Compensatory Guaiacyl Lignin Biosynthesis at the Expense of Syringyl Lignin in 4CL1-Knockout Poplar.

The lignin biosynthetic pathway is highly conserved in angiosperms, yet pathway manipulations give rise to a variety of taxon-specific outcomes. Knockout of lignin-associated 4-coumarate:CoA ligases (4CLs) in herbaceous species mainly reduces guaiacyl (G) lignin and enhances cell wall saccharification. Here we show that CRISPR-knockout of 4CL1 in poplar (Populus tremula × alba) preferentially reduced syringyl (S) lignin, with negligible effects on biomass recalcitrance. Concordant with reduced S-lignin was downregulation of ferulate 5-hydroxylases (F5Hs). Lignification was largely sustained by 4CL5, a low-affinity paralog of 4CL1 typically with only minor xylem expression or activity. Levels of caffeate, the preferred substrate of 4CL5, increased in line with significant upregulation of caffeoyl shikimate esterase1 Upregulation of caffeoyl-CoA O-methyltransferase1 and downregulation of F5Hs are consistent with preferential funneling of 4CL5 products toward G-lignin biosynthesis at the expense of S-lignin. Thus, transcriptional and metabolic adaptations to 4CL1-knockout appear to have enabled 4CL5 catalysis at a level sufficient to sustain lignification. Finally, genes involved in sulfur assimilation, the glutathione-ascorbate cycle, and various antioxidant systems were upregulated in the mutants, suggesting cascading responses to perturbed thioesterification in lignin biosynthesis.

Network-based integration of systems genetics data reveals pathways associated with lignocellulosic biomass accumulation and processing.

As a consequence of their remarkable adaptability, fast growth, and superior wood properties, eucalypt tree plantations have emerged as key renewable feedstocks (over 20 million ha globally) for the production of pulp, paper, bioenergy, and other lignocellulosic products. However, most biomass properties such as growth, wood density, and wood chemistry are complex traits that are hard to improve in long-lived perennials. Systems genetics, a process of harnessing multiple levels of component trait information (e.g., transcript, protein, and metabolite variation) in populations that vary in complex traits, has proven effective for dissecting the genetics and biology of such traits. We have applied a network-based data integration (NBDI) method for a systems-level analysis of genes, processes and pathways underlying biomass and bioenergy-related traits using a segregating Eucalyptus hybrid population. We show that the integrative approach can link biologically meaningful sets of genes to complex traits and at the same time reveal the molecular basis of trait variation. Gene sets identified for related woody biomass traits were found to share regulatory loci, cluster in network neighborhoods, and exhibit enrichment for molecular functions such as xylan metabolism and cell wall development. These findings offer a framework for identifying the molecular underpinnings of complex biomass and bioprocessing-related traits. A more thorough understanding of the molecular basis of plant biomass traits should provide additional opportunities for the establishment of a sustainable bio-based economy.

Exome resequencing and GWAS for growth, ecophysiology, and chemical and metabolomic composition of wood of Populus trichocarpa.

BACKGROUND: Populus trichocarpa is an important forest tree species for the generation of lignocellulosic ethanol. Understanding the genomic basis of biomass production and chemical composition of wood is fundamental in supporting genetic improvement programs. Considerable variation has been observed in this species for complex traits related to growth, phenology, ecophysiology and wood chemistry. Those traits are influenced by both polygenic control and environmental effects, and their genome architecture and regulation are only partially understood. Genome wide association studies (GWAS) represent an approach to advance that aim using thousands of single nucleotide polymorphisms (SNPs). Genotyping using exome capture methodologies represent an efficient approach to identify specific functional regions of genomes underlying phenotypic variation. RESULTS: We identified 813 K SNPs, which were utilized for genotyping 461 P. trichocarpa clones, representing 101 provenances collected from Oregon and Washington, and established in California. A GWAS performed on 20 traits, considering single SNP-marker tests identified a variable number of significant SNPs (p-value < 6.1479E-8) in association with diameter, height, leaf carbon and nitrogen contents, and δ15N. The number of significant SNPs ranged from 2 to 220 per trait. Additionally, multiple-marker analyses by sliding-windows tests detected between 6 and 192 significant windows for the analyzed traits. The significant SNPs resided within genes that encode proteins belonging to different functional classes as such protein synthesis, energy/metabolism and DNA/RNA metabolism, among others. CONCLUSIONS: SNP-markers within genes associated with traits of importance for biomass production were detected. They contribute to characterize the genomic architecture of P. trichocarpa biomass required to support the development and application of marker breeding technologies.

Expression profiles of cell-wall related genes vary broadly between two common maize inbreds during stem development.

BACKGROUND: The cellular machinery for cell wall synthesis and metabolism is encoded by members of large multi-gene families. Maize is both a genetic model for grass species and a potential source of lignocellulosic biomass from crop residues. Genetic improvement of maize for its utility as a bioenergy feedstock depends on identification of the specific gene family members expressed during secondary wall development in stems. RESULTS: High-throughput sequencing of transcripts expressed in developing rind tissues of stem internodes provided a comprehensive inventory of cell wall-related genes in maize (Zea mays, cultivar B73). Of 1239 of these genes, 854 were expressed among the internodes at ≥95 reads per 20 M, and 693 of them at ≥500 reads per 20 M. Grasses have cell wall compositions distinct from non-commelinid species; only one-quarter of maize cell wall-related genes expressed in stems were putatively orthologous with those of the eudicot Arabidopsis. Using a slope-metric algorithm, five distinct patterns for sub-sets of co-expressed genes were defined across a time course of stem development. For the subset of genes associated with secondary wall formation, fifteen sequence motifs were found in promoter regions. The same members of gene families were often expressed in two maize inbreds, B73 and Mo17, but levels of gene expression between them varied, with 30% of all genes exhibiting at least a 5-fold difference at any stage. Although presence-absence and copy-number variation might account for much of these differences, fold-changes of expression of a CADa and a FLA11 gene were attributed to polymorphisms in promoter response elements. CONCLUSIONS: Large genetic variation in maize as a species precludes the extrapolation of cell wall-related gene expression networks even from one common inbred line to another. Elucidation of genotype-specific expression patterns and their regulatory controls will be needed for association panels of inbreds and landraces to fully exploit genetic variation in maize and other bioenergy grass species.

Transgenic miR156 switchgrass in the field: growth, recalcitrance and rust susceptibility.

Sustainable utilization of lignocellulosic perennial grass feedstocks will be enabled by high biomass production and optimized cell wall chemistry for efficient conversion into biofuels. MicroRNAs are regulatory elements that modulate the expression of genes involved in various biological functions in plants, including growth and development. In greenhouse studies, overexpressing a microRNA (miR156) gene in switchgrass had dramatic effects on plant architecture and flowering, which appeared to be driven by transgene expression levels. High expressing lines were extremely dwarfed, whereas low and moderate-expressing lines had higher biomass yields, improved sugar release and delayed flowering. Four lines with moderate or low miR156 overexpression from the prior greenhouse study were selected for a field experiment to assess the relationship between miR156 expression and biomass production over three years. We also analysed important bioenergy feedstock traits such as flowering, disease resistance, cell wall chemistry and biofuel production. Phenotypes of the transgenic lines were inconsistent between the greenhouse and the field as well as among different field growing seasons. One low expressing transgenic line consistently produced more biomass (25%-56%) than the control across all three seasons, which translated to the production of 30% more biofuel per plant during the final season. The other three transgenic lines produced less biomass than the control by the final season, and the two lines with moderate expression levels also exhibited altered disease susceptibilities. Results of this study emphasize the importance of performing multiyear field studies for plants with altered regulatory transgenes that target plant growth and development.

Identification and overexpression of gibberellin 2-oxidase (GA2ox) in switchgrass (Panicum virgatum L.) for improved plant architecture and reduced biomass recalcitrance.

Gibberellin 2-oxidases (GA2oxs) are a group of 2-oxoglutarate-dependent dioxygenases that catalyse the deactivation of bioactive GA or its precursors through 2ß-hydroxylation reaction. In this study, putatively novel switchgrass C20 GA2ox genes were identified with the aim of genetically engineering switchgrass for improved architecture and reduced biomass recalcitrance for biofuel. Three C20 GA2ox genes showed differential regulation patterns among tissues including roots, seedlings and reproductive parts. Using a transgenic approach, we showed that overexpression of two C20 GA2ox genes, that is PvGA2ox5 and PvGA2ox9, resulted in characteristic GA-deficient phenotypes with dark-green leaves and modified plant architecture. The changes in plant morphology appeared to be associated with GA2ox transcript abundance. Exogenous application of GA rescued the GA-deficient phenotypes in transgenic lines. Transgenic semi-dwarf lines displayed increased tillering and reduced lignin content, and the syringyl/guaiacyl lignin monomer ratio accompanied by the reduced expression of lignin biosynthetic genes compared to nontransgenic plants. A moderate increase in the level of glucose release in these transgenic lines might be attributed to reduced biomass recalcitrance as a result of reduced lignin content and lignin composition. Our results suggest that overexpression of GA2ox genes in switchgrass is a feasible strategy to improve plant architecture and reduce biomass recalcitrance for biofuel.

Genetic Determinants for Enzymatic Digestion of Lignocellulosic Biomass Are Independent of Those for Lignin Abundance in a Maize Recombinant Inbred Population.

Biotechnological approaches to reduce or modify lignin in biomass crops are predicated on the assumption that it is the principal determinant of the recalcitrance of biomass to enzymatic digestion for biofuels production. We defined quantitative trait loci (QTL) in the Intermated B73 × Mo17 recombinant inbred maize (Zea mays) population using pyrolysis molecular-beam mass spectrometry to establish stem lignin content and an enzymatic hydrolysis assay to measure glucose and xylose yield. Among five multiyear QTL for lignin abundance, two for 4-vinylphenol abundance, and four for glucose and/or xylose yield, not a single QTL for aromatic abundance and sugar yield was shared. A genome-wide association study for lignin abundance and sugar yield of the 282-member maize association panel provided candidate genes in the 11 QTL of the B73 and Mo17 parents but showed that many other alleles impacting these traits exist among this broader pool of maize genetic diversity. B73 and Mo17 genotypes exhibited large differences in gene expression in developing stem tissues independent of allelic variation. Combining these complementary genetic approaches provides a narrowed list of candidate genes. A cluster of SCARECROW-LIKE9 and SCARECROW-LIKE14 transcription factor genes provides exceptionally strong candidate genes emerging from the genome-wide association study. In addition to these and genes associated with cell wall metabolism, candidates include several other transcription factors associated with vascularization and fiber formation and components of cellular signaling pathways. These results provide new insights and strategies beyond the modification of lignin to enhance yields of biofuels from genetically modified biomass.

Ectopic expression of a loblolly pine class II 4-coumarate:CoA ligase alters soluble phenylpropanoid metabolism but not lignin biosynthesis in Populus.

4-Coumarate:CoA ligase (4CL) catalyzes the formation of hydroxycinnamoyl-CoA esters for phenylpropanoid biosynthesis. Phylogenetically distinct Class I and Class II 4CL isoforms occur in angiosperms, and support lignin and non-lignin phenylpropanoid biosynthesis, respectively. In contrast, the few experimentally characterized gymnosperm 4CLs are associated with lignin biosynthesis and belong to the conifer-specific Class III. Here we report a new Pinus taeda isoform Pinta4CL3 that is phylogenetically more closely related to Class II angiosperm 4CLs than to Class III Pinta4CL1. Like angiosperm Class II 4CLs, Pinta4CL3 transcript levels were detected in foliar and root tissues but were absent in xylem, and recombinant Pinta4CL3 exhibited a substrate preference for 4-coumaric acid. Constitutive expression of Pinta4CL3 in transgenic Populus led to significant increases of hydroxycinnamoyl-quinate esters at the expense of hydroxycinnamoyl-glucose esters in green tissues. In particular, large increases of cinnamoyl-quinate in transgenic leaves suggested in vivo utilization of cinnamic acid by Pinta4CL3. Lignin was unaffected in transgenic Populus, consistent with Pinta4CL3 involvement in biosynthesis of non-structural phenylpropanoids. We discuss the in vivo cinnamic acid utilization activity of Pinta4CL3 and its adaptive significance in conifer defense. Together with phylogenetic inference, our data support an ancient origin of Class II 4CLs that pre-dates the angiosperm-gymnosperm split.

Two-year field analysis of reduced recalcitrance transgenic switchgrass.

Switchgrass (Panicum virgatum L.) is a leading candidate for a dedicated lignocellulosic biofuel feedstock owing to its high biomass production, wide adaptation and low agronomic input requirements. Lignin in cell walls of switchgrass, and other lignocellulosic feedstocks, severely limits the accessibility of cell wall carbohydrates to enzymatic breakdown into fermentable sugars and subsequently biofuels. Low-lignin transgenic switchgrass plants produced by the down-regulation of caffeic acid O-methyltransferase (COMT), a lignin biosynthetic enzyme, were analysed in the field for two growing seasons. COMT transcript abundance, lignin content and the syringyl/guaiacyl lignin monomer ratio were consistently lower in the COMT-down-regulated plants throughout the duration of the field trial. In general, analyses with fully established plants harvested during the second growing season produced results that were similar to those observed in previous greenhouse studies with these plants. Sugar release was improved by up to 34% and ethanol yield by up to 28% in the transgenic lines relative to controls. Additionally, these results were obtained using senesced plant material harvested at the end of the growing season, compared with the young, green tissue that was used in the greenhouse experiments. Another important finding was that transgenic plants were not more susceptible to rust (Puccinia emaculata). The results of this study suggest that lignin down-regulation in switchgrass can confer real-world improvements in biofuel yield without negative consequences to biomass yield or disease susceptibility.

Lignin content in natural Populus variants affects sugar release.

The primary obstacle to producing renewable fuels from lignocellulosic biomass is a plant's recalcitrance to releasing sugars bound in the cell wall. From a sample set of wood cores representing 1,100 individual undomesticated Populus trichocarpa trees, 47 extreme phenotypes were selected across measured lignin content and ratio of syringyl and guaiacyl units (S/G ratio). This subset was tested for total sugar release through enzymatic hydrolysis alone as well as through combined hot-water pretreatment and enzymatic hydrolysis using a high-throughput screening method. The total amount of glucan and xylan released varied widely among samples, with total sugar yields of up to 92% of the theoretical maximum. A strong negative correlation between sugar release and lignin content was only found for pretreated samples with an S/G ratio < 2.0. For higher S/G ratios, sugar release was generally higher, and the negative influence of lignin was less pronounced. When examined separately, only glucose release was correlated with lignin content and S/G ratio in this manner, whereas xylose release depended on the S/G ratio alone. For enzymatic hydrolysis without pretreatment, sugar release increased significantly with decreasing lignin content below 20%, irrespective of the S/G ratio. Furthermore, certain samples featuring average lignin content and S/G ratios exhibited exceptional sugar release. These facts suggest that factors beyond lignin and S/G ratio influence recalcitrance to sugar release and point to a critical need for deeper understanding of cell-wall structure before plants can be rationally engineered for reduced recalcitrance and efficient biofuels production.

Association genetics of chemical wood properties in black poplar (Populus nigra).

Black poplar (Populus nigra) is a potential feedstock for cellulosic ethanol production, although breeding for this specific end use is required. Our goal was to identify associations between single nucleotide polymorphism (SNP) markers within candidate genes encoding cellulose and lignin biosynthetic enzymes, with chemical wood property phenotypic traits, toward the aim of developing genomics-based breeding technologies for bioethanol production. Pyrolysis molecular beam mass spectrometry was used to determine contents of five- and six-carbon sugars, lignin, and syringyl : guaiacyl ratio. The association population included 599 clones from 17 half-sib families, which were successfully genotyped using 433 SNPs from 39 candidate genes. Statistical analyses were performed to estimate genetic parameters, linkage disequilibrium (LD), and single marker and haplotype-based associations. A moderate to high heritability was observed for all traits. The LD, across all candidate genes, showed a rapid decay with physical distance. Analysis of single marker-phenotype associations identified six significant marker-trait pairs, whereas nearly 280 haplotypes were associated with phenotypic traits, in both an individual and multiple trait-specific manner. The rapid decay of LD within candidate genes in this population and the genetic associations identified suggest a close relationship between the associated SNPs and the causative polymorphisms underlying the genetic variation of lignocellulosic traits in black poplar.

Rapid determination of sugar content in biomass hydrolysates using nuclear magnetic resonance spectroscopy.

Large populations of potential cellulosic biomass feedstocks are currently being screened for fuel and chemical applications. The monomeric sugar content, released through hydrolysis, is of particular importance and is currently measured with time-consuming HPLC methods. A method for sugar detection is presented here that employs (1)H NMR spectra regressed against primary HPLC sugar concentration data to build partial least squares (PLS) models. The PLS2 model is able to predict concentrations of both major sugar components, like glucose and xylose, and minor sugars, such as arabinose and mannose, in biomass hydrolysates. The model was built with 65 samples from a variety of different biomass species and covers a wide range of sugar concentrations. Model predictions were validated with a set of 15 samples which were all within error of both HPLC and NMR integration measurements. The data collection time for these NMR measurements is less than 20 min, offering a significant improvement to the 1 h acquisition time that is required for HPLC.

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.

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.

Solid-state 13C NMR assignment of carbon resonances on metallic and semiconducting single-walled carbon nanotubes.

Solid-state (13)C NMR spectroscopy was used to investigate the chemical shift of nanotube carbons on m- and s-SWNTs (metallic and semiconducting single-walled nanotubes) for samples with widely varying s-SWNT content, including samples highly enriched with nearly 100% m- and s-SWNTs. High-resolution (13)C NMR was found to be a sensitive probe for m- and s-SWNTs in mixed SWNT samples with diameters of approximately 1.3 nm. The two highly enriched m- and s-SWNT samples clearly exhibited features for m- and s-SNWT (13)C nuclei (approximately 123 and 122 ppm, respectively) and were successfully fit with a single Gaussian, while five mixed samples required two Gaussians for a satisfactory fit.

Association genetics of traits controlling lignin and cellulose biosynthesis in black cottonwood (Populus trichocarpa, Salicaceae) secondary xylem.

• An association genetics approach was used to examine individual genes and alleles at the loci responsible for complex traits controlling lignocellulosic biosynthesis in black cottonwood (Populus trichocarpa). Recent interest in poplars as a source of renewable energy, combined with the vast genomic resources available, has enabled further examination of their genetic diversity. • Forty candidate genes were resequenced in a panel of 15 unrelated individuals to identify single nucleotide polymorphisms (SNPs). Eight hundred and seventy-six SNPs were successfully genotyped in a clonally replicated population (448 clones). The association population (average of 2.4 ramets per clone) was phenotyped using pyrolysis molecular beam mass spectrometry. Both single-marker and haplotype-based association tests were implemented to identify associations for composite traits representing lignin content, syringyl : guaiacyl ratio and C6 sugars. • Twenty-seven highly significant, unique, single-marker associations (false discovery rate Q < 0.10) were identified across 40 candidate genes in three composite traits. Twenty-three significant haplotypes within 11 genes were discovered in two composite traits. • Given the rapid decay of within-gene linkage disequilibrium and the high coverage of amplicons across each gene, it is likely that the numerous polymorphisms identified are in close proximity to the causative SNPs and the haplotype associations reflect information present in the associations between markers.

Genetic resources for maize cell wall biology.

Grass species represent a major source of food, feed, and fiber crops and potential feedstocks for biofuel production. Most of the biomass is contributed by cell walls that are distinct in composition from all other flowering plants. Identifying cell wall-related genes and their functions underpins a fundamental understanding of growth and development in these species. Toward this goal, we are building a knowledge base of the maize (Zea mays) genes involved in cell wall biology, their expression profiles, and the phenotypic consequences of mutation. Over 750 maize genes were annotated and assembled into gene families predicted to function in cell wall biogenesis. Comparative genomics of maize, rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana) sequences reveal differences in gene family structure between grass species and a reference eudicot species. Analysis of transcript profile data for cell wall genes in developing maize ovaries revealed that expression within families differed by up to 100-fold. When transcriptional analyses of developing ovaries before pollination from Arabidopsis, rice, and maize were contrasted, distinct sets of cell wall genes were expressed in grasses. These differences in gene family structure and expression between Arabidopsis and the grasses underscore the requirement for a grass-specific genetic model for functional analyses. A UniformMu population proved to be an important resource in both forward- and reverse-genetics approaches to identify hundreds of mutants in cell wall genes. A forward screen of field-grown lines by near-infrared spectroscopic screen of mature leaves yielded several dozen lines with heritable spectroscopic phenotypes. Pyrolysis-molecular beam mass spectrometry confirmed that several nir mutants had altered carbohydrate-lignin compositions.

Silencing Folylpolyglutamate Synthetase1 (FPGS1) in Switchgrass (Panicum virgatum L.) Improves Lignocellulosic Biofuel Production.

Switchgrass (Panicum virgatum L.) is a lignocellulosic perennial grass with great potential in bioenergy field. Lignocellulosic bioenergy crops are mostly resistant to cell wall deconstruction, and therefore yield suboptimal levels of biofuel. The one-carbon pathway (also known as C1 metabolism) is critical for polymer methylation, including that of lignin and hemicelluloses in cell walls. Folylpolyglutamate synthetase (FPGS) catalyzes a biochemical reaction that leads to the formation of folylpolyglutamate, an important cofactor for many enzymes in the C1 pathway. In this study, the putatively novel switchgrass PvFPGS1 gene was identified and its functional role in cell wall composition and biofuel production was examined by RNAi knockdown analysis. The PvFPGS1-downregulated plants were analyzed in the field over three growing seasons. Transgenic plants with the highest reduction in PvFPGS1 expression grew slower and produced lower end-of-season biomass. Transgenic plants with low-to-moderate reduction in PvFPGS1 transcript levels produced equivalent biomass as controls. There were no significant differences observed for lignin content and syringyl/guaiacyl lignin monomer ratio in the low-to-moderately reduced PvFPGS1 transgenic lines compared with the controls. Similarly, sugar release efficiency was also not significantly different in these transgenic lines compared with the control lines. However, transgenic plants produced up to 18% more ethanol while maintaining congruent growth and biomass as non-transgenic controls. Severity of rust disease among transgenic and control lines were not different during the time course of the field experiments. Altogether, the unchanged lignin content and composition in the low-to-moderate PvFPGS1-downregulated lines may suggest that partial downregulation of PvFPGS1 expression did not impact lignin biosynthesis in switchgrass. In conclusion, the manipulation of PvFPGS1 expression in bioenergy crops may be useful to increase biofuel potential with no growth penalty or increased susceptibility to rust in feedstock.

Identification of functional single nucleotide polymorphism of Populus trichocarpa PtrEPSP-TF and determination of its transcriptional effect.

In plants, the phenylpropanoid pathway is responsible for the synthesis of a diverse array of secondary metabolites that include lignin monomers, flavonoids, and coumarins, many of which are essential for plant structure, biomass recalcitrance, stress defense, and nutritional quality. Our previous studies have demonstrated that Populus trichocarpa PtrEPSP-TF, an isoform of 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, has transcriptional activity and regulates phenylpropanoid biosynthesis in Populus. In this study, we report the identification of single nucleotide polymorphism (SNP) of PtrEPSP-TF that defines its functionality. Populus natural variants carrying this SNP were shown to have reduced lignin content. Here, we demonstrated that the SNP-induced substitution of 142nd amino acid (PtrEPSP-TFD142E) dramatically impairs the DNA-binding and transcriptional activity of PtrEPSP-TF. When introduced to a monocot species rice (Oryza sativa) in which an EPSP synthase isoform with the DNA-binding helix-turn-helix (HTH) motif is absent, the PtrEPSP-TF, but not PtrEPSP-TFD142E, activated genes in the phenylpropanoid pathway. More importantly, heterologous expression of PtrEPSP-TF uncovered five new transcriptional regulators of phenylpropanoid biosynthesis in rice. Collectively, this study identifies the key amino acid required for PtrEPSP-TF functionality and provides a strategy to uncover new transcriptional regulators in phenylpropanoid biosynthesis.

Correction to: Multiple levers for overcoming the recalcitrance of lignocellulosic biomass.

[This corrects the article DOI: 10.1186/s13068-019-1353-7.].