Project Research Publications: Feedstocks

  • Casler, M.D., Vermerris, W. and Dixon, R.A. (2015). Replication concepts for bioenergy research experiments. BioEnergy Research 8(1):1-16. DOI: 10.1007/s12155-015-9580-7

      • Abstract:

      • While there are some large and fundamental differences among disciplines related to the conversion of biomass to bioenergy, all scientific endeavors involve the use of biological feedstocks. As such, nearly every scientific experiment conducted in this area, regardless of the specific discipline, is subject to random variation, some of which is unpredictable and unidentifiable (i.e., pure random variation such as variation among plots in an experiment, individuals within a plot, or laboratory samples within an experimental unit) while some is predictable and identifiable (repeatable variation, such as spatial or temporal patterns within an experimental field, a glasshouse or growth chamber, or among laboratory containers). Identifying the scale and sources of this variation relative to the specific hypotheses of interest is a critical component of designing good experiments that generate meaningful and believable hypothesis tests and inference statements. Many bioenergy feedstock experiments are replicated at an incorrect scale, typically by sampling feedstocks to estimate laboratory error or by completely ignoring the errors associated with growing feedstocks in an agricultural area at a field or farmland (micro- or macro-region) scale. As such, actual random errors inherent in experimental materials are frequently underestimated, with unrealistically low standard errors of statistical parameters (e.g., means), leading to improper inferences. The examples and guidelines set forth in this paper and many of the references cited are intended to form the general policy and guidelines for replication of bioenergy feedstock experiments to be published in BioEnergy Research.

      • https://doi.org/10.1007/s12155-015-9580-7

  • Felderhoff, T.J., McIntyre, L.M., Saballos, A. and Vermerris, W. (2016). Using genotyping by sequencing to map two novel anthracnose resistance loci in Sorghum bicolor. G3 (Genes Genomes Genetics) 6(7):1935-1946. DOI: 10.1534/g3.116.030510

      • Abstract:

      • Colletotrichum sublineola is an aggressive fungal pathogen that causes anthracnose in sorghum [Sorghum bicolor (L.) Moench]. The obvious symptoms of anthracnose are leaf blight and stem rot. Sorghum, the fifth most widely grown cereal crop in the world, can be highly susceptible to the disease, most notably in hot and humid environments. In the southeastern United States the acreage of sorghum has been increasing steadily in recent years, spurred by growing interest in producing biofuels, bio-based products, and animal feed. Resistance to anthracnose is, therefore, of paramount importance for successful sorghum production in this region. To identify anthracnose resistance loci present in the highly resistant cultivar 'Bk7', a biparental mapping population of F3:4 and F4:5 sorghum lines was generated by crossing 'Bk7' with the susceptible inbred 'Early Hegari-Sart'. Lines were phenotyped in three environments and in two different years following natural infection. The population was genotyped by sequencing. Following a stringent custom filtering protocol, totals of 5186 and 2759 informative SNP markers were identified in the two populations. Segregation data and association analysis identified resistance loci on chromosomes 7 and 9, with the resistance alleles derived from 'Bk7'. Both loci contain multiple classes of defense-related genes based on sequence similarity and gene ontologies. Genetic analysis following an independent selection experiment of lines derived from a cross between 'Bk7' and sweet sorghum 'Mer81-4' narrowed the resistance locus on chromosome 9 substantially, validating this QTL. As observed in other species, sorghum appears to have regions of clustered resistance genes. Further characterization of these regions will facilitate the development of novel germplasm with resistance to anthracnose and other diseases.

      • https://doi.org/10.1534/g3.116.030510

  • Kadam, S., Abril, A., Dhanapal, A.P., Koester, R.P., Vermerris, W., Jose, S. and Fritschi, F.B. (2017). Characterization and regulation of aquaporin genes of sorghum [Sorghum bicolor (L.) Moench] in response to waterlogging stress. Frontiers in Plant Science 8:862. DOI: 10.3389/fpls.2017.00862

      • Abstract:

      • Waterlogging is a significant environmental constraint to crop production, and a better understanding of plant responses is critical for the improvement of crop tolerance to waterlogged soils. Aquaporins (AQPs) are a class of channel-forming proteins that play an important role in water transport in plants. This study aimed to examine the regulation of AQP genes under waterlogging stress and to characterize the genetic variability of AQP genes in sorghum (Sorghum bicolor). Transcriptional profiling of AQP genes in response to waterlogging stress in nodal root tips and nodal root basal regions of two tolerant and two sensitive sorghum genotypes at 18 and 96 h after waterlogging stress imposition revealed significant gene-specific pattern with regard to genotype, root tissue sample, and time point. For some tissue sample and time point combinations, PIP2-6, PIP2-7, TIP2-2, TIP4-4, and TIP5-1 expression was differentially regulated in tolerant compared to sensitive genotypes. The differential response of these AQP genes suggests that they may play a tissue specific role in mitigating waterlogging stress. Genetic analysis of sorghum revealed that AQP genes were clustered into the same four subfamilies as in maize (Zea mays) and rice (Oryza sativa) and that residues determining the AQP channel specificity were largely conserved across species. Single nucleotide polymorphism (SNP) data from 50 sorghum accessions were used to build an AQP gene-based phylogeny of the haplotypes. Phylogenetic analysis based on single nucleotide polymorphisms of sorghum AQP genes placed the tolerant and sensitive genotypes used for the expression study in distinct groups. Expression analyses suggested that selected AQPs may play a pivotal role in sorghum tolerance to water logging stress. Further experimentation is needed to verify their role and to leverage phylogenetic analyses and AQP expression data to improve waterlogging tolerance in sorghum.

      • https://doi.org/10.3389/fpls.2017.00862

  • Jun, S.Y., Walker, A.M., Kim, H., Ralph, J., Vermerris, W., Sattler, S.E. and Kang, C.H. (2017). The enzyme activity and substrate specificity of the major cinnamyl alcohol dehydrogenases in sorghum (Sorghum bicolor), SbCAD2 and SbCAD4. Plant Physiology 174(4):2128-2145. DOI: 10.1104/pp.17.00576

      • Abstract:

      • Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in monolignol biosynthesis, reducing sinapaldehyde, coniferaldehyde, and p-coumaraldehyde to their corresponding alcohols in an NADPH-dependent manner. Because of its terminal location in monolignol biosynthesis, the variation in substrate specificity and activity of CAD can result in significant changes in overall composition and amount of lignin. Our in-depth characterization of two major CAD isoforms, SbCAD2 (Brown midrib 6 [bmr6]) and SbCAD4, in lignifying tissues of sorghum (Sorghum bicolor), a strategic plant for generating renewable chemicals and fuels, indicates their similarity in both structure and activity to Arabidopsis (Arabidopsis thaliana) CAD5 and Populus tremuloides sinapyl alcohol dehydrogenase, respectively. This first crystal structure of a monocot CAD combined with enzyme kinetic data and a catalytic model supported by site-directed mutagenesis allows full comparison with dicot CADs and elucidates the potential signature sequence for their substrate specificity and activity. The L119W/G301F-SbCAD4 double mutant displayed its substrate preference in the order coniferaldehyde > p-coumaraldehyde > sinapaldehyde, with higher catalytic efficiency than that of both wild-type SbCAD4 and SbCAD2. As SbCAD4 is the only major CAD isoform in bmr6 mutants, replacing SbCAD4 with L119W/G301F-SbCAD4 in bmr6 plants could produce a phenotype that is more amenable to biomass processing.

      • https://doi.org/10.1104/pp.17.005760

  • Rao, P.S., Vinutha, K.S., Kumar, G.S.A., Chiranjeevi, T., Uma, A., Lal, P., Prakasham, R.S., Singh, H.P., Rao, R.S., Chopra, S. and Jose, S. (2016). Sorghum: A Multipurpose Bioenergy Crop. In: Ciampitti, I. and Prasad, V. (eds.), Sorghum: State of the Art and Future Perspectives, Agronomy Monograph 58. ASA and CSSA, Madison, WI. DOI: 10.2134/agronmonogr58.2014.0074

      • Abstract:

      • Bioethanol and biodiesel produced from renewable energy sources are gaining importance in light of volatile fossil fuel prices, depleting oil reserves, and increasing greenhouse effects associated with the use of fossil fuels. Among several alternative renewable energy sources, energy derived from plant biomass is found to be promising and sustainable. Sorghum [Sorghum bicolor (L.) Moench] is a resilient dryland cereal crop with wide adaptation having high water, nutrient, and radiation use efficiencies. This crop is expected to enhance food, feed, fodder, and fuel security. Sweet sorghum is similar to grain sorghum but has the ability to accumulate sugars in the stalks without much reduction in grain production. Hence, it is used as a first-generation biofuel feedstock, where the grain and stalk sugars can be used for producing bioenergy, while energy sorghum or biomass sorghum is increasingly viewed as a potential feedstock for lignocellulosic biofuel production. Although the commercial use of sweet sorghum for bioethanol production has been demonstrated in China and India, the viability of large-scale lignocellulosic conversion of sorghum biomass to biofuels is yet to be demonstrated. This chapter dwells on sorghum feedstock characteristics, biofuel production models, sustainability indicators, and commercialization.

      • https://doi.org/10.2134/agronmonogr58.2014.0074

  • Sattler, S.A., Walker, A.M., Vermerris, W., Sattler, S.E. and Kang, C.H. (2017). Structural and biochemical characterization of cinnamoyl-CoA reductases. Plant Physiology 173(3):1031-1044. DOI: 10.1104/pp.16.01671

      • Abstract:

      • Cinnamoyl-coenzyme A reductase (CCR) catalyzes the reduction of hydroxycinnamoyl-coenzyme A (CoA) esters using NADPH to produce hydroxycinnamyl aldehyde precursors in lignin synthesis. The catalytic mechanism and substrate specificity of cinnamoyl-CoA reductases from sorghum (Sorghum bicolor), a strategic plant for bioenergy production, were deduced from crystal structures, site-directed mutagenesis, and kinetic and thermodynamic analyses. Although SbCCR1 displayed higher affinity for caffeoyl-CoA or p-coumaroyl-CoA than for feruloyl-CoA, the enzyme showed significantly higher activity for the latter substrate. Through molecular docking and comparisons between the crystal structures of the Vitis vinifera dihydroflavonol reductase and SbCCR1, residues threonine-154 and tyrosine-310 were pinpointed as being involved in binding CoA-conjugated phenylpropanoids. Threonine-154 of SbCCR1 and other CCRs likely confers strong substrate specificity for feruloyl-CoA over other cinnamoyl-CoA thioesters, and the T154Y mutation in SbCCR1 led to broader substrate specificity and faster turnover. Through data mining using our structural and biochemical information, four additional putative CCR genes were discovered from sorghum genomic data. One of these, SbCCR2, displayed greater activity toward p-coumaroyl-CoA than did SbCCR1, which could imply a role in the synthesis of defense-related lignin. Taken together, these findings provide knowledge about critical residues and substrate preference among CCRs and provide, to our knowledge, the first three-dimensional structure information for a CCR from a monocot species.

      • https://doi.org/10.1104/pp.16.01671

  • Shukla, S., Felderhoff, T.J., Saballos, A. and Vermerris, W. (2017). The relationship between plant height and sugar accumulation in the stems of sweet sorghum (Sorghum bicolor (L.) Moench). Field Crops Research 203:181-191. DOI: 10.1016/j.fcr.2016.12.004

      • Abstract:

      • Sweet sorghum (Sorghum bicolor (L.) Moench) is a tall, seed-propagated C4 grass with stems that contain saccharine juice. The sugars in the juice can be easily extracted with a press, which generates large amounts of bagasse. Sweet sorghum has potential as a multi-purpose crop whereby depending on the available infrastructure and market demands, all fermentable sugars from juice and biomass can be converted to renewable fuels and chemicals, or the juice can be processed to syrup, fuels, or chemicals, while the bagasse is either burned or used as fodder. Large-scale industrial production of sweet sorghum requires large amounts of seed, but due to their height, sweet sorghums are not compatible with existing combine harvesters. In addition, grain yield is often limited. The availability of hybrid seed that can be produced on short seed parents would enable combine harvesting and offer greater seed yield. This requires the availability of short, sweet inbred lines. All known sweet sorghums, however, are tall, and prior research identified a positive correlation between height and sugar accumulation. Since the physiological mechanisms underlying sugar accumulation in sweet sorghum are not well understood, it is unknown whether the apparent association between sugar accumulation in the stem and plant height is the result of selection, or dictated by physiological or genetic constraints. Three experiments were conducted to examine this relationship. First, the role of shading on sugar accumulation was examined in breeding populations with contrasting heights. Second, the sugar concentration was compared between short plants harboring the unstable dwarf3 (dw3) allele and their tall Dw3 revertants. Third, tall photoperiod-sensitive lines were compared with their matching short, photoperiod-insensitive lines. The results from these three experiments indicated that high sugar concentration in sweet sorghum is not conditional on the plants being tall, making the development of short, sweet inbred lines feasible. This information will also significantly benefit studies aimed at identifying QTL for sugar yield in sweet sorghum.

      • https://doi.org/10.1016/j.fcr.2016.12.004

  • Ten, E. and Vermerris, W. (2015). Recent developments in polymers derived from industrial lignin. Journal of Applied Polymer Science 132(24):42069. DOI: 10.1002/app.42069

      • Abstract:

      • Lignin is an aromatic polymer that makes up 15–30% of the cell walls of terrestrial plants. While lignin's role in facilitating water transport through the vasculature, providing rigidity and acting as a defense against pests and pathogens is important for the plant's survival, industries that process plant biomass for the production of biofuels and bio-based chemicals have historically primarily been interested in the cell wall polysaccharides, especially cellulose. Consequently, lignin is generated in large quantities as a by-product that is often burned to generate heat and electricity, or that is used in low-value applications. It is becoming clear that, rather than treating it as waste, lignin is very suitable for the production of enhanced composites, carbon fibers, and nanomaterials, which offers both economic and environmental benefits. This review highlights recent uses of these polymers as adsorbents, flocculants, adhesives, anti-oxidants, energy storing films, and vehicles for drug delivery and gene therapy.

      • https://doi.org/10.1002/app.42069

  • Vermerris, W. and Abril, A. (2015). Enhancing cellulose utilization for fuels and chemicals by genetic modification of plant cell wall architecture. Current Opinion in Biotechnology 32:104–112. DOI: 10.1016/j.copbio.2014.11.024

      • Abstract:

      • Cellulose from plant biomass can serve as a sustainable feedstock for fuels, chemicals and polymers that are currently produced from petroleum. In order to enhance economic feasibility, the efficiency of cell wall deconstruction needs to be enhanced. With the use of genetic and biotechnological approaches cell wall composition can be modified in such a way that interactions between the major cell wall polymers — cellulose, hemicellulosic polysaccharides and lignin — are altered. Some of the resulting plants are compromised in their growth and development, but this may be caused in part by the plant's overcompensation for metabolic perturbances. In other cases novel structures have been introduced in the cell wall without negative effects. The first field studies with engineered bioenergy crops look promising, while detailed structural analyses of cellulose synthase offer new opportunities to modify cellulose itself.

      • https://doi.org/10.1016/j.copbio.2014.11.024

  • Walker, A.M., Sattler, S.A., Regner, M., Jones, J.P., Ralph, J., Vermerris, W., Sattler, S.E. and Kang, C.H. (2016). The structure and catalytic mechanism of Sorghum bicolor caffeoyl-CoA O-methyltransferase. Plant Physiology 172(1):78-92. DOI: 10.1104/pp.16.00845

      • Abstract:

      • Caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) is an S-adenosyl methionine (SAM)-dependent O-methyltransferase responsible for methylation of the meta-hydroxyl group of caffeoyl-coenzyme A (CoA) on the pathway to monolignols, with their ring methoxylation status characteristic of guaiacyl or syringyl units in lignin. In order to better understand the unique class of type 2 O-methyltransferases from monocots, we have characterized CCoAOMT from sorghum (Sorghum bicolor; SbCCoAOMT), including the SAM binary complex crystal structure and steady-state enzyme kinetics. Key amino acid residues were validated with site-directed mutagenesis. Isothermal titration calorimetry data indicated a sequential binding mechanism for SbCCoAOMT, wherein SAM binds prior to caffeoyl-CoA, and the enzyme showed allosteric behavior with respect to it. 5-Hydroxyferuloyl-CoA was not a substrate for SbCCoAOMT. We propose a catalytic mechanism in which lysine-180 acts as a catalytic base and deprotonates the reactive hydroxyl group of caffeoyl-CoA. This deprotonation is facilitated by the coordination of the reactive hydroxyl group by Ca2+ in the active site, lowering the pKa of the 3′-OH group. Collectively, these data give a new perspective on the catalytic mechanism of CCoAOMTs and provide a basis for the functional diversity exhibited by type 2 plant OMTs that contain a unique insertion loop (residues 208–231) conferring affinity for phenylpropanoid-CoA thioesters. The structural model of SbCCoAOMT can serve as the basis for protein engineering approaches to enhance the nutritional, agronomic, and industrially relevant properties of sorghum.

      • https://doi.org/10.1104/pp.16.00845

  • Weerasekara, C. (2017). MS Thesis. Nitrogen and Harvest Impact on Warm-Season Grass Biomass Yield Feedstock Quality. Center for Agroforestry, University of Missouri, Columbia, MO. Thesis supervisor: Jose, S.

      • Abstract: Not Available