Switchgrass Lines Research Articles

MiR156-PvSPL2 controls culm development by transcriptional repression of switchgrass CYTOKININ OXIDASE/DEHYDROGENASE4.

Culm development in grasses can be controlled by both miR156 and cytokinin. However, the crosstalk between the miR156-SPL module and the cytokinin metabolic pathway remains largely unknown. Here, we found CYTOKININ OXIDASE/DEHYDROGENASE4 (PvCKX4) plays a negative regulatory role in culm development of the bioenergy grass Panicum virgatum (switchgrass). Overexpression of PvCKX4 in switchgrass reduced the internode diameter and length without affecting tiller number. Interestingly, we also found that PvCKX4 was always upregulated in miR156 overexpressing (miR156OE) transgenic switchgrass lines. Additionally, upregulation of either miR156 or PvCKX4 in switchgrass reduced the content of isopentenyl adenine (iP) without affecting trans-zeatin (tZ) accumulation. It is consistent with the evidence that the recombinant PvCKX4 protein exhibited much higher catalytic activity against iP than tZ in vitro. Furthermore, our results showed that miR156-targeted SPL2 bound directly to the promoter of PvCKX4 to repress its expression. Thus, alleviating the SPL2-mediated transcriptional repression of PvCKX4 through miR156 overexpression resulted in a significant increase in cytokinin degradation and impaired culm development in switchgrass. On the contrary, suppressing PvCKX4 in miR156OE transgenic plants restored iP content, internode diameter, and length to wild-type levels. Most strikingly, the double transgenic lines retained the same increased tiller numbers as the miR156OE transgenic line, which yielded more biomass than the wild type. These findings indicate that the miR156-SPL module can control culm development through transcriptional repression of PvCKX4 in switchgrass, which provides a promising target for precise design of shoot architecture to yield more biomass from grasses.

The Genetic Architecture of Nitrogen Use Efficiency in Switchgrass (Panicum virgatum L.).

Switchgrass (Panicum virgatum L.) has immense potential as a bioenergy crop with the aim of producing biofuel as an end goal. Nitrogen (N)-related sustainability traits, such as nitrogen use efficiency (NUE) and nitrogen remobilization efficiency (NRE), are important factors affecting switchgrass quality and productivity. Hence, it is imperative to develop nitrogen use-efficient switchgrass accessions by exploring the genetic basis of NUE in switchgrass. For that, we used 331 diverse field-grown switchgrass accessions planted under low and moderate N fertility treatments. We performed a genome wide association study (GWAS) in a holistic manner where we not only considered NUE as a single trait but also used its related phenotypic traits, such as total dry biomass at low N and moderate N, and nitrogen use index, such as NRE. We have evaluated the phenotypic characterization of the NUE and the related traits, highlighted their relationship using correlation analysis, and identified the top ten nitrogen use-efficient switchgrass accessions. Our GWAS analysis identified 19 unique single nucleotide polymorphisms (SNPs) and 32 candidate genes. Two promising GWAS candidate genes, caffeoyl-CoA O-methyltransferase (CCoAOMT) and alfin-like 6 (AL6), were further supported by linkage disequilibrium (LD) analysis. Finally, we discussed the potential role of nitrogen in modulating the expression of these two genes. Our findings have opened avenues for the development of improved nitrogen use-efficient switchgrass lines.

Haploidy and aneuploidy in switchgrass mediated by misexpression of CENH3.

Cross bred species such as switchgrass may benefit from advantageous breeding strategies requiring inbred lines. Doubled haploid production methods offer several ways that these lines can be produced that often involve uniparental genome elimination as the rate limiting step. We have used a centromere-mediated genome elimination strategy in which modified CENH3 is expressed to induce the process. Transgenic tetraploid switchgrass lines coexpressed Cas9, a poly-cistronic tRNA-gRNA tandem array containing eight guide RNAs that target two CENH3 genes, and different chimeric versions of CENH3 with alterations to the N-terminal tail region. Genotyping of CENH3 genes in transgenics identified edits including frameshift mutations and deletions in one or both copies of the two CENH3 genes. Flow cytometry of T1 seedlings identified two T0 lines that produced five haploid individuals representing an induction rate of 0.5% and 1.4%. Eight different T0 lines produced aneuploids at rates ranging from 2.1 to 14.6%. A sample of aneuploid lines were sequenced at low coverage and aligned to the reference genome, revealing missing chromosomes and chromosome arms.

Down-regulation of PvSAMS impairs S-adenosyl-L-methionine and lignin biosynthesis, and improves cell wall digestibility in switchgrass.

S-adenosyl- l-methionine (SAM) is the methyl donor involved in the biosynthesis of guaiacyl (G) and syringyl (S) lignins in vascular plants. SAM is synthesized from methionine through the catalysis of the enzyme S-adenosylmethionine synthase (SAMS). However, the detailed function of SAMS in lignin biosynthesis has not been widely investigated in plants, particularly in monocot species. In this study, we identified PvSAMS genes from switchgrass (Panicum virgatum L.), an important dual-purpose fodder and biofuel crop, and generated numerous transgenic switchgrass lines through PvSAMS RNA interference technology. Down-regulation of PvSAMS reduced the contents of SAM, G-lignins, and S-lignins in the transgenic switchgrass. The methionine and glucoside derivatives of caffeoyl alcohol were found to accumulate in the transgenic plants. Moreover, down-regulation of PvSAMS in switchgrass resulted in brownish stems associated with reduced lignin content and improved cell wall digestibility. Furthermore, transcriptomic analysis revealed that most sulfur deficiency-responsive genes were differentially expressed in the transgenic switchgrass, leading to a significant increase in total sulfur content; thus implying an important role of SAMS in the methionine cycle, lignin biosynthesis, and sulfur assimilation. Taken together, our results suggest that SAMS is a valuable target in lignin manipulation, and that manipulation of PvSAMS can simultaneously regulate the biosynthesis of SAM and methylated monolignols in switchgrass.

Overexpression of PvWOX3a in switchgrass promotes stem development and increases plant height

Switchgrass (Panicum virgatum L.) is an important perennial, noninvasive, tall ornamental grass that adds color and texture to gardens and landscapes. Moreover, switchgrass has been considered a forage and bioenergy crop because of its vigorous growth, low-input requirements, and broad geography. Here, we identified PvWOX3a from switchgrass, which encodes a WUSCHEL-related homeobox transcription factor. Transgenic overexpression of PvWOX3a in switchgrass increased stem length, internode diameter, and leaf blade length and width, all of which contributed to a 95% average increase in dry weight biomass compared with control plants. Yeast one-hybrid and transient dual-luciferase assays showed that PvWOX3a can repress the expression of gibberellin 2-oxidase and cytokinin oxidase/dehydrogenase through apparently direct interaction with their promoter sequences. These results suggested that overexpression of PvWOX3a could increase gibberellin and cytokinin levels in transgenic switchgrass plants, which promotes cell division, elongation, and vascular bundle development. We also overexpressed PvWOX3a in a transgenic miR156-overexpressing switchgrass line that characteristically exhibited more tillers, thinner internodes, and narrower leaf blades. Double transgenic switchgrass plants displayed significant increases in internode length and diameter, leaf blade width, and plant height but retained a tiller number comparable to that of plants expressing miR156 alone. Ultimately, the double transgenic switchgrass plants produced 174% more dry-weight biomass and 162% more solubilized sugars on average than control plants. These findings indicated that PvWOX3a is a viable potential genetic target for engineering improved shoot architecture and biomass yield of horticulture, fodder, and biofuel crops.

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

BackgroundLignin 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.ResultsThe 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.ConclusionThis 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.

First Report of Rust Disease Caused by Puccinia emaculata on Switchgrass in Georgia.

Switchgrass (Panicum virgatum), native to North America, has been selected by the United States Department of Energy as a model herbaceous bioenergy crop. However, there are several foliar diseases that may impact switchgrass biomass yield and quality. In the spring of 2008, different switchgrass cultivars and breeding lines of two major ecotypes, upland and lowland, were included in the Southern Switchgrass Diversity Panel (SSDP) planted in Watkinsville, GA. In the summer of 2009, brown to black rectangular lesions in a linear pattern were observed on the leaves of most of switchgrass lines. The spores were consecutively collected from the field in 2011 and 2012, and then dehydrated, stored at -80°C. In 2016, one field isolate from year 2011 was rehydrated, recovered, and inoculated on the seedlings of switchgrass cultivars Alamo, Kanlow, Summer, and Blackwell planted in disease-free magenta boxes. The inoculated plants were kept in the dark at room temperature for 8 hours and then moved to a growth chamber at 28°C with a 16h light/8h dark cycle. Uredinia formed on the abaxial leaf surface seven days post inoculation (dpi) and then developed primarily on the adaxial leaf surfaces. The morphology of uredinia on the switchgrass cultivars in the magenta boxes was identical to what was previously observed on the susceptible switchgrass lines in the field. Uredinia were dark brown, elongated, and caulicolous. Urediniospores were cinnamon brown, globose to broadly ellipsoid, with a width of 21.7 to 28.1 μm (average 24.2 μm) and length of 27.3 to 30.8 μm (average 28.1 μm) (n=50). The thickness of cell wall was 1.0 to 2.0 μm (average 1.4 μm) (n=50). Urediniospores were isolated and single-spore inoculation was made on the leaves of Alamo, Kanlow, Summer, and Blackwell using a detached-leaf assay. Uredinia appeared on the abaxial surface of leaves 7 dpi and the morphology of isolated urediniospores was consistent with the original inoculum. Teliospores were observed in the field in late summer and were two-celled, appeared dark brown in color with an oblong to ellipsoid shape and averaged 33.2 ± 4.1 μm in length (n=50). The apical cell width and basal cell width averaged 17.5 ± 1.3 μm and 15.7 ± 1.9 μm (n=50), respectively. Urediniospore and teliospore morphology confirmed the identification of collected rust spores as Puccinia emaculata Schwein. (Ramachar and Cummins 1965). Genomic DNA was extracted from a uredinium derived from a single urediniospore on the infected detached leaf of cultivar Blackwell. A 1.2 kb fragment of 18S rDNA was amplified using primers designed by Frazier et al. (2013). The 18S rDNA sequence (GenBank: MN699697.1) was blasted in GenBank and the sequence showed 99.92% identity with the 18S rDNA of P. emaculata (GenBank: EU915294.1). Based on the morphological and genetic information, the rust pathogen was identified as P. emaculata (Ramachar and Cummins 1965). P. emaculata has also been reported in Tennessee, Arkansas and Virginia (Zale et al. 2008; Hirsch et al. 2010; Frazier et al. 2013), but to our knowledge, this is the first report of P. emaculata on switchgrass in Georgia. Switchgrass rust is currently affecting the growth of many switchgrass cultivars with various levels of susceptibility. This pathogen reduces switchgrass biomass yield, and therefore the identification and control of it is vital for switchgrass biofuel production in the US.

Nitrogen Fixation and Resorption Efficiency Differences Among Twelve Upland and Lowland Switchgrass Cultivars

In nitrogen (N)-limited terrestrial ecosystems, plants employ various strategies to acquire and conserve N, including translocation of N in perennial tissues and stimulation of N fixation in roots and soils. Switchgrass (Panicum virgatum) is a genotypically and phenotypically diverse perennial grass with two distinct ecotypes (lowland and upland) and numerous genotypes. It grows well in low-N soils, likely because of its ability to translocate N and to associate with N-fixing microbes, but little is known about variation in these traits among cultivars or even ecotypes. We measured N translocation, N fixation potential in roots and soils, soil net N mineralization, soil net nitrification, and biomass yields in 12 switchgrass cultivars grown in a replicated block experiment in southwestern Michigan, United States. Lowland cultivars had higher yields, rates of N translocation, soil net N mineralization, and N fixation potentials on washed, nonsterile roots, while upland cultivars exhibited higher N fixation potentials in root-free soil. N resorption efficiencies averaged 53 ± 5% (± standard error) for lowland versus 29 ± 3% for upland cultivars. Additionally, there were significant among-cultivar differences for all response variables except mineralization and nitrification, with differences likely explained by cultivar-specific physiologies and microbial communities. The ideal cultivar for biofuels is one that can maintain high yields with minimal fertilizer addition, and there appear to be several cultivars that meet these criteria. In addition, results suggest substantial N cycle differences among cultivars that might be exploited by breeders to create new or improved high-yielding, N-conserving switchgrass lines.

Simultaneous regulation of F5H in COMT-RNAi transgenic switchgrass alters effects of COMT suppression on syringyl lignin biosynthesis.

SummaryFerulate 5‐hydroxylase (F5H) catalyses the hydroxylation of coniferyl alcohol and coniferaldehyde for the biosynthesis of syringyl (S) lignin in angiosperms. However, the coordinated effects of F5H with caffeic acid O‐methyltransferase (COMT) on the metabolic flux towards S units are largely unknown. We concomitantly regulated F5H expression in COMT‐down‐regulated transgenic switchgrass (Panicum virgatum L.) lines and studied the coordination of F5H and COMT in lignin biosynthesis. Down‐regulation of F5H in COMT‐RNAi transgenic switchgrass plants further impeded S lignin biosynthesis and, consequently, increased guaiacyl (G) units and reduced 5‐OH G units. Conversely, overexpression of F5H in COMT‐RNAi transgenic plants reduced G units and increased 5‐OH units, whereas the deficiency of S lignin biosynthesis was partially compensated or fully restored, depending on the extent of COMT down‐regulation in switchgrass. Moreover, simultaneous regulation of F5H and COMT expression had different effects on cell wall digestibility of switchgrass without biomass loss. Our results indicate that up‐regulation and down‐regulation of F5H expression, respectively, have antagonistic and synergistic effects on the reduction in S lignin resulting from COMT suppression. The coordinated effects between lignin genes should be taken into account in future studies aimed at cell wall bioengineering.

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

SummaryCell 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 (TFs) 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 TFs identified in this study, and seven TFs identified in previous studies, as bait genes. Six TFs (PvMYB58/63, PvMYB42/85, PvMYB4, PvWRKY12, PvSND2 and PvSWN2) 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 TFs in controlling secondary wall formation. RNA‐seq analysis of four of the transgenic switchgrass lines revealed downstream target genes of the secondary wall‐related TFs and crosstalk with other biological pathways. In vitro transactivation assays further confirmed the regulation of specific lignin pathway genes by four of the TFs. Our meta‐analysis provides a hierarchical network of TFs and their potential target genes for future manipulation of secondary cell wall formation for lignin modification in switchgrass.

Overexpression of a rice BAHD acyltransferase gene in switchgrass (Panicum virgatum L.) enhances saccharification

BackgroundSwitchgrass (Panicum virgatum L.) is a promising bioenergy feedstock because it can be grown on marginal land and produces abundant biomass. Recalcitrance of the lignocellulosic components of the switchgrass cell wall to enzymatic degradation into simple sugars impedes efficient biofuel production. We previously demonstrated that overexpression of OsAT10, a BAHD acyltransferase gene, enhances saccharification efficiency in rice.ResultsHere we show that overexpression of the rice OsAT10 gene in switchgrass decreased the levels of cell wall-bound ferulic acid (FA) in green leaf tissues and to a lesser extent in senesced tissues, and significantly increased levels of cell wall-bound p-coumaric acid (p-CA) in green leaves but decreased its level in senesced tissues of the T0 plants under greenhouse conditions. The engineered switchgrass lines exhibit an approximate 40% increase in saccharification efficiency in green tissues and a 30% increase in senesced tissues.ConclusionOur study demonstrates that overexpression of OsAT10, a rice BAHD acyltransferase gene, enhances saccharification of lignocellulosic biomass in switchgrass.

Antioxidant metabolism variation associated with alkali-salt tolerance in thirty switchgrass (Panicum virgatum) lines.

Soil salinization is a major factor limiting crop growth and development in many areas. Switchgrass (Panicum virgatum L.) is an important warm-season grass species used for biofuel production. The objective of this study was to investigate antioxidant metabolism, proline,and protein variation associated with alkali-salt tolerance among 30 switchgrass lines and identify metabolic markers for evaluating alkali-salt tolerance of switchgrass lines. The grass lines were transplanted into plastic pots containing fine sand. When the plants reached E5 developmental stage, they were subjected to either alkali-salt stress treatment (150 mM Na+ and pH of 9.5) or control (no alkali-salt stress) for 20 d. The 30 switchgrass lines differed in alkali-salt tolerance as determined by the level of leaf malondialdehyde (MDA), antioxidant enzyme activity [(superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX)], proline and protein. Alkali-salt stress increased MDA, proline, SOD, reduced CAT activity, but its effect on protein and APX varied depending on lines. Wide variations in the five parameters existed among the 30 lines. In general, the lines with higher CAT activity and lower proline content under alkali-salt stress had less MDA, exhibiting better alkali-salt tolerance. Among the five parameters, CAT can be considered as valuable metabolic markers for assessment of switchgrass tolerance to alkali-salt stress.

Alteration of S-adenosylhomocysteine levels affects lignin biosynthesis in switchgrass.

SummaryMethionine (Met) synthesized from aspartate is a fundamental amino acid needed to produce S‐adenosylmethionine (SAM) that is an important cofactor for the methylation of monolignols. As a competitive inhibitor of SAM‐dependent methylation, the effect of S‐adenosylhomocysteine (SAH) on lignin biosynthesis, however, is still largely unknown in plants. Expression levels of Cystathionine γ‐synthase (PvCGS) and S‐adenosylhomocysteine hydrolase 1 (PvSAHH1) were down‐regulated by RNAi technology, respectively, in switchgrass, a dual‐purpose forage and biofuel crop. The transgenic switchgrass lines were subjected to studying the impact of SAH on lignin biosynthesis. Our results showed that down‐regulation of PvCGS in switchgrass altered the accumulation of aspartate‐derived and aromatic amino acids, reduced the content of SAH, enhanced lignin biosynthesis and stunted plant growth. In contrast, down‐regulation of PvSAHH1 raised SAH levels in switchgrass, impaired the biosynthesis of both guaiacyl and syringyl lignins and therefore significantly increased saccharification efficiency of cell walls. This work indicates that SAH plays a crucial role in monolignol methylation in switchgrass. Genetic regulation of either PvCGS or PvSAHH1 expression in switchgrass can change intracellular SAH contents and SAM to SAH ratios and therefore affect lignin biosynthesis. Thus, our study suggests that genes involved in Met metabolism are of interest as new valuable targets for cell wall bioengineering in future.

Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum)

BackgroundSwitchgrass (Panicum virgatum), a robust perennial C4-type grass, has been evaluated and designated as a model bioenergy crop by the U.S. DOE and USDA. Conventional breeding of switchgrass biomass is difficult because it displays self-incompatible hindrance. Therefore, direct genetic modifications of switchgrass have been considered the more effective approach to tailor switchgrass with traits of interest. Successful transformations have demonstrated increased biomass yields, reduction in the recalcitrance of cell walls and enhanced saccharification efficiency. Several tissue culture protocols have been previously described to produce transgenic switchgrass lines using different nutrient-based media, co-cultivation approaches, and antibiotic strengths for selection.ResultsAfter evaluating the published protocols, we consolidated these approaches and optimized the process to develop a more efficient protocol for producing transgenic switchgrass. First, seed sterilization was optimized, which led to a 20% increase in yield of induced calluses. Second, we have selected a N6 macronutrient/B5 micronutrient (NB)-based medium for callus induction from mature seeds of the Alamo cultivar, and chose a Murashige and Skoog-based medium to regenerate both Type I and Type II calluses. Third, Agrobacterium-mediated transformation was adopted that resulted in 50–100% positive regenerated transformants after three rounds (2 weeks/round) of selection with antibiotic. Genomic DNA PCR, RT-PCR, Southern blot, visualization of the red fluorescent protein and histochemical β-glucuronidase (GUS) staining were conducted to confirm the positive switchgrass transformants. The optimized methods developed here provide an improved strategy to promote the production and selection of callus and generation of transgenic switchgrass lines.ConclusionThe process for switchgrass transformation has been evaluated and consolidated to devise an improved approach for transgenic switchgrass production. With the optimization of seed sterilization, callus induction, and regeneration steps, a reliable and effective protocol is established to facilitate switchgrass engineering.

Development and use of a switchgrass (Panicum virgatum L.) transformation pipeline by the BioEnergy Science Center to evaluate plants for reduced cell wall recalcitrance

BackgroundThe mission of the BioEnergy Science Center (BESC) was to enable efficient lignocellulosic-based biofuel production. One BESC goal was to decrease poplar and switchgrass biomass recalcitrance to biofuel conversion while not affecting plant growth. A transformation pipeline (TP), to express transgenes or transgene fragments (constructs) in these feedstocks with the goal of understanding and decreasing recalcitrance, was considered essential for this goal. Centralized data storage for access by BESC members and later the public also was essential.ResultsA BESC committee was established to codify procedures to evaluate and accept genes into the TP. A laboratory information management system (LIMS) was organized to catalog constructs, plant lines and results from their analyses. One hundred twenty-eight constructs were accepted into the TP for expression in switchgrass in the first 5 years of BESC. Here we provide information on 53 of these constructs and the BESC TP process. Eleven of the constructs could not be cloned into an expression vector for transformation. Of the remaining constructs, 22 modified expression of the gene target. Transgenic lines representing some constructs displayed decreased recalcitrance in the field and publications describing these results are tabulated here. Transcript levels of target genes and detailed wall analyses from transgenic lines expressing six additional tabulated constructs aimed toward modifying expression of genes associated with wall structure (xyloglucan and lignin components) are provided. Altered expression of xyloglucan endotransglucosylase/hydrolases did not modify lignin content in transgenic plants. Simultaneous silencing of two hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferases was necessary to decrease G and S lignin monomer and total lignin contents, but this reduced plant growth.ConclusionsA TP to produce plants with decreased recalcitrance and a LIMS for data compilation from these plants were created. While many genes accepted into the TP resulted in transgenic switchgrass without modified lignin or biomass content, a group of genes with potential to improve lignocellulosic biofuel yields was identified. Results from transgenic lines targeting xyloglucan and lignin structure provide examples of the types of information available on switchgrass lines produced within BESC. This report supplies useful information when developing coordinated, large-scale, multi-institutional reverse genetic pipelines to improve crop traits.

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)‐to‐moderate (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‐temperate 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.

Bioavailability of Carbohydrate Content in Natural and Transgenic Switchgrasses for the Extreme Thermophile Caldicellulosiruptor bescii.

Improving access to the carbohydrate content of lignocellulose is key to reducing recalcitrance for microbial deconstruction and conversion to fuels and chemicals. Caldicellulosiruptor bescii completely solubilizes naked microcrystalline cellulose, yet this transformation is impeded within the context of the plant cell wall by a network of lignin and hemicellulose. Here, the bioavailability of carbohydrates to C. bescii at 70°C was examined for reduced lignin transgenic switchgrass lines COMT3(+) and MYB Trans, their corresponding parental lines (cultivar Alamo) COMT3(-) and MYB wild type (WT), and the natural variant cultivar Cave-in-Rock (CR). Transgenic modification improved carbohydrate solubilization by C. bescii to 15% (2.3-fold) for MYB and to 36% (1.5-fold) for COMT, comparable to the levels achieved for the natural variant, CR (36%). Carbohydrate solubilization was nearly doubled after two consecutive microbial fermentations compared to one microbial step, but it never exceeded 50% overall. Hydrothermal treatment (180°C) prior to microbial steps improved solubilization 3.7-fold for the most recalcitrant line (MYB WT) and increased carbohydrate recovery to nearly 50% for the least recalcitrant lines [COMT3(+) and CR]. Alternating microbial and hydrothermal steps (T→M→T→M) further increased bioavailability, achieving carbohydrate solubilization ranging from 50% for MYB WT to above 70% for COMT3(+) and CR. Incomplete carbohydrate solubilization suggests that cellulose in the highly lignified residue was inaccessible; indeed, residue from the T→M→T→M treatment was primarily glucan and inert materials (lignin and ash). While C. bescii could significantly solubilize the transgenic switchgrass lines and natural variant tested here, additional or alternative strategies (physical, chemical, enzymatic, and/or genetic) are needed to eliminate recalcitrance.IMPORTANCE Key to a microbial process for solubilization of plant biomass is the organism's access to the carbohydrate content of lignocellulose. Economically viable routes will characteristically minimize physical, chemical, and biological pretreatment such that microbial steps contribute to the greatest extent possible. Recently, transgenic versions of plants and trees have been developed with the intention of lowering the barrier to lignocellulose conversion, with particular focus on lignin content and composition. Here, the extremely thermophilic bacterium Caldicellulosiruptor bescii was used to solubilize natural and genetically modified switchgrass lines, with and without the aid of hydrothermal treatment. For lignocellulose conversion, it is clear that the microorganism, plant biomass substrate, and processing steps must all be considered simultaneously to achieve optimal results. Whether switchgrass lines engineered for low lignin or natural variants with desirable properties are used, conversion will depend on microbial access to crystalline cellulose in the plant cell wall.

Generation of octoploid switchgrass in three cultivars by colchicine treatment

Polyploidization is regularly regarded as an important tool for the generation of innovative germplasm resources and plant breeding. In this study, autopolyploid switchgrass lines were produced from calli derived from immature inflorescence of tetraploid switchgrass (Panicum virgatum L.), both lowland cv. Alamo and Kanlow, and upland cv. Ranlow, through colchicine treatments. Ploidy levels of plantlets regenerated from the calli were screened by flow cytometry. Growth characteristics of the tetraploid and octoploid switchgrass cultivars were further investigated in a field experiment. Compared with the tetraploid plants of the three switchgrass cultivars, the corresponding octoploid plants showed significant reduction in plant height, number of tillers per plant and fresh matter, but an increase in leaf length and width.

Transcriptional analysis of defense mechanisms in upland tetraploid switchgrass to greenbugs

BackgroundAphid infestation of switchgrass (Panicum virgatum) has the potential to reduce yields and biomass quality. Although switchgrass-greenbug (Schizaphis graminum; GB) interactions have been studied at the whole plant level, little information is available on plant defense responses at the molecular level.ResultsThe global transcriptomic response of switchgrass cv Summer to GB was monitored by RNA-Seq in infested and control (uninfested) plants harvested at 5, 10, and 15 days after infestation (DAI). Differentially expressed genes (DEGs) in infested plants were analyzed relative to control uninfested plants at each time point. DEGs in GB-infested plants induced by 5-DAI included an upregulation of reactive burst oxidases and several cell wall receptors. Expression changes in genes linked to redox metabolism, cell wall structure, and hormone biosynthesis were also observed by 5-DAI. At 10-DAI, network analysis indicated a massive upregulation of defense-associated genes, including NAC, WRKY, and MYB classes of transcription factors and potential ancillary signaling molecules such as leucine aminopeptidases. Molecular evidence for loss of chloroplastic functions was also detected at this time point. Supporting these molecular changes, chlorophyll content was significantly decreased, and ROS levels were elevated in infested plants 10-DAI. Total peroxidase and laccase activities were elevated in infested plants at 10-DAI relative to control uninfested plants. The net result appeared to be a broad scale defensive response that led to an apparent reduction in C and N assimilation and a potential redirection of nutrients away from GB and towards the production of defensive compounds, such as pipecolic acid, chlorogenic acid, and trehalose by 10-DAI. By 15-DAI, evidence of recovery in primary metabolism was noted based on transcript abundances for genes associated with carbon, nitrogen, and nutrient assimilation.ConclusionsExtensive remodeling of the plant transcriptome and the production of ROS and several defensive metabolites in an upland switchgrass cultivar were observed in response to GB feeding. The early loss and apparent recovery in primary metabolism by 15-DAI would suggest that these transcriptional changes in later stages of GB infestation could underlie the recovery response categorized for this switchgrass cultivar. These results can be exploited to develop switchgrass lines with more durable resistance to GB and potentially other aphids.

Study of traits and recalcitrance reduction of field-grown COMT down-regulated switchgrass

BackgroundThe native recalcitrance of plants hinders the biomass conversion process using current biorefinery techniques. Down-regulation of the caffeic acid O-methyltransferase (COMT) gene in the lignin biosynthesis pathway of switchgrass reduced the thermochemical and biochemical conversion recalcitrance of biomass. Due to potential environmental influences on lignin biosynthesis and deposition, studying the consequences of physicochemical changes in field-grown plants without pretreatment is essential to evaluate the performance of lignin-altered plants. We determined the chemical composition, cellulose crystallinity and the degree of its polymerization, molecular weight of hemicellulose, and cellulose accessibility of cell walls in order to better understand the fundamental features of why biomass is recalcitrant to conversion without pretreatment. The most important is to investigate whether traits and features are stable in the dynamics of field environmental effects over multiple years.ResultsField-grown COMT down-regulated plants maintained both reduced cell wall recalcitrance and lignin content compared with the non-transgenic controls for at least 3 seasons. The transgenic switchgrass yielded 35–84% higher total sugar release (enzymatic digestibility or saccharification) from a 72-h enzymatic hydrolysis without pretreatment and also had a 25–32% increase in enzymatic sugar release after hydrothermal pretreatment. The COMT-silenced switchgrass lines had consistently lower lignin content, e.g., 12 and 14% reduction for year 2 and year 3 growing season, respectively, than the control plants. By contrast, the transgenic lines had 7–8% more xylan and galactan contents than the wild-type controls. Gel permeation chromatographic results revealed that the weight-average molecular weights of hemicellulose were 7–11% lower in the transgenic than in the control lines. In addition, we found that silencing of COMT in switchgrass led to 20–22% increased cellulose accessibility as measured by the Simons’ stain protocol. No significant changes were observed on the arabinan and glucan contents, cellulose crystallinity, and cellulose degree of polymerization between the transgenic and control plants. With the 2-year comparative analysis, both the control and transgenic lines had significant increases in lignin and glucan contents and hemicellulose molecular weight across the growing seasons.ConclusionsThe down-regulation of COMT in switchgrass resulting in a reduced lignin content and biomass recalcitrance is stable in a field-grown trial for at least three seasons. Among the determined affecting factors, the reduced biomass recalcitrance of the COMT-silenced switchgrass, grown in the field conditions for two and three seasons, was likely related to the decreased lignin content and increased biomass accessibility, whereas the cellulose crystallinity and degree of its polymerization and hemicellulose molecular weights did not contribute to the reduction of recalcitrance significantly. This finding suggests that lignin down-regulation in lignocellulosic feedstock confers improved saccharification that translates from greenhouse to field trial and that lignin content and biomass accessibility are two significant factors for developing a reduced recalcitrance feedstock by genetic modification.