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Publications

When it comes to interdisciplinary collaboration, the titles of GLBRC publications speak for themselves. Each new year of operation has seen more publications from multiple labs that span the four Research Areas, accelerating the Center's production of the basic research that generates technology to convert cellulosic biomass to advanced biofuels.

Publications

Does plant biomass manipulation in static chambers affect nitrous oxide emissions from soils?

Sarah M. Collier; Andrew P. Dean; Lawrence G. Oates; Matthew D. Ruark; Randall D. Jackson

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2016

One of the most widespread approaches for measurement of greenhouse gas emissions from soils involves the use of static chambers. This method is relatively inexpensive, is easily replicated, and is ideally suited to plot-based experimental systems. Among its limitations is the loss of detection sensitivity with increasing chamber height, which creates challenges for deployment in systems including tall vegetation. It is not always possible to avoid inclusion of plants within chambers or to extend chamber height to fully accommodate plant growth. Thus, in many systems, such as perennial forages and biomass crops, plants growing within static chambers must either be trimmed or folded during lid closure. Currently, data on how different types of biomass manipulation affect measured results is limited. Here, we compare the effects of cutting vs. folding of biomass on nitrous oxide measurements in switchgrass (Panicum virgatum L.) and alfalfa (Medicago sativa L.) systems. We report only limited evidence of treatment effects during discrete sampling events and little basis for concern that effects may intensify over time as biomass manipulation is repeatedly imposed. However, nonsignificant treatment effects that were consistently present amounted to significant overall trends in three out of the four systems studied. Such minor disparities in flux could amount to considerable quantities over time, suggesting that caution should be exercised when comparing cumulative emission values from studies using different biomass manipulation strategies.

Draft assembly of elite inbred line PH207 provides insights into genomic and transcriptome diversity in maize

Candice N. Hirsch; Cory D. Hirsch; Alex B. Brohammer; Megan J. Bowman; Ilya Soifer; Omer Barad; Doron Shem-Tov; Kobi Baruch; Fei Lu; Alvaro G. Hernandez; Christopher J. Fields; Chris L. Wright; Klaus Koehler; Nathan M. Springer; Edward Buckler; Robin Buell; Natalia de Leon; Shawn M. Kaeppler; Kevin L. Childs; Mark A. Mikel

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2016

Intense artificial selection over the last 100 years has produced elite maize (Zea mays) inbred lines that combine to produce high-yielding hybrids. To further our understanding of how genome and transcriptome variation contribute to the production of high-yielding hybrids, we generated a draft genome assembly of the inbred line PH207 to complement and compare with the existing B73 reference sequence. B73 is a founder of the Stiff Stalk germplasm pool, while PH207 is a founder of Iodent germplasm, both of which have contributed substantially to the production of temperate commercial maize and are combined to make heterotic hybrids. Comparison of these two assemblies revealed over 2,500 genes present in only one of the two genotypes and 136 gene families that have undergone extensive expansion or contraction. Transcriptome profiling revealed extensive expression variation, with as many as 10,564 differentially expressed transcripts and 7,128 transcripts expressed in only one of the two genotypes in a single tissue. Genotype-specific genes were more likely to have tissue/condition-specific expression and lower transcript abundance. The availability of a high-quality genome assembly for the elite maize inbred PH207 expands our knowledge of the breadth of natural genome and transcriptome variation in elite maize inbred lines across heterotic pools.

Dynamic evolution of nitric oxide detoxifying flavohemoglobins, a family of single-protein metabolic modules in bacteria and eukaryotes

Jennifer H. Wisecaver; William G. Alexander; Sean B. King; Chris Todd Hittinger; Antonis Rokas

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2016

Due to their functional independence, proteins that comprise standalone metabolic units, which we name single-protein metabolic modules, may be particularly prone to gene duplication (GD) and horizontal gene transfer (HGT). Flavohemoglobins (flavoHbs) are prime examples of single-protein metabolic modules, detoxifying nitric oxide—a ubiquitous toxin whose antimicrobial properties many life forms exploit in competition and to defend against infection—to nitrate—a common source of nitrogen for organisms. FlavoHbs appear widespread in bacteria and have been identified in a handful of microbial eukaryotes, but how the distribution of this ecologically and biomedically important protein family evolved remains unknown. Reconstruction of the evolutionary history of 3,318 flavoHb protein sequences covering the family’s known diversity showed evidence of recurrent HGT at multiple evolutionary scales including intra-bacterial HGT, as well as HGT from bacteria to eukaryotes. One of the most striking examples of HGT is the acquisition of a flavoHb by the dandruff- and eczema-causing fungus Malassezia from Corynebacterium Actinobacteria, a transfer that growth experiments show are capable of mediating NO resistance in fungi. Other flavoHbs arose via GD; for example, many filamentous fungi poss­ess two flavoHbs that are differentially targeted to the cytosol and mitochondria, likely conferring protection against external and internal sources of NO, respectively. Previous studies of cytotoxic aerolysins and antibacterial lysozymes also reported high rates of GD and HGT, raising the hypothesis that such single-protein metabolic modules might be frequent actors in host-microbe arms races due to their functional independence and propensity for GD and HGT.

Dynamics of biomass partitioning, stem gene expression, cell wall biosynthesis, and sucrose accumulation during development of Sorghum bicolor

Brian McKinley; William Rooney; Curtis Wilkerson; John Mullet

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2016

Biomass accumulated preferentially in leaves of the sweet sorghum Della until floral initiation, then stems until anthesis, followed by panicles until grain maturity, and apical tillers. Sorghum stem RNA-seq transcriptome profiles and composition data were collected for ~100 days of development beginning at floral initiation. The analysis identified >200 differentially expressed genes involved in stem growth, cell wall biology, and sucrose accumulation. Genes encoding expansins and xyloglucan endotransglucosylase/hydrolases were differentially expressed in growing stem internodes. Genes encoding enzymes involved in the synthesis of cellulose, lignin, and glucuronoarabinoxylan were expressed at elevated levels in stems until ~7 days before anthesis and then down regulated. CESA genes involved in primary and secondary cell wall synthesis showed different temporal patterns of expression. Following floral initiation, the level of sucrose and other non-structural carbohydrates increased to ~50% of the stem's dry weight. Stem sucrose accumulation was inversely correlated with >100-fold down-regulation of SbVIN1, a gene encoding a vacuolar invertase. Accumulation of stem sucrose was also correlated with cessation of leaf and stem growth at anthesis, decreased expression of genes involved in stem cell wall synthesis, and ~10-fold lower expression of SbSUS4, a gene encoding sucrose synthase that generates UDP-glucose from sucrose for cell wall biosynthesis. Genes for mixed linkage glucan synthesis (CSLF) and turnover were expressed at high levels in stems throughout development. Overall, the stem transcription profile resource and the genes and regulatory dynamics identified in this study will be useful for engineering sorghum stem composition for improved conversion to biofuels and bioproducts. This article is protected by copyright. All rights reserved.

Ecosystem water-use efficiency of annual corn and perennial grasslands: contributions from land-use history and species composition

Michael Abraha; Ilya Gelfand; Stephen K. Hamilton; Changliang Shao; Yahn-Jauh Su; Philip Robertson; Jiquan Chen

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2016

Carbon and water exchanges between vegetated land surfaces and the atmosphere reveal the ecosystem-scale water-use efficiency (WUE) of primary production. We examined the interacting influence of dominant plant functional groups (C3 and C4) and land-use history on WUEs of annual corn and perennial (restored prairie, switchgrass and smooth brome grass) grasslands in the US Midwest from 2010 through 2013. To this end, we determined ecosystem-level (eWUE) and intrinsic (iWUE) WUEs using eddy covariance and plant carbon isotope ratios, respectively. Corn, switchgrass, and restored prairie were each planted on lands previously managed as grasslands under the USDA Conservation Reserve Program (CRP), or as corn/soybean rotation under conventional agriculture (AGR), while a field of smooth brome grass remained in CRP management. The iWUEs of individual C3 plant species varied little across years. Corn had the highest (4.1) and smooth brome grass the lowest (2.3) overall eWUEs (g C kg−1 H2O) over the 4 years. Corn and switchgrass did not consistently show a significant difference in seasonal eWUE between former CRP and AGR lands, whereas restored prairie had significantly higher seasonal eWUE on former AGR than on former CRP land due to a greater shift from C3 to C4 species on the former AGR land following a drought in 2012. Thus, differences in grassland eWUE were largely determined by the relative dominance of C3 and C4 species within the plant communities. In this humid temperate climate with common short-term and occasional long-term droughts, it is likely that mixed grasslands will become increasingly dominated by C4 grasses over time, with higher yields and eWUE than C3 plants. These results inform models of the interaction between carbon and water cycles in grassland ecosystems under current and future climate and management scenarios.

Effective alkaline metal-catalyzed oxidative delignification of hybrid poplar

Aditya Bhalla; Namita Bansal; Ryan J. Stoklosa; Mackenzie Fountain; John Ralph; David B. Hodge; Eric L. Hegg

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2016

Strategies to improve copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment of hybrid poplar were investigated. These improvements included a combination of increasing hydrolysis yields, while simultaneously decreasing process inputs through (i) more efficient utilization of H2O2 and (ii) the addition of an alkaline extraction step prior to the metal-catalyzed AHP pretreatment. We hypothesized that utilizing this improved process could substantially lower the chemical inputs needed during pretreatment.

Electron partitioning in anoxic phototrophic bacteria

Melanie A. Spero; Saheed Imam; Daniel R. Noguera; Timothy J. Donohue

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2016

Photosynthetic cells make major contributions to many important processes on this planet, including solar energy capture, nitrogen or carbon dioxide sequestration and production of useful biocommodities. The sheer number of photosynthetic cells also makes them significant contributors to global nutrient cycling, especially in aquatic ecosystems. For each of these activities, photosynthetic cells need efficient systems for production and distribution of reducing power among the myriad of cellular pathways that depend on reductant. This chapter focuses on the partitioning of reductant in purple nonsulfur photosynthetic bacteria. It summarizes known membrane and cytoplasmic enzymes and pathways that need the reductant produced via photochemical activity (quinol in these organisms). These observations illustrate that quinol is used to provide reducing power to a variety of crucial cellular processes (cellular biosynthesis, maintenance of a proton motive force) and key assimilatory pathways (carbon dioxide and nitrogen fixation), depending on the availability of nutrients. We also summarize data illustrating that cells use a variety of pathways to recycle excess reductant. Finally, we illustrate how recent use of genomic and computational approaches to the analysis of these and other photosynthetic organisms has provided testable predictions and considerable new insight into the partitioning of reductant among that is produced from solar energy capture.

Enhancing digestibility and ethanol yield of Populus wood via expression of an engineered monolignol 4-O-methyltransferase

Yuanheng Cai; Kewei Zhang; Hoon Kim; Guichuan Hou; Xuebin Zhang; Huijun Yang; Huan Feng; Lisa Miller; John Ralph; Chang-Jun Liu

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2016

Producing cellulosic biofuels and bio-based chemicals from woody biomass is impeded by the presence of lignin polymer in the plant cell wall. Manipulating the monolignol biosynthetic pathway offers a promising approach to improved processability, but often impairs plant growth and development. Here, we show that expressing an engineered 4-O-methyl- transferase that chemically modifies the phenolic moiety of lignin monomeric precursors, thus preventing their incorporation into the lignin polymer, substantially alters hybrid aspens’ lignin content and structure. Woody biomass derived from the transgenic aspens shows a 62% increase in the release of simple sugars and up to a 49% increase in the yield of ethanol when the woody biomass is subjected to enzymatic digestion and yeast-mediated fermentation. Moreover, the cell wall structural changes do not affect growth and biomass production of the trees. Our study provides a useful strategy for tailoring woody biomass for bio-based applications.

Evolution and ecology of Actinobacteria and their bioenergy applications

Gina R. Lewin; Camila Carlos; Marc G. Chevrette; Heidi A. Horn; Brandon R. McDonald; Robert J. Stankey; Brian G. Fox; Cameron R. Currie

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2016

Evolution of VRN2/Ghd7-like genes in vernalization-mediated repression of grass flowering

Daniel P. Woods; Meghan A. McKeown; Yinxin Dong; Jill C. Preston; Richard M. Amasino

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2016

Flowering of many plant species is coordinated with seasonal environmental cues such as temperature and photoperiod. Vernalization provides competence to flower after prolonged cold exposure, and a vernalization requirement prevents flowering from occurring prior to winter. In winter wheat and barley, three genes VRN1, VRN2, and FT form a regulatory loop that regulates the initiation of flowering. Prior to cold exposure, VRN2 represses FT. During cold, VRN1 expression increases, resulting in the repression of VRN2, which in turn allows activation of FT during long days to induce flowering. Here we test whether the circuitry of this regulatory loop is conserved across Pooideae,consistent with their niche transition from the tropics to the temperate zone. Our phylogenetic analyses of VRN2-like genes reveal a duplication event occurred before the diversification of the grasses that gave rise to a CO9 and VRN2/GhD7 clade, and supports orthology between wheat/barley VRN2 and rice GhD7. Our Brachypodium distachyon VRN1 and VRN2 knockdown and overexpression experiments demonstrate functional conservation of grass VRN1 and VRN2 in the promotion and repression of flowering, respectively. However, expression analyses in a range of pooids demonstrate that the cold repression of VRN2 is unique to core Pooideae such as wheat and barley. Furthermore, VRN1 knock down in Brachypodium demonstrates that the VRN1 mediated suppression of VRN2 is not conserved. Thus, the VRN1-VRN2 feature of the regulatory loop appears to have evolved late in the diversification of temperate grasses.

Evolution of high cellulolytic activity in symbiotic Streptomyces through selection of expanded gene content and coordinated gene expression

Adam J. Book; Gina R. Lewin; Bradon R. McDonald; Taichi E. Takasuka; Evelyn Wendt-Pienkowski; Drew T. Doering; Steven Suh; Kenneth F. Raffa; Brian G. Fox; Cameron R. Currie

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2016

Cellulose deconstruction helps shape the global carbon cycle; this study shows that high cellulolytic ability evolved in select lineages of the bacterial genus Streptomyces through key changes in gene content and transcriptional regulation.

Formaldehyde stabilization facilitates lignin monomer production during biomass depolymerization

Li Shuai; Masoud Talebi Amiri; Ydna M. Questell-Santiago; Florent Héroguel; Yanding Li; Hoon Kim; Richard Meilan; Clint Chapple; John Ralph; Jeremy S. Luterbacher

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2016

Genome sequence and analysis of a stress-tolerant, wild-derived strain of Saccharomyces cerevisiae used in biofuels research

Sean J. McIlwain; David Peris; Maria Sardi; Oleg V. Moskvin; Fujie Zhan; Kevin S. Myers; Nicholas M. Riley; Alyssa Buzzell; Lucas S. Parreiras; Irene M. Ong; Robert Landick; Joshua J. Coon; Audrey P. Gasch; Trey K. Sato; Chris Todd Hittinger

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2016

The genome sequences of more than 100 strains of the yeast Saccharomyces cerevisiae have been published. Unfortunately, most of these genome assemblies contain dozens to hundreds of gaps at repetitive sequences, including transposable elements, tRNAs, and subtelomeric regions, which is where novel genes generally reside. Relatively few strains have been chosen for genome sequencing based on their biofuel production potential, leaving an additional knowledge gap. Here, we describe the nearly complete genome sequence of GLBRCY22-3 (Y22-3), a strain of S. cerevisiae derived from the stress-tolerant wild strain NRRL YB-210 and subsequently engineered for xylose metabolism. After benchmarking several genome assembly approaches, we developed a pipeline to integrate Pacific Biosciences (PacBio) and Illumina sequencing data and achieved one of the highest quality genome assemblies for any S. cerevisiae strain. Specifically, the contig N50 is 693 kbp, and the sequences of most chromosomes, the mitochondrial genome, and the 2-micron plasmid are complete. Our annotation predicts 92 genes that are not present in the reference genome of the laboratory strain S288c, over 70% of which were expressed. We predicted functions for 43 of these genes, 28 of which were previously uncharacterized and unnamed. Remarkably, many of these genes are predicted to be involved in stress tolerance and carbon metabolism and are shared with a Brazilian bioethanol production strain, even though the strains differ dramatically at most genetic loci. The Y22-3 genome sequence provides an exceptionally high-quality resource for basic and applied research in bioenergy and genetics.

Genomic analysis and D-xylose fermentation of three novel Spathaspora species: Spathaspora girioi sp. nov., Spathaspora Hagerdaliae f. a., sp. nov. and Spathaspora gorwiae f. a., sp. nov.

Mariana R. Lopes; Camila G. Morais; Jacek Kominek; Raquel M. Cadete; Marco A. Soares; Ana Paula T. Uetanabaro; Cesar Fonseca; Marc-Andre Lachance; Chris T. Hittinger; Carlos A. Rosa

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2016

Three novel D-xylose-fermenting yeast species of Spathaspora clade were recovered from rotting wood in regions of the Atlantic Rainforest ecosystem in Brazil. Differentiation of new species was based on analyses of the gene encoding the D1/D2 sequences of large subunit of rRNA and on 642 conserved, single-copy, orthologous genes from genome sequence assemblies from the newly described species and 15 closely-related Debaryomycetaceae/Metschnikowiaceae species. Spathaspora girioi sp. nov. produced unconjugated asci with a single elongated ascospore with curved ends; ascospore formation was not observed for the other two species. The three novel species ferment D-xylose with different efficiencies. Spathaspora hagerdaliae sp. nov. and Sp. girioi sp. nov. showed xylose reductase (XR) activity strictly dependent on NADPH, whereas Sp. gorwiae sp. nov. had XR activity that used both NADH and NADPH as co-factors. The genes that encode enzymes involved in D-xylose metabolism (XR, xylitol dehydrogenase and xylulokinase) were also identified for these novel species. The type strains are Sp. girioi sp. nov. UFMG-CM-Y302(T) (=CBS 13476), Sp. hagerdaliae f.a., sp. nov. UFMG-CM-Y303(T) (=CBS 13475) and Sp. gorwiae f.a., sp. nov. UFMG-CM-Y312(T) (=CBS 13472).

Horizontally acquired genes in early-diverging pathogenic fungi enable the use of host nucleosides and nucleotides

Wiliam G. Alexander; Jennifer H. Wisecaver; Antonis Rokas; Chris T. Hittinger

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2016

Horizontal gene transfer (HGT) among bacteria, archaea, and viruses is widespread, but the extent of transfers from these lineages into eukaryotic organisms is contentious. Here we systematically identify hundreds of genes that were likely acquired horizontally from a variety of sources by the early-diverging fungal phyla Microsporidia and Cryptomycota. Interestingly, the Microsporidia have acquired via HGT several genes involved in nucleic acid synthesis and salvage, such as those encoding thymidine kinase (TK), cytidylate kinase, and purine nucleotide phosphorylase. We show that these HGT-derived nucleic acid synthesis genes tend to function at the interface between the metabolic networks of the host and pathogen. Thus, these genes likely play vital roles in diversifying the useable nucleic acid components available to the intracellular parasite, often through the direct capture of resources from the host. Using an in vivo viability assay, we also demonstrate that one of these genes, TK, encodes an enzyme that is capable of activating known prodrugs to their active form, which suggests a possible treat- ment route for microsporidiosis. We further argue that interfacial genes with well-understood activities, especially those horizontally transferred from bacteria or viruses, could provide medical treat- ments for microsporidian infections.

How did nature engineer the highest surface lipid accumulation among plants? Exceptional expression of acyl-lipid-associated genes for the assembly of extracellular triacylglycerol by Bayberry (Myrica pensylvanica) fruits

Jeffrey P. Simpson; Nicholas Thrower; John B. Ohlrogge

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2016

Bayberry (Myrica pensylvanica) fruits are covered with a remarkably thick layer of crystalline wax consisting of triacylglycerol (TAG) and diacylglycerol (DAG) esterified exclusively with saturated fatty acids. As the only plant known to accumulate soluble glycerolipids as a major component of surface waxes, bayberry represents a novel system to investigate neutral lipid biosynthesis and lipid secretion by vegetative plant cells. The assembly of bayberry wax is distinct from conventional TAG and other surface waxes, and instead proceeds through a pathway related to cutin synthesis (Simpson and Ohlrogge, 2016). In this study, microscopic examination revealed that the fruit tissue that produces and secretes wax (bayberry knobs) is fully developed before wax accumulates and that wax is secreted to the surface without cell disruption. Comparison of transcript expression to genetically related tissues (bayberry leaves, M. rubra fruits), cutin-rich tomato and cherry fruit epidermis, and to oil-rich mesocarp and seeds, revealed exceptionally high expression of 13 transcripts for acyl-lipid metabolism together with down-regulation of fatty acid oxidases and desaturases. The predicted protein sequences of the most highly expressed lipid-related enzyme-encoding transcripts in bayberry knobs are 100% identical to the sequences from bayberry leaves, which do not produce surface DAG or TAG. Together, these results indicate that TAG biosynthesis and secretion in bayberry is achieved by both up and down-regulation of a small subset of genes related to the biosynthesis of cutin and saturated fatty acids, and also implies that modifications in gene expression, rather than evolution of new gene functions, was the major mechanism by which bayberry evolved its specialized lipid metabolism. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

Identification of 4-O-5-units in softwood lignins via definitive lignin models and NMR

Fengxia Yue; Fachuang Lu; Sally Ralph; John Ralph

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2016

Lignins are complex and heterogeneous natural polymers in which the major units are characterized by certain prominent interunit linkages. Previous attempts to identify and quantify 4-O-5-linked units in softwood lignins by NMR were not successful. In this work, various lignin model compounds, including the tetramers formed by the 4-O-5-coupling of beta-O-4-, beta-beta-, and beta-5-model dimers, were synthesized. Such compounds are better able to model the corresponding structures in lignins than those used previously. 4-O-5-Linked structures could be clearly observed and identified in real softwood lignin samples by comparison of their 2D HSQC NMR spectra with those from the model compounds. When comparing NMR data of phenol-acetylated versus phenol-etherified model compounds with those of acetylated lignins, it was apparent that most of the 4-O-5-linked structures in softwood lignins are present as free-phenolic end units.

Identification of multiple lipid genes with modifications in expression and sequence associated with the evolution of hydroxy fatty acid accumulation in Physaria fendleri

Patrick J. Horn; Jinjie Liu; Jean-Christophe Cocuron; Kate McGlew; Nicholas A. Thrower; Matt Larson; Chaofu Lu; Ana Paula Alonso; John Ohlrogge

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2016

Inconvenient truths about landowner (Un)willingness to grow dedicated bioenergy crops

Bradford L. Barham; Daniel F. Mooney; Scott M. Swinton

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2016

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