Very fine roots differ among switchgrass (Panicum virgatum L.) cultivars and differentially affect soil pores and carbon processes
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Lee, Jin Ho (ORCID:0000000323809892)↗
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Anti-fungal bioactive terpenoids in the bioenergy crop switchgrass (Panicum virgatum) may contribute to ecotype-specific microbiome composition
Abstract Plant derived bioactive small molecules have attracted attention of scientists across fundamental and applied scientific disciplines. We seek to understand the influence of these phytochemicals on rhizosphere and root-associated fungi. We hypothesize that – consistent with accumulating evidence that switchgrass genotype impacts microbiome assembly – differential terpenoid accumulation contributes to switchgrass ecotype-specific microbiome composition. An initial in vitro Petri plate-based disc diffusion screen of 18 switchgrass root derived fungal isolates revealed differential responses to upland- and lowland-isolated metabolites. To identify specific fungal growth-modulating metabolites, we tested fractions from root extracts on three ecologically important fungal isolates – Linnemania elongata, Trichoderma sp. and Fusarium sp. Saponins and diterpenoids were identified as the most prominent antifungal metabolites. Finally, analysis of liquid chromatography-purified terpenoids revealed fungal inhibition structure – activity relationships (SAR). Saponin antifungal activity was primarily determined by the number of sugar moieties – saponins glycosylated at a single core position were inhibitory whereas saponins glycosylated at two core positions were inactive. Saponin core hydroxylation and acetylation were also associated with reduced activity. Diterpenoid activity required the presence of an intact furan ring for strong fungal growth inhibition. These results inform future breeding and biotechnology strategies for crop protection with reduced pesticide application.
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Genomic prediction of regional-scale performance in switchgrass ( Panicum virgatum ) by accounting for genotype-by-environment variation and yield surrogate traits
Switchgrass is a potential crop for bioenergy or carbon capture schemes, but further yield improvements through selective breeding are needed to encourage commercialization. To identify promising switchgrass germplasm for future breeding efforts, we conducted multisite and multitrait genomic prediction with a diversity panel of 630 genotypes from 4 switchgrass subpopulations (Gulf, Midwest, Coastal, and Texas), which were measured for spaced plant biomass yield across 10 sites. Our study focused on the use of genomic prediction to share information among traits and environments. Specifically, we evaluated the predictive ability of cross-validation (CV) schemes using only genetic data and the training set (cross-validation 1: CV1), a subset of the sites (cross-validation 2: CV2), and/or with 2 yield surrogates (flowering time and fall plant height). We found that genotype-by-environment interactions were largely due to the north–south distribution of sites. The genetic correlations between the yield surrogates and the biomass yield were generally positive (mean height r = 0.85; mean flowering time r = 0.45) and did not vary due to subpopulation or growing region (North, Middle, or South). Genomic prediction models had CV predictive abilities of –0.02 for individuals using only genetic data (CV1), but 0.55, 0.69, 0.76, 0.81, and 0.84 for individuals with biomass performance data from 1, 2, 3, 4, and 5 sites included in the training data (CV2), respectively. To simulate a resource-limited breeding program, we determined the predictive ability of models provided with the following: 1 site observation of flowering time (0.39); 1 site observation of flowering time and fall height (0.51); 1 site observation of fall height (0.52); 1 site observation of biomass (0.55); and 5 site observations of biomass yield (0.84). The ability to share information at a regional scale is very encouraging, but further research is required to accurately translate spaced plant biomass to commercial-scale sward biomass performance.
09 BIOMASS FUELS↗
Iron incorporation both intra- and extra-cellularly improves the yield and saccharification of switchgrass (Panicum virgatum L.) biomass
Abstract Background Pretreatments are commonly used to facilitate the deconstruction of lignocellulosic biomass to its component sugars and aromatics. Previously, we showed that iron ions can be used as co-catalysts to reduce the severity of dilute acid pretreatment of biomass. Transgenic iron-accumulating Arabidopsis and rice plants exhibited higher iron content in grains, increased biomass yield, and importantly, enhanced sugar release from the biomass. Results In this study, we used intracellular ferritin (FerIN) alone and in combination with an improved version of cell wall-bound carbohydrate-binding module fused iron-binding peptide (IBPex) specifically targeting switchgrass, a bioenergy crop species. The FerIN switchgrass improved by 15% in height and 65% in yield, whereas the FerIN/IBPex transgenics showed enhancement up to 30% in height and 115% in yield. The FerIN and FerIN/IBPex switchgrass had 27% and 51% higher in planta iron accumulation than the empty vector (EV) control, respectively, under normal growth conditions. Improved pretreatability was observed in FerIN switchgrass (~ 14% more glucose release than the EV), and the FerIN/IBPex plants showed further enhancement in glucose release up to 24%. Conclusions We conclude that this iron-accumulating strategy can be transferred from model plants and applied to bioenergy crops, such as switchgrass. The intra- and extra-cellular iron incorporation approach improves biomass pretreatability and digestibility, providing upgraded feedstocks for the production of biofuels and bioproducts.
09 BIOMASS FUELS↗
Genetic modification of the shikimate pathway to reduce lignin content in switchgrass ( Panicum virgatum L.) significantly impacts plant microbiomes
QsuB transgenic switchgrass associated 16S microbiome of different compartments
actinobacteria↗
Genetic modification of the shikimate pathway to reduce lignin content in switchgrass ( Panicum virgatum L.) significantly impacts plant microbiomes
QsuB transgenic switchgrass associated ITS microbiome of different compartments
actinobacteria↗
The influence of tillage and fertilizer on the flux and source of nitrous oxide with reference to atmospheric variation using laser spectroscopy
Nitrous oxide (N 2 O) is the third most important long-lived greenhouse gas and agriculture is the largest source of N 2 O emissions. Curbing N 2 O emissions requires understanding influences on the flux and sources of N 2 O. We measured flux and evaluated microbial sources of N 2 O using site preference ( S P ; the intramolecular distribution of 15 N in N 2 O) in flux chambers from a grassland tilling and agricultural fertilization experiments and atmosphere. We identified values greater than that of the average atmosphere to reflect nitrification and/or fungal denitrification and those lower than atmosphere as increased denitrification. Our spectroscopic approach was based on an extensive calibration with 18 standards that yielded S P accuracy and reproducibility of 0.7 ‰ and 1.0 ‰, respectively, without preconcentration. Chamber samples from the tilling experiment taken ~ monthly over a year showed a wide range in N 2 O flux (0–1.9 g N 2 O-N ha -1 d -1 ) and S P (- 1.8 to 25.1 ‰). Flux and S P were not influenced by tilling but responded to sampling date. Large fluxes occurred in October and May in no-till when soils were warm and moist and during a spring thaw, an event likely representing release of N 2 O accumulated under snow cover. These high fluxes could not be ascribed to a single microbial process as S P differed among chambers. However, the year-long S P and flux data for no-till showed a slight direct relationship suggesting that nitrification increased with flux. The comparative data in till showed an inverse relationship indicating that high flux events are driven by denitrification. Corn ( Zea mays ) showed high fluxes and S P values indicative of nitrification ~ 4 wk after fertilization with subsequent declines in S P indicating denitrification. Although there was no effect of fertilizer treatment on flux or S P in switchgrass ( Panicum virgatum) , high fluxes occurred ~1 month after fertilization. In both treatments, S P was indicative of denitrification in many instances, but evidence of nitrification/fungal denitrification also prevailed. At 2 m atmospheric N 2 O S P had a range of 31.1 ‰ and 14.6 ‰ in the grassland tilling and agricultural fertilization experiments, respectively. These data suggest the influence of soil microbial processes on atmospheric N 2 O and argue against the use of the global average atmospheric S P in isotopic modeling approaches.
Environmental Sciences & Ecology↗
Multiple highly expressed phospho enol pyruvate carboxylase genes have divergent enzyme kinetic properties in two C4 grasses
Abstract Background and Aims Phosphoenolpyruvate (PEP) carboxylase (PEPC) catalyses the irreversible carboxylation of PEP with bicarbonate to produce oxaloacetate. This reaction powers the carbon-concentrating mechanism (CCM) in plants that perform C4 photosynthesis. This CCM is generally driven by a single PEPC gene product that is highly expressed in the cytosol of mesophyll cells. We found two C4 grasses, Panicum miliaceum and Echinochloa colona, that each have two highly expressed PEPC genes. We characterized the kinetic properties of the two most abundant PEPCs in E. colona and P. miliaceum to better understand how the enzyme’s amino acid structure influences its function. Methods Coding sequences of the two most abundant PEPC proteins in E. colona and P. miliaceum were synthesized by GenScript and were inserted into bacteria expression plasmids. Point mutations resulting in substitutions at conserved amino acid residues (e.g. N-terminal serine and residue 890) were created via site-directed PCR mutagenesis. The kinetic properties of semi-purified plant PEPCs from Escherichia coli were analysed using membrane-inlet mass spectrometry and a spectrophotometric enzyme-coupled reaction. Key Results The two most abundant P. miliaceum PEPCs (PmPPC1 and PmPPC2) have similar sequence identities (>95 %), and as a result had similar kinetic properties. The two most abundant E. colona PEPCs (EcPPC1 and EcPPC2) had identities of ~78 % and had significantly different kinetic properties. The PmPPCs and EcPPCs had different responses to allosteric inhibitors and activators, and substitutions at the conserved N-terminal serine and residue 890 resulted in significantly altered responses to allosteric regulators. Conclusions The two, significantly expressed C4Ppc genes in P. miliaceum were probably the result of genomes combining from two closely related C4Panicum species. We found natural variation in PEPC’s sensitivity to allosteric inhibition that seems to bypass the conserved 890 residue, suggesting alternative evolutionary pathways for increased malate tolerance and other kinetic properties.
DiMario, Robert J. (ORCID:0000000250566868)↗
Genetics and Genomics of Pathogen Resistance in Switchgrass
This project investigates the genetic and genomic bases of resistance/tolerance to disease in switchgrass (Panicum virgatum) populations developed as bioenergy feedstock. The focus is placed populations adapted to the Central Great Plains derived from the hybridization of upland and lowland ecotypes, represented by cultivars Summer and Kanlow, respectively. Resistance was investigated against the rust disease caused by the fungus Puccinia novo-panici (syn. P emaculata) and mosaic disease caused by the virus complex of Panicum mosaic virus and its satellite. Objectives of the project were: 1) to elucidate the genetic and genomic bases of resistance/tolerance in locally adapted upland and lowland switchgrass breeding populations with contrasting disease symptoms under field conditions; 2) determine the molecular underpinnings of why cv Kanlow has broad resistance/tolerance to pathogens; and 3) discover the molecular differences that permit systemic viral infections in some switchgrass plants but not in other genetically-related plants.
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Corresponding Standard Reference Material Data used in Partial Least Squares Regression Models for Sugar Composition Estimates in Biomass in: Economic Impact of Yield and Composition Variation in Bioenergy Crops: Populus trichocarpa
Corresponding Standard Reference Material Data used in Partial Least Squares Regression Models for Sugar Composition Estimates in Biomass in: Economic Impact of Yield and Composition Variation in Bioenergy Crops: Populus trichocarpa (for corresponding manuscript: DOI: 10.1002/bbb.2148) PDF Files: Images of 1H NMR spectra for neutralized 2-stage acid hydrolysates of 4 NIST Standard Reference Material biomass samples (Monterey Pine 8493, Sugarcane Bagasse 8491, Wheat Straw 8494, and Eastern Cottonwood/Poplar 8492) and 2 Center for Bioenergy Innovation reference biomass samples (Poplar - Populus trichocarpa and Switchgrass - Panicum Virgatum). Suppression of the water peak was achieved using a NOESY-1D with presaturation, a recycle delay of 5 s, and a total of 64 scans. Spectra were acquired at 298 K and processed with automatic phase correction, baseline correction, and chemical shift referencing to TSP-d4. Images show all 1H data from 10 to 1ppm with inset spectra of region of interest (4.0 to 3.1 ppm). Text Files: Spectra for neutralized 2-stage acid hydrolysates of 4 NIST Standard Reference Material biomass samples (Monterey Pine 8493, Sugarcane Bagasse 8491, Wheat Straw 8494, and Eastern Cottonwood/Poplar 8492) and 2 Center for Bioenergy Innovation reference biomass samples (Poplar - Populus trichocarpa and Switchgrass - Panicum Virgatum) were converted into text files for plotting. Files contain 8192 points of raw spectral data from 12.23 to -2.78 ppm. The text file contains 4 columns of data and includes: Point number, Intensity, Hz, and ppm. Xcel Spreadsheet: HPLC measured monomeric sugar concentrations and bucketed 1H NMR data used to build monomeric sugar composition prediction models. Sugar composition in biomass determined from HPLC analyses are given in mg sugar/mg of biomass. Spectral bucketing was performed using Bruker’s AMIX software. Spectra were divided into 0.005 ppm buckets in the region of 3.10– 4.15 ppm for a total of 210 buckets. Headers for the bucketed data are the chemical shift in ppm of the center of the bucket. Bucketed data was used to build partial least squares models for subsequent predictions in The Unscrambler v. 10.5(CAMO A/S, Trondheim, Norway). The formation of methanol during hydrolysis interferes with the quantitative NMR analysis of sugars, so the methanol peak centered at 3.37 ppm and spanning four buckets (3.2925 – 3.2775 ppm) was set to zero for all spectra.
09 BIOMASS FUELS↗
A generalist–specialist trade-off between switchgrass cytotypes impacts climate adaptation and geographic range
Polyploidy results from whole-genome duplication and is a unique form of heritable variation with pronounced evolutionary implications. Different ploidy levels, or cytotypes, can exist within a single species, and such systems provide an opportunity to assess how ploidy variation alters phenotypic novelty, adaptability, and fitness, which can, in turn, drive the development of unique ecological niches that promote the coexistence of multiple cytotypes. Switchgrass, Panicum virgatum, is a widespread, perennial C4 grass in North America with multiple naturally occurring cytotypes, primarily tetraploids (4×) and octoploids (8×). Using a combination of genomic, quantitative genetic, landscape, and niche modeling approaches, we detect divergent levels of genetic admixture, evidence of niche differentiation, and differential environmental sensitivity between switchgrass cytotypes. Taken together, these findings support a generalist (8×)–specialist (4×) trade-off. Our results indicate that the 8× represent a unique combination of genetic variation that has allowed the expansion of switchgrass’ ecological niche and thus putatively represents a valuable breeding resource.
09 BIOMASS FUELS↗
Whole plant traits of coastal dune vegetation and implications for interactions with dune dynamics
Abstract Coastal dunes are important protective features against sea level rise and coastal storms. Interactions between dune plant aboveground structures and sediment trapping that allow for dune building and maintenance are well established. More recently, studies documenting belowground biomass for promoting erosion resistance in dominant dune species have been conducted, yet a knowledge gap remains regarding species‐specific characterization of whole plants, specifically with respect to roots, rhizomes, and belowground stems. Our objective was to quantify above‐ and belowground traits of four dominant dune grasses to document the potential for species‐specific effects on dune growth, maintenance, and erosion resistance. We examined above‐ and belowground traits among four prominent dune grasses of the Atlantic and Gulf Coasts of North America: Ammophila breviligulata , Panicum amarum , Spartina patens , and Uniola paniculata . Whole plant samples of each species were collected from the foredune at the US Army Engineer Research and Development Center's Field Research Facility in Duck, North Carolina, USA, and quantified for several above‐ and belowground traits (e.g., stem height, rhizome number and length, root surface area by diameter class, root tensile strength, and mycorrhizal percent infection). Belowground factors known to impact important dune processes, such as rhizome length, mycorrhizal percent infection, and root traits, differed substantially among species. When visualized in multivariate space, all species significantly differed in suites of above‐ and belowground traits. When considering belowground only, Ammophila and Spartina were similar, despite differences in biomass allocation. Species separated along axes related to mycorrhizal association, biomass allocation, and root construction. The four co‐occurring dune grass species were dissimilar in suites of plant traits. Belowground trait differences were driven by those describing root construction, biomass allocation, and mycorrhizal infection. Dissimilarity in above‐ and belowground suites of traits may demonstrate different approaches for surviving the dune environment. Incorporating belowground traits into modeling will enhance predictions of dune response to climate change through interactions between vegetation and dune dynamics that facilitate coastal resistance and resilience.
54 ENVIRONMENTAL SCIENCES↗
Biomass Quality Responses to Selection for Increased Biomass Yield in Perennial Energy Grasses
Perennial grasses are candidates for biomass cropping systems that are focused on providing a wide range of ecosystem services in addition to sustainable bioenergy production. Switchgrass (Panicum virgatum L.) and big bluestem (Andropogon gerardii Vitman) are two of the dominant grasses of the tallgrass prairie and both are candidates for development of multifunctional diverse production systems. Breeding programs for both species are aimed at increasing biomass yield as a mechanism of improving economic sustainability without increasing production costs. The objective of this study was to determine if long-term selection for increased biomass yield in these two species has had any adverse impacts on biomass quality, or the ability to convert biomass into bioenergy. Check cultivars and improved breeding populations of both species were evaluated for a wide range of biomass quality traits at 13 locations in the North Central and Northeastern USA, and a subset of these populations were also subjected to more intensive and detailed fermentation analyses. In general, lignin and ferulates either remained constant or decreased following selection for increased biomass yield in the various genetic pedigrees. These changes resulted in some increases in predicted ethanol production and in vitro digestibility. Here, the prediction of increased digestibility was confirmed by higher glucose release by pretreatment and deconstruction of an advanced lowland population. However, bioreactor fermentations with two different biofuel-producing microbes showed no differences in ethanol production. Overall, these studies indicated that the improved switchgrass and big bluestem populations had greater biomass yields without significantly reducing biomass quality or conversion efficiency into ethanol, suggesting that selection can achieve increases in biomass productivity while maintaining consistent biomass quality.
09 BIOMASS FUELS↗
Influence of living grass Roots and endophytic fungal hyphae on soil hydraulic properties
Soil hydraulic properties are often estimated based on laboratory data or pedotransfer functions dependent on soil physical properties, which often do not consider potential impacts of soil roots or fungal hyphae. Here, we first review current knowledge of how these soil biotic components affect hydraulic properties, then we conducted laboratory experiments to specifically test if the presence of roots and mycorrhizal fungi had a significant effect on the hydraulic properties of two soils with contrasting textures: Flint sand and Hamblen silt loam. Soil cores were seeded with (Panicum virgatum) and grown in a greenhouse over three separate growth periods. The endophytic fungus Serendipita indica was injected as liquid inoculant into designated mycorrhizal cores. Saturated hydraulic conductivity (K sat ) measurements were made with a constant head permeameter, and soil water retention curves were obtained by the evaporation method, supplemented at the dry end for Hamblen silt loam with water activity meter data. Retention curve parameters were obtained by fitting the van Genuchten equation to the resulting measurements. Mean root volume ratios were higher in the mycorrhizal inoculated treatment than in the uninoculated treatment for both soils. For Flint sand, analysis of variance revealed that K sat was reduced by the presence of roots as compared to bare soil. This was likely due to roots clogging soil pores. Results also indicated the presence of roots changed the shape of the water retention curve for Flint sand by increasing water content at saturation and by reducing the slope of the curve. These changes suggested roots created additional porosity and broadened the pore-size distribution. The presence of mycorrhizal fungi accentuated the root effects. The influence of roots and mycorrhizal fungi on hydraulic properties was less obvious for the Hamblen silt loam, as none of the treatments differed from each other at p < 0.05. The results highlight the necessity to consider the impact of root and fungal structures on models of soil hydraulic properties.
59 BASIC BIOLOGICAL SCIENCES↗
Genomic mechanisms of climate adaptation in polyploid bioenergy switchgrass
Long-term climate change and periodic environmental extremes threaten food and fuel security and global crop productivity. Although molecular and adaptive breeding strategies can buffer the effects of climatic stress and improve crop resilience, these approaches require sufficient knowledge of the genes that underlie productivity and adaptation—knowledge that has been limited to a small number of well-studied model systems. Here we present the assembly and annotation of the large and complex genome of the polyploid bioenergy crop switchgrass ( Panicum virgatum ). Analysis of biomass and survival among 732 resequenced genotypes, which were grown across 10 common gardens that span 1,800 km of latitude, jointly revealed extensive genomic evidence of climate adaptation. Climate–gene–biomass associations were abundant but varied considerably among deeply diverged gene pools. Furthermore, we found that gene flow accelerated climate adaptation during the postglacial colonization of northern habitats through introgression of alleles from a pre-adapted northern gene pool. The polyploid nature of switchgrass also enhanced adaptive potential through the fractionation of gene function, as there was an increased level of heritable genetic diversity on the nondominant subgenome. In addition to investigating patterns of climate adaptation, the genome resources and gene–trait associations developed here provide breeders with the necessary tools to increase switchgrass yield for the sustainable production of bioenergy.
09 BIOMASS FUELS↗
Correlational selection and genetic architecture shape the evolution of the leaf economics spectrum in a perennial grass
The generality of the worldwide leaf economics spectrum (LES) has made it a pillar of trait-based ecological research. Yet, few studies have examined the processes shaping the evolution of the LES within species, in part, because most species occupy only a small portion of the LES. Here, to address this gap, we took advantage of the distinct leaf economics strategies present in different ecotypes of the phenotypically diverse perennial grass Panicum virgatum (switchgrass) to generate a genetic mapping population, which we planted in common gardens at three sites spanning 12 degrees of latitude in the central United States. With this genetic mapping population, we evaluated two potentially interacting causes of LES evolution: 1) genetic architecture, where multiple traits are influenced by either the same gene (pleiotropy) or by genes in close physical proximity (genetic linkage), and 2) correlational selection, where selection acts on traits in combination rather than in isolation. We found that shared genetic architecture influenced covariation between photosynthetic rate (A MASS ) and leaf nitrogen (N MASS ) and between A MASS and leaf mass per area (LMA). We also found that correlational selection favored the trait combinations predicted by the LES (e.g., high LMA with low N MASS or low LMA with high N MASS ) and disfavored other, mismatched trait combinations at two of the three sites. Together, these results demonstrate how the evolution of an integrated LES within species can arise from multiple evolutionary causes.
59 BASIC BIOLOGICAL SCIENCES↗
A Pleiotropic Flowering Time QTL Exhibits Gene-by-Environment Interaction for Fitness in a Perennial Grass
Appropriate flowering time is a crucial adaptation impacting fitness in natural plant populations. Although the genetic basis of flowering variation has been extensively studied, its mechanisms in nonmodel organisms and its adaptive value in the field are still poorly understood. Here, we report new insights into the genetic basis of flowering time and its effect on fitness in Panicum hallii, a native perennial grass. Genetic mapping in populations derived from inland and coastal ecotypes identified flowering time quantitative trait loci (QTL) and many exhibited extensive QTL-by-environment interactions. Patterns of segregation within recombinant hybrids provide strong support for directional selection driving ecotypic divergence in flowering time. A major QTL on chromosome 5 (q-FT5) was detected in all experiments. Fine-mapping and expression studies identified a gene with orthology to a rice FLOWERING LOCUS T-like 9 (PhFTL9) as the candidate underlying q-FT5. We used a reciprocal transplant experiment to test for local adaptation and the specific impact of q-FT5 on performance. We did not observe local adaptation in terms of fitness tradeoffs when contrasting ecotypes in home versus away habitats. However, we observed that the coastal allele of q-FT5 conferred a fitness advantage only in its local habitat but not at the inland site. Sequence analyses identified an excess of low-frequency polymorphisms at the PhFTL9 promoter in the inland lineage, suggesting a role for either selection or population expansion on promoter evolution. Together, our findings demonstrate the genetic basis of flowering variation in a perennial grass and provide evidence for conditional neutrality underlying flowering time divergence.
54 ENVIRONMENTAL SCIENCES↗