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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Local adaptation of switchgrass drives trait relations to yield and differential responses to climate and soil environments

Abstract Switchgrass, a potential biofuel crop, is a genetically diverse species with phenotypic plasticity enabling it to grow in a range of environments. Two primary divergent ecotypes, uplands and lowlands, exhibit trait combinations representative of acquisitive and conservative growth allocation strategies, respectively. Whether these ecotypes respond differently to various types of environmental drivers remains unclear but is crucial to understanding how switchgrass varieties will respond to climate change. We grew two upland, two lowland, and two intermediate/hybrid cultivars of switchgrass at three sites along a latitudinal gradient in the central United States. Over a 4‐year period, we measured plant functional traits and biomass yields and evaluated genotype‐by‐environment (G × E) interaction effects by analyzing switchgrass responses to soil and climate variables. We found substantial evidence of G × E interactions on biomass yield, primarily due to deviations in the response of the southern lowland cultivar Alamo, which produced more biomass in hotter and drier environments relative to other cultivars. While lowland cultivars had the highest potential for yield, their yields were more variable year‐to‐year compared to other cultivars, suggesting greater sensitivity to environmental perturbations. Models comparing soil and climate principal components as explanatory variables revealed soil properties, especially nutrients, to be most effective at predicting switchgrass biomass yield. Also, positive correlations between biomass yield and conservative plant traits, such as high stem mass and tiller height, became stronger at lower latitudes where the climate is hotter and drier, regardless of ecotype. Lowland cultivars, however, showed a greater predisposition to exhibit these conservative traits. These results suggest switchgrass trait allocation trade‐offs that prioritize aboveground biomass production are more tightly associated in hot, dry environments and that lowland cultivars may exhibit a more specialized strategy relative to other cultivars. Altogether, this research provides essential knowledge for improving the viability of switchgrass as a biofuel crop.

09 BIOMASS FUELS↗
DOE OSTI2015

The WRKY transcription factor family and senescence in switchgrass

Early aerial senescence in switchgrass (Panicum virgatum) can significantly limit biomass yields. WRKY transcription factors that can regulate senescence could be used to reprogram senescence and enhance biomass yields. All potential WRKY genes present in the version 1.0 of the switchgrass genome were identified and curated using manual and bioinformatic methods. Expression profiles of WRKY genes in switchgrass flag leaf RNA-Seq datasets were analyzed using clustering and network analyses tools to identify both WRKY and WRKY-associated gene co-expression networks during leaf development and senescence onset. We identified 240 switchgrass WRKY genes including members of the RW5 and RW6 families of resistance proteins. Weighted gene co-expression network analysis of the flag leaf transcriptomes across development readily separated clusters of co-expressed genes into thirteen modules. A visualization highlighted separation of modules associated with the early and senescence-onset phases of flag leaf growth. The senescence-associated module contained 3000 genes including 23 WRKYs. Putative promoter regions of senescence-associated WRKY genes contained several cis-element-like sequences suggestive of responsiveness to both senescence and stress signaling pathways. A phylogenetic comparison of senescence-associated WRKY genes from switchgrass flag leaf with senescence-associated WRKY genes from other plants revealed notable hotspots in Group I, IIb, and IIe of the phylogenetic tree. We have identified and named 240 WRKY genes in the switchgrass genome. Twenty three of these genes show elevated mRNA levels during the onset of flag leaf senescence. Eleven of the WRKY genes were found in hotspots of related senescence-associated genes from multiple species and thus represent promising targets for future switchgrass genetic improvement. Overall, individual WRKY gene expression profiles could be readily linked to developmental stages of flag leaves.

Biotechnology & Applied Microbiology↗
DOE OSTI2016

Identification and Characterization of Switchgrass Histone H3 and CENH3 Genes

Switchgrass is one of the most promising energy crops and only recently has been employed for biofuel production. The draft genome of switchgrass was recently released; however, relatively few switchgrass genes have been functionally characterized. CENH3 , the major histone protein found in centromeres, along with canonical H3 and other histones, plays an important role in maintaining genome stability and integrity. Despite their importance, the histone H3 genes of switchgrass have remained largely uninvestigated. In this study, we identified 17 putative switchgrass histone H3 genes in silico. Of these genes, 15 showed strong homology to histone H3 genes including six H3.1 genes, three H3.3 genes, four H3.3 -like genes and two H3.1 -like genes. The remaining two genes were found to be homologous to CENH3 . RNA-seq data derived from lowland cultivar Alamo and upland cultivar Dacotah allowed us to identify SNPs in the histone H3 genes and compare their differential gene expression. Interestingly, we also found that overexpression of switchgrass histone H3 and CENH3 genes in N. benthamiana could trigger cell death of the transformed plant cells. Localization and deletion analyses of the histone H3 and CENH3 genes revealed that nuclear localization of the N-terminal tail is essential and sufficient for triggering the cell death phenotype. Lastly, our results deliver insight into the mechanisms underlying the histone-triggered cell death phenotype and provide a foundation for further studying the variations of the histone H3 and CENH3 genes in switchgrass.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Biosynthesis and Emission of Stress-Induced Volatile Terpenes in Roots and Leaves of Switchgrass ( Panicum virgatum L.)

Switchgrass ( Panicum virgatum L.), a perennial C4 grass, represents an important species in natural and anthropogenic grasslands of North America. Its resilience to abiotic and biotic stress has made switchgrass a preferred bioenergy crop. However, little is known about the mechanisms of resistance of switchgrass against pathogens and herbivores. Volatile compounds such as terpenes have important activities in plant direct and indirect defense. Here, we show that switchgrass leaves emit blends of monoterpenes and sesquiterpenes upon feeding by the generalist insect herbivore Spodoptera frugiperda (fall armyworm) and in a systemic response to the treatment of roots with defense hormones. Belowground application of methyl jasmonate also induced the release of volatile terpenes from roots. To correlate the emission of terpenes with the expression and activity of their corresponding biosynthetic genes, we identified a gene family of 44 monoterpene and sesquiterpene synthases (mono- and sesqui-TPSs) of the type-a, type-b, type-g, and type-e subfamilies, of which 32 TPSs were found to be functionally active in vitro. The TPS genes are distributed over the K and N subgenomes with clusters occurring on several chromosomes. Synteny analysis revealed syntenic networks for approximately 30–40% of the switchgrass TPS genes in the genomes of Panicum hallii, Setaria italica , and Sorghum bicolor , suggesting shared TPS ancestry in the common progenitor of these grass lineages. Eighteen switchgrass TPS genes were substantially induced upon insect and hormone treatment and the enzymatic products of nine of these genes correlated with compounds of the induced volatile blends. In accordance with the emission of volatiles, TPS gene expression was induced systemically in response to belowground treatment, whereas this response was not observed upon aboveground feeding of S. frugiperda . Our results demonstrate complex above and belowground responses of induced volatile terpene metabolism in switchgrass and provide a framework for more detailed investigations of the function of terpenes in stress resistance in this monocot crop

Plant Sciences↗
DOE OSTI2015

Transcriptome Profiling of Rust Resistance in Switchgrass Using RNA-Seq Analysis

Switchgrass rust caused by Puccinia emaculata is a major limiting factor for switchgrass (Panicum virgatum L.) production, especially in monoculture. Natural populations of switchgrass displayed diverse reactions to P. emaculata when evaluated in an Ardmore, OK, field. In order to identify the differentially expressed genes during the rust infection process and the mechanisms of switchgrass rust resistance, transcriptome analysis using RNA-Seq was conducted in two pseudo-F 1 parents ('PV281' and 'NFGA472'), and three moderately resistant and three susceptible progenies selected from a three-generation, four-founder switchgrass population (K5 x A4) x (AP13 x VS16). On average, 23.5 million reads per sample (leaf tissue was collected at 0, 24, and 60 h post-inoculation (hpi)) were obtained from paired-end (2 x 100 bp) sequencing on the Illumina HiSeq2000 platform. Furthermore, mapping of the RNA-Seq reads to the switchgrass reference genome (AP13 ver. 1.1 assembly) constructed a total of 84,209 transcripts from 98,007 gene loci among all of the samples. Further analysis revealed that host defense- related genes, including the nucleotide binding site-leucinerich repeat domain containing disease resistance gene analogs, play an important role in resistance to rust infection. Rust-induced gene (RIG) transcripts inherited across generations were identified. The rust-resistant gene transcripts can be a valuable resource for developing molecular markers for rust resistance. Finally we identified the rust-resistant genotypes and gene transcripts which can expedite rust-resistant cultivar development in switchgrass.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Extensive Genetic Diversity is Present within North American Switchgrass Germplasm

Switchgrass ( Panicum virgatum L ) is a perennial native North American grass present in two ecotypes: upland, found primarily in the northern range of switchgrass habitats, and lowland, found largely in the southern reaches of switchgrass habitats. Previous studies focused on a diversity panel of primarily northern switchgrass, so to expand our knowledge of genetic diversity in a broader set of North American switchgrass, exome capture sequence data were generated for 632 additional, primarily lowland individuals. In total, over 37 million single nucleotide polymorphisms (SNPs) were identified and a set of 1.9 million high-confidence SNPs were obtained from 1169 individuals from 140 populations (67 upland, 65 lowland, 8 admixed) were used in downstream analyses of genetic diversity and population structure. Seven separate population groups were identified with moderate genetic differentiation [mean fixation index (Fst) estimate of 0.06] between the lowland and the upland populations. Ecotype-specific and population-specific SNPs were identified for use in germplasm evaluations. Relative to rice ( Oryza sativa L.), maize ( Zea mays L.), soybean [ Glycine max (L.) Merr.], and Medicago truncatula Gaertn., analyses of nucleotide diversity revealed a high degree of genetic diversity (0.0135) across all individuals, consistent with the outcrossing mode of reproduction and the polyploidy of switchgrass. This study supports the hypothesis that repeated glaciation events, ploidy barriers, and restricted gene flow caused by flowering time differences have resulted in distinct gene pools across ecotypes and geographic regions. These data provide a resource to associate alleles with traits of interest for forage, restoration, and biofuel feedstock efforts in switchgrass.

09 BIOMASS FUELS↗
DOE OSTI2017

Data from: Extensive genetic diversity is present within North American switchgrass germplasm

Switchgrass (Panicum virgatum L.) is a perennial native North American grass present in two ecotypes: upland, found primarily in the northern range of switchgrass habitats, and lowland, found largely in the southern reaches of switchgrass habitats. Previous studies focused on a diversity panel of primarily northern switchgrass, so to expand our knowledge of genetic diversity in a broader set of North American switchgrass, exome capture sequence data were generated for 632 additional, primarily lowland individuals. In total, over 37 million single nucleotide polymorphisms (SNPs) were identified and a set of 1.9 million high-confidence SNPs were obtained from 1169 individuals from 140 populations (67 upland, 65 lowland, 8 admixed) were used in downstream analyses of genetic diversity and population structure. Seven separate population groups were identified with moderate genetic differentiation [mean fixation index (Fst) estimate of 0.06] between the lowland and the upland populations. Ecotype-specific and population-specific SNPs were identified for use in germplasm evaluations. Relative to rice (Oryza sativa L.), maize (Zea mays L.), soybean [Glycine max (L.) Merr.], and Medicago truncatula Gaertn., analyses of nucleotide diversity revealed a high degree of genetic diversity (0.0135) across all individuals, consistent with the outcrossing mode of reproduction and the polyploidy of switchgrass. This study supports the hypothesis that repeated glaciation events, ploidy barriers, and restricted gene flow caused by flowering time differences have resulted in distinct gene pools across ecotypes and geographic regions. These data provide a resource to associate alleles with traits of interest for forage, restoration, and biofuel feedstock efforts in switchgrass.

09 BIOMASS FUELS↗
DOE OSTI2016

Antimicrobial and anti-inflammatory activity of switchgrass-derived extractives

Switchgrass is an increasingly important biofuel crop, but knowledge of switchgrass fungal pathogens is not extensive. The purpose of this research was to identify the fungal pathogens that decrease crop yield of switchgrass grown in Tennessee and to investigate a potential sustainable disease management strategy from a value-added by-product of the switchgrass biofuel conversion process. The specific objectives were 1) to identify and characterize prevalent fungal pathogens of switchgrass in Tennessee, 2) assess switchgrass seed produced in the United States for seedborne fungal pathogens, and 3) evaluate switchgrass extractives for antimicrobial activity against plant pathogens.

09 BIOMASS FUELS↗
DOE OSTI2015

Reduction of ethanol yield from switchgrass infected with rust caused by Puccinia emaculata

Switchgrass ( Panicum virgatum ) is an important biofuel crop candidate thought to have low disease susceptibility. As switchgrass production becomes more prevalent, monoculture and production fields in close proximity to one another may increase the spread and severity of diseases such as switchgrass rust caused by the pathogen Puccinia emaculata . The objective of this research was to examine the impact of rust on ethanol yield in switchgrass. In 2010 and 2012, naturally infected leaves from field-grown Alamo and Kanlow in Knoxville, TN (2010, 2012) and Crossville, TN (2012) were visually categorized as exhibiting low, medium, or high disease based on the degree of chlorosis and sporulation. P. emaculata was isolated from each disease range to confirm infection. Samples from 2010 were acid/heat pretreated and subjected to two runs of simultaneous saccharification and fermentation (SSF) with Saccharomyces cerevisiae D 5 A to measure ethanol yield. Near-infrared spectroscopy (NIRS) was used to estimate ethanol yield for 2012 samples. SSF and NIRS data were analyzed separately using ANOVA. Disease level effects were significant within both models (P < 0.05) and both models explained a large amount of variation in ETOH (SSF: R 2 = 0.99, NIRS: R 2 = 0.99). In the SSF dataset, ethanol was reduced by 35 % in samples exhibiting medium disease symptoms and by 55 % in samples exhibiting high disease symptoms. In the NIRS dataset, estimated ethanol was reduced by 10 % in samples exhibiting medium disease symptoms and by 21 % in samples exhibiting high disease symptoms. Lastly, results indicate that switchgrass rust will likely have a negative impact on ethanol yield in switchgrass grown as a biofuel crop.

09 BIOMASS FUELS↗
DOE OSTI2017

Light and temperature effects on miR156 transgenic switchgrass flowering: A simulated latitudinal study

The control of flowering in perennial grasses is an important trait, especially among biofuel feedstocks. Lignocellulosic biomass may be increased commensurate with decreased or delayed flowering as the plant allocates energy for stems and leaves harvested for bioenergy at the end of the growing season. For transgenic feedstocks, such as switchgrass ( Panicum virgatum L. ) grown in its geographic center of distribution, it is foreseeable that regulators may require greatly decreased gene flow frequencies to enable commercialization. Transgenic switchgrass with various overexpression levels of a rice microRNA gene, miR156, when grown in field conditions, holds promise for decreased flowering, yielding high biomass, and altered cell wall traits, which renders it as a potential crossing partner for further breeding with switchgrass lines for decreased recalcitrance. In the current research, we simulated a latitudinal cline in controlled growth chamber experiments for various individual sites from the tropics to cool-temperate conditions which included weekly average high and low temperatures and day lengths over the switchgrass growing season for each simulated site: Guayaquil, Ecuador; Laredo, Texas, USA; and Brattleboro, Vermont, USA. Flowering and reproduction among transgenic lines with low (T-14 and T-35)--tomoderate (T-27 and T-37) overexpression of miR156 were assessed. Lower simulated latitudes (higher temperatures with low-variant day length) and long growing seasons promoted flowering of the miR156 transgenic switchgrass lines. Tropical conditions rescued the flowering phenotype in all transgenic lines except T-27. Higher numbers of plants in lines T-35 and T-37 and the controls produced panicles, which also occurred earlier in the study as temperatures increased and day length decreased. Line T-14 was the exception as more clonal replicates flowered in the cool-temperate (Vermont) conditions. Increased biomass was found in transgenic lines T-35 and T-37 in tropical conditions. No difference in biomass was found in subtropical (Texas) chambers, and two lines (T-14 and T-35) produced less biomass than the control in cool-emperate conditions. Our findings suggest that switchgrass plants engineered to overexpress miR156 for delayed flowering to promote bioconfinement and biomass production may be used for plant breeding at tropical sites.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Discrete element modeling of switchgrass particles under compression and rotational shear

Switchgrass is a perennial herbaceous plant regarded as a biomass energy crop in the United States for its highadaptability and yield potential. Processing and handling of switchgrass particles are challenging due to the erratic mechanical and flow behavior originating from their intrinsic particulate properties. Here, we present a bonded-sphere discrete element model designed specifically for switchgrass particles. The model simultaneously captures three key particulate features, i.e., fibrous particle shapes, a wide range of particle sizes, and particle deformability. Realistic yet computationally efficient particle shape templates are created based on the image analysis data of switchgrass specimens. A fitting procedure is proposed to ensure both the particle width and length distributions are captured, a unique requirement for fibrous particles. Two full-scale numerical models, i.e., a uniaxial compression model and a Schulze ring shear model, are developed using information fromphysical experiments. The model is calibrated using experimental data of chopped-small switchgrass specimens, and then, is validated using data of chopped-large specimens in both compression and ring-shear tests. Numerical results show that the numerical models capture bulk densities accurately (with an error of 3%) while slightly underestimate the bulk friction angle. Furthermore, an extensive sensitivity analysis reveals that (1) switchgrass particles with rougher edges (due to different processing techniques) exhibit a higher shear strength and a lower flowability; (2) stiffer particles yield a lower bulk density (up to 21% lower) compared to more deformable particles, indicating particle deformability should be incorporated when modeling biomass flow in a preprocessing system.

09 BIOMASS FUELS↗
DOE OSTI2016

Understanding Multiscale Structural Changes During Dilute Acid Pretreatment of Switchgrass and Poplar

Biofuels produced from lignocellulosic biomass hold great promise as a renewable alternative energy and fuel source. To realize a cost and energy efficient approach, a fundamental understanding of the deconstruction process is critically necessary to reduce biomass recalcitrance. Here, the structural and morphological changes over multiple scales (5–6000 Å) in herbaceous (switchgrass) and woody (hybrid poplar) biomass during dilute sulfuric acid pretreatment were explored using neutron scattering and X-ray diffraction. Switchgrass undergoes a larger increase (20–84 Å) in the average diameter of the crystalline core of the elementary cellulose fibril than hybrid poplar (19–50 Å). Switchgrass initially forms lignin aggregates with an average size of 90 Å that coalesce to 200 Å, which is double that observed for hybrid poplar, 55–130 Å. Switchgrass shows a smooth-to-rough transition in the cell wall surface morphology unlike the diffuse-to-smooth transition of hybrid poplar. Yet, switchgrass and hybrid poplar pretreated under the same experimental conditions result in pretreated switchgrass producing higher glucose yields (~76 wt %) than pretreated hybrid poplar (~60 wt %). This observation shows that other aspects like cellulose allomorph transitions, cellulose accessibility, cellular biopolymer spatial distribution, and enzyme–substrate interactions may be more critical in governing the enzymatic hydrolysis efficiency.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Responsiveness of miscanthus and switchgrass yields to stand age and nitrogen fertilization: A meta‐regression analysis

Abstract Optimal management of the perennial bioenergy crops, miscanthus and switchgrass, requires an understanding of their responsiveness to nitrogen (N) fertilizer at different maturity stages across locations and growing conditions. Earlier studies that have examined the yield response of these crops to N and stand age using field experiments or meta‐analysis techniques provide mixed evidence. We extend earlier studies by applying a multi‐level mixed‐effects (MLME) meta‐regression model to conduct a more extensive multivariate regression of yield response of these crops to N and stand age, while controlling for climate and location conditions and unobserved factors related to study design. Our findings are based on 1403 and 2811 yield observations for miscanthus and switchgrass, respectively, from experiments conducted between 2002 and 2019 across the rainfed region in the United States. We find statistically significant evidence that an additional year of maturity increases miscanthus and switchgrass yields but at a decreasing rate; yields peak at the 7th and 6th year respectively, for the observed range of applied N rates and stands. We also find that an increase in N application increases yield by a statistically significant level, but at a declining rate; the magnitude of the yield response to N is, however, small and varies with the age of the crop. The impact of N is larger on older compared to younger and middle‐aged stands of miscanthus. In contrast, the impact of N on switchgrass is larger on middle‐aged compared to younger and older stands of switchgrass. We do not find a statistically significant effect of soil productivity on yield for either crop. This analysis provides a basis for developing N application recommendations and optimal rotation age for miscanthus and switchgrass and shows that these energy crops can grow just as productively on low productivity land as on high productivity land.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Gene regulatory networks for lignin biosynthesis in switchgrass (Panicum virgatum )

Summary Cell wall recalcitrance is the major challenge to improving saccharification efficiency in converting lignocellulose into biofuels. However, information regarding the transcriptional regulation of secondary cell wall biogenesis remains poor in switchgrass ( Panicum virgatum ), which has been selected as a biofuel crop in the United States. In this study, we present a combination of computational and experimental approaches to develop gene regulatory networks for lignin formation in switchgrass. To screen transcription factors ( TF s) involved in lignin biosynthesis, we developed a modified method to perform co‐expression network analysis using 14 lignin biosynthesis genes as bait (target) genes. The switchgrass lignin co‐expression network was further extended by adding 14 TF s identified in this study, and seven TF s identified in previous studies, as bait genes. Six TF s (Pv MYB 58/63, Pv MYB 42/85, Pv MYB 4, Pv WRKY 12, Pv SND 2 and Pv SWN 2) were targeted to generate overexpressing and/or down‐regulated transgenic switchgrass lines. The alteration of lignin content, cell wall composition and/or plant growth in the transgenic plants supported the role of the TF s in controlling secondary wall formation. RNA ‐seq analysis of four of the transgenic switchgrass lines revealed downstream target genes of the secondary wall‐related TF s and crosstalk with other biological pathways. In vitro transactivation assays further confirmed the regulation of specific lignin pathway genes by four of the TF s. Our meta‐analysis provides a hierarchical network of TF s and their potential target genes for future manipulation of secondary cell wall formation for lignin modification in switchgrass.

transcription factors↗
DOE OSTI2012

evaluation of the bioconversion of genetically modified switchgrass using simultaneous saccharification and fermentation ans a consolidated bioprocessing approach

Abstract Background: The inherent recalcitrance of lignocellulosic biomass is one of the major economic hurdles for the production of fuels and chemicals from biomass. Additionally, lignin is recognized as having a negative impact on enzymatic hydrolysis of biomass, and as a result much interest has been placed on modifying the lignin pathway to improve bioconversion of lignocellulosic feedstocks. Results: Previous results showed down-regulation of the caffeic acid 3-O-methyl transferase (COMT) gene in the lignin pathway yielded switchgrass (Panicum virgatum) that was more susceptible to bioconversion after dilute acid pretreatment. Here we examined the response of these plant lines to milder pretreatment conditions with yeast-based SSF, CBP with Clostridium thermocellum, and fermentations with the cellulolytic extreme thermophiles, Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis. Unlike the S. cerevisiae SSF conversions, fermentations of pretreated down-regulated COMT transgenic switchgrass with C. thermocellum showed an apparent inhibition of fermentation not observed in the wild-type switchgrass. This inhibition can be eliminated by hot water extraction of the pretreated biomass which resulted in superior conversion yield with transgenic versus wild-type switchgrass for C. thermocellum, also exceeding the yeast-based SSF yield. Further fermentation evaluation of the transgenic switchgrass indicated differential inhibition for the Caldicellulosiruptor strains, which could not be rectified by additional processing conditions. Gas chromatography-mass spectrometry metabolite profiling was used to examine the fermentation broth to elucidate the relative abundance of lignin derived aromatic compounds. The types and abundance of fermentation-derived lignin constituents varied between C. thermocellum and each of the Caldicellulosiruptor strains. Conclusions: The down-regulation of the COMT gene improves the bioconversion of switchgrass relative to the wild-type regardless of the pretreatment condition or fermentation microorganism. However, bacterial fermentations demonstrated strain-dependent sensitivity to the COMT transgenic biomass, likely due to additional soluble lignin pathway-derived constituents resulting from the COMT gene disruption. Removal of these inhibitory constituents permitted completion of fermentation by C. thermocellum, but not by the Caldicellulosiruptor strains. The reason for this difference needs to be explored further.

Caldicellulosiruptor bescii↗
DOE OSTI2022

Identification of microRNAs responsive to arbuscular mycorrhizal fungi in Panicum virgatum (switchgrass)

Background: MicroRNAs (miRNAs) are important post-transcriptional regulators involved in the control of a range of processes, including symbiotic interactions in plants. MiRNA involvement in arbuscular mycorrhizae (AM) symbiosis has been mainly studied in model species, and our study is the first to analyze global miRNA expression in the roots of AM colonized switchgrass ( Panicum virgatum ), an emerging biofuel feedstock. AM symbiosis helps plants gain mineral nutrition from the soil and may enhance switchgrass biomass production on marginal lands. Our goals were to identify miRNAs and their corresponding target genes that are controlling AM symbiosis in switchgrass. Results: Through genome-wide analysis of next-generation miRNA sequencing reads generated from switchgrass roots, we identified 122 mature miRNAs, including 28 novel miRNAs. By comparing miRNA expression profiles of AM-inoculated and control switchgrass roots, we identified 15 AM-responsive miRNAs across lowland accession “Alamo”, upland accession “Dacotah”, and two upland/lowland F 1 hybrids. We used degradome sequencing to identify target genes of the AM-responsive miRNAs revealing targets of miRNAs residing on both K and N subgenomes. Notably, genes involved in copper ion binding were targeted by downregulated miRNAs, while upregulated miRNAs mainly targeted GRAS family transcription factors. Conclusion: Through miRNA analysis and degradome sequencing, we revealed that both upland and lowland switchgrass genotypes as well as upland-lowland hybrids respond to AM by altering miRNA expression. We demonstrated complex GRAS transcription factor regulation by the miR171 family, with some miR171 family members being AM responsive while others remained static. Copper miRNA downregulation was common amongst the genotypes tested and we identified superoxide dismutases and laccases as targets, suggesting that these Cu-miRNAs are likely involved in ROS detoxification and lignin deposition, respectively. Other prominent targets of the Cu miRNAs were blue copper proteins. Overall, the potential effect of AM colonization on lignin deposition pathways in this biofuel crop highlights the importance of considering AM and miRNA in future biofuel crop development strategies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Expression of a bacterial 3-dehydroshikimate dehydratase (QsuB) reduces lignin and improves biomass saccharification efficiency in switchgrass (Panicum virgatum L.)

Abstract Background Lignin deposited in plant cell walls negatively affects biomass conversion into advanced bioproducts. There is therefore a strong interest in developing bioenergy crops with reduced lignin content or altered lignin structures. Another desired trait for bioenergy crops is the ability to accumulate novel bioproducts, which would enhance the development of economically sustainable biorefineries. As previously demonstrated in the model plant Arabidopsis, expression of a 3-dehydroshikimate dehydratase in plants offers the potential for decreasing lignin content and overproducing a value-added metabolic coproduct (i.e., protocatechuate) suitable for biological upgrading. Results The 3-dehydroshikimate dehydratase QsuB from Corynebacterium glutamicum was expressed in the bioenergy crop switchgrass ( Panicum virgatum L.) using the stem-specific promoter of an O-methyltransferase gene ( pShOMT ) from sugarcane. The activity of pShOMT was validated in switchgrass after observation in-situ of beta-glucuronidase (GUS) activity in stem nodes of plants carrying a pShOMT::GUS fusion construct. Under controlled growth conditions, engineered switchgrass lines containing a pShOMT::QsuB construct showed reductions of lignin content, improvements of biomass saccharification efficiency, and accumulated higher amount of protocatechuate compared to control plants. Attempts to generate transgenic switchgrass lines carrying the QsuB gene under the control of the constitutive promoter pZmUbi-1 were unsuccessful, suggesting possible toxicity issues associated with ectopic QsuB expression during the plant regeneration process. Conclusion This study validates the transfer of the QsuB engineering approach from a model plant to switchgrass. We have demonstrated altered expression of two important traits: lignin content and accumulation of a co-product. We found that the choice of promoter to drive QsuB expression should be carefully considered when deploying this strategy to other bioenergy crops. Field-testing of engineered QsuB switchgrass are in progress to assess the performance of the introduced traits and agronomic performances of the transgenic plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Pollen-mediated gene flow from transgenic to non-transgenic switchgrass (Panicum virgatum L.) in the field

Background: Switchgrass is C4 perennial grass species that is being developed as a cellulosic bioenergy feedstock. It is wind-pollinated and considered to be an obligate outcrosser. Genetic engineering has been used to alter cell walls for more facile bioprocessing and biofuel yield. Gene flow from transgenic cultivars would likely be of regulatory concern. In this study we investigated pollen-mediated gene flow from transgenic to nontransgenic switchgrass in a 3-year field experiment performed in Oliver Springs, Tennessee, U.S.A. using a modified Nelder wheel design. The planted area (0.6 ha) contained sexually compatible pollen source and pollen receptor switchgrass plants. One hundred clonal switchgrass ‘Alamo’ plants transgenic for an orange-fluorescent protein (OFP) and hygromycin resistance were used as the pollen source; whole plants, including pollen, were orange-fluorescent. To assess pollen movement, pollen traps were placed at 10 m intervals from the pollen-source plot in the four cardinal directions extending to 20 m, 30 m, 30 m, and 100 m to the north, south, west, and east, respectively. To assess pollination rates, nontransgenic ‘Alamo 2’ switchgrass clones were planted in pairs adjacent to pollen traps. Results: In the eastward direction there was a 98% decrease in OFP pollen grains from 10 to 100 m from the pollen-source plot (Poisson regression, F1,8 = 288.38, P < 0.0001). At the end of the second and third year, 1,820 F1 seeds were collected from pollen recipient-plots of which 962 (52.9%) germinated and analyzed for their transgenic status. Transgenic progeny production detected in each pollen-recipient plot decreased with increased distance from the edge of the transgenic plot (Poisson regression, F1,15 = 12.98, P < 0.003). The frequency of transgenic progeny detected in the eastward plots (the direction of the prevailing wind) ranged from 79.2% at 10 m to 9.3% at 100 m. Conclusions: In these experiments we found transgenic pollen movement and hybridization rates to be inversely associated with distance. However, these data suggest pollen-mediated gene flow is likely to occur up to, at least, 100 m. This study gives baseline data useful to determine isolation distances and other management practices should transgenic switchgrass be grown commercially in relevant environments.

09 BIOMASS FUELS↗