated from clusters of G- or S:G-based polymers. Thioacidolysis generally showed higher recoveries of thioethylated products from S:G or S:G:H polymers made with higher H content, indicating an increase in the linear ether linkages. CONCLUSIONS: Overall, the experimental results support theoretical predictions for the reactivity and structural influences of the H monomer on the formation of lignin-like polymers.